Advances in Parasitology Volume 59

CONTRIBUTORS TO VOLUME 59 M. S. ABRAHAMSEN, Department of Veterinary and Biomedical Science, College of Veterinary Medi...

Author: John R. Baker | R. Muller | David Rollinson

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CONTRIBUTORS TO VOLUME 59 M. S. ABRAHAMSEN, Department of Veterinary and Biomedical Science, College of Veterinary Medicine, University of Minnesota, 1988 Fitch Avenue, St Paul, MN 55108, USA and Biomedical Genomics Center, University of Minnesota, 1988 Fitch Avenue, St Paul, MN 55108, USA C. ELSO, Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, CA 94550-9234, USA S. ENOMOTO, Faculty of Genetics Program, Texas A&M University, College Station, TX 77843-4467, USA and Department of Veterinary and Biomedical Science, College of Veterinary Medicine, University of Minnesota, 1988 Fitch Avenue, St Paul, MN 55108, USA S. FOOTE, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia E. HANDMAN, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia B. HANELT, Department of Biology, 167 Castetter Hall, University of New Mexico, Albuquerque, NM 87131-0001, USA N. S. HIJJAWI, World Health Organization Collaborating Centre for the Molecular Epidemiology of Parasitic Infections, School (not Division) of Veterinary and Division of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, WA 6150, Australia

ADVANCES IN PARASITOLOGY VOL 59 ISSN: 0065-308X DOI: 10.1016/S0065-308X(05)59005-5

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CONTRIBUTORS TO VOLUME 59

R. A. MATTHEWS, School of Biological Sciences, University of Plymouth, Drake Circus, Plymouth PL4 8AA, UK M. E. OLSON, Department of Microbiology and Infectious Diseases, University of Calgary, 3900 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1 A. SCHMIDT-RAESA, Zoomorphologie und Systematik, Fakulta¨t fu¨r Biologie, Universita¨t Bielefeld, Postfach 100131, D-33501 Bielefeld, Germany F. THOMAS, GEMI/UMR, CNRS-IRD 2724, 911 Avenue Agropolis, B.P. 5045, 34032 Montpellier Cedex 1, France R. C. A. THOMPSON, World Health Organization Collaborating Centre for the Molecular Epidemiology of Parasitic Infections, School (not Division) of Veterinary and Division of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, WA 6150, Australia G. ZHU, Department of Veterinary Pathobiology and Faculty of Genetics Program, Texas A&M University, College Station, TX 77843-4467, USA

PREFACE The opening chapter in this volume, by Emanuela Handman and Colleen Elso of the Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia and Simon Foote of the Laurence Livermore National Laboratory, California, USA, echoes that of the previous volume in describing the interactions between host and parasite in Leishmania. The present review shows how recent research on human and animal genome sequences and on gene mapping and cloning is enabling the identification of disease susceptibility genes, which will help to prevent and treat the various forms of leishmaniasis. The authors describe how the mouse has proved to be a particularly fruitful host since there are inbred strains with different susceptibilities to infection, particularly to T-cell responses. Combined with clonal parasite lines, unaccountable variations in parasite virulence can be removed and the genes involved in host response to infection will be increasingly obtainable. The second paper by Andrew Thompson and Nawal Hyjjawi of Murdoch University, Australia, Merle Olsen of the University of Calgary, Canada, Guan Zhu and Shinichiro Enomoto of Texas A & M University, USA and Mitchell Abrahamansen of the University of Minnesota, USA, provides a comprehensive review of all aspects of recent research on Cryptosporidium in a variety of hosts. Most work over the last 10 years has concentrated on the development and application of molecular tools to solve questions about the epidemiology and zoonotic potential of this parasite. However, recent developments in in vitro cultivation, life cycle (including previously unknown stages), propagation and elucidation of the complete genome sequence of one species is helping to answer important questions on its biology and host–parasite relationships.


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Cryptosporidium has been found to differ greatly from all other coccidians in life cycle, morphology and biochemistry and shows similarities to the gregarines; the authors think it is clear that its phylogenetic affinities need re-evaluation. R. A. (Tony) Matthews, from the School of Biological Sciences in the University of Plymouth, UK, has contributed a review of the perhaps little-known but economically important ciliate Ichthyophthirius multifiliis. This organism, commonly known as ‘ich’ (pronounced ‘ik’) for obvious reasons, is an endoparasite of freshwater teleost fish, causing the disease known colloquially as ‘white spot.’ The ciliate has a direct life cycle, with free-living and endoparasitic stages, the latter in the fishes’ epidermis where it reproduces by binary fission. In the encysted free-living stage, repeated binary fission gives rise to numerous tomites, which leave the cyst and re-enter the epidermis of other fish. Surprisingly, sexual reproduction (conjugation), though likely, has not yet been conclusively demonstrated. This review describes the life cycle and morphology of the various stages in detail, and discusses the immunology, pathogenesis and control and treatment of the infection, including attempts, not yet fully successful, to develop a commercial vaccine. There is one metazoan phylum, whose members have a parasitic way of life, which has received scant attention from research biologists. Hopefully, the closing paper in this volume on the biology of the Nematomorpha will help to address the balance. There are two main groups of nematomorphs (also known as hair worms): the nectonematids are parasites of marine invertebrates and the gordiids are parasites of terrestrial arthropods. The adults are free-living and can be found in marine and freshwaters, respectively. Pulling together their complementary expertise Ben Hanelt from the University of New Mexico, USA, Fre´de´ric Thomas from Montpellier, France and Andreas Schmidt-Rhaesa from the University of Bielefeld, Germany provide a comprehensive overview of the Nematomorpha paying special attention to recent advances concerning morphology, taxonomy and systematics, life cycle and ecology and

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the influence of the parasite on host behaviour. There is much of interest here and maybe the review will encourage others to look at the neglected hairworms. John Baker Ralph Muller David Rollinson

Contents CONTRIBUTORS TO VOLUME 59 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Genes and Susceptibility to Leishmaniasis E. Handman, C. Elso and S. Foote 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Leishmania and Leishmaniasis . . . . . . . . . . . . . . . . . . . . . . . . Animal Models of Leishmaniasis . . . . . . . . . . . . . . . . . . . . . . The Immunology of Murine Cutaneous Leishmaniasis and its Role in Host Resistance to Disease . . . . . . . . . . . . . . . . . . . . . The Immunology of Human Cutaneous Leishmaniasis and its Role in Host Resistance to Disease . . . . . . . . . . . . . . . . . . . . . The Genetic Tools for Analysis of Host Response to Infection in the Mouse. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Genetic Tools for Analysis of Host Response in Humans . . The Genetics of Host Response to Leishmaniasis . . . . . . . . . . . Environmental Factors Influencing Leishmaniasis. . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Cryptosporidium and Cryptosporidiosis R. C. A. Thompson, M. E. Olson, G. Zhu, S. Enomoto, M. S. Abrahamsen and N. S. Hijjawi 1. 2.

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phylogenetic Relationships and Taxonomy . . . . . . . . . . . . . . .


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Life Cycle and Development. . . . . . . . . . Host–Parasite Relationship. . . . . . . . . . . The Regulation of Biochemical Processes. Epidemiology and Transmission . . . . . . . Control. . . . . . . . . . . . . . . . . . . . . . . . . Perspectives for the Future . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . .

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Ichthyophthirius multifiliis Fouquet and Ichthyophthiriosis in Freshwater Teleosts R. A. Matthews 1. 2. 3. 4. 5. 6. 7. 8.

Abstract . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . Free-Living Stages . . . . . . . . . . . . . . . Infection of the Fish Host . . . . . . . . . Trophont . . . . . . . . . . . . . . . . . . . . . Immunity to Ichthyophthirius multifiliis Ichthyophthiriosis . . . . . . . . . . . . . . . Control and Treatment . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

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Biology of the Phylum Nematomorpha B. Hanelt, F. Thomas and A. Schmidt-Rhaesa 1. 2. 3. 4. 5.

Abstract . . . . . . . . . . . . . . . Morphology . . . . . . . . . . . . Taxonomy and Systematics . Life Cycle and Ecology . . . . Host Behavioural Alterations General Conclusion . . . . . . . Acknowledgements . . . . . . . References . . . . . . . . . . . . .

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INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONTENTS OF VOLUMES IN THIS SERIES . . . . . . . . . . . . . . . . . . . . . . . . . . The Colour Plate Section appears between pages 177 and 178.

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Genes and Susceptibility to Leishmaniasis Emanuela Handman1,, Colleen Elso2 and Simon Foote1 1

The Walter and Eliza Hall Institute of Medical Research, Post Office, Royal Melbourne Hospital, Victoria 3050, Australia 2 Lawrence Livermore National Laboratory, 7000 East Ave, Livermore, CA 94550, USA

Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Leishmania and Leishmaniasis . . . . . . . . . . . . . . . . . . . . . Animal Models of Leishmaniasis . . . . . . . . . . . . . . . . . . . . The Immunology of Murine Cutaneous Leishmaniasis and its Role in Host Resistance to Disease . . . . . . . . . . . . . . . . . . 4.1. The Innate Immune Response in Murine Leishmaniasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. The Role of the Adaptive Immune Response in Leishmaniasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. The Role of Cytokines and Other Immunomodulatory Molecules in Murine Leishmaniasis . . . . . . . . . . . . . . 4.4. Transcription Factors Involved in the Th1/Th2 Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. The Immunology of Human Cutaneous Leishmaniasis and its Role in Host Resistance to Disease . . . . . . . . . . . . . . . . . . 6. The Genetic Tools for Analysis of Host Response to Infection in the Mouse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Multiple Strain Comparisons . . . . . . . . . . . . . . . . . . . 6.2. Mapping Host Response Loci in the Mouse . . . . . . . . 6.3. Intercrosses or Backcrosses . . . . . . . . . . . . . . . . . . . 6.4. Recombinant Inbred Strains (RIS) . . . . . . . . . . . . . . . 6.5. Recombinant Congenic Strains (RCS) . . . . . . . . . . . . 1. 2. 3. 4.

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Author for correspondence.


Copyright r 2005 Elsevier Ltd All rights of reproduction in any form reserved

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6.6. Common Haplotype Mapping . . . . . . . . . . . . . . . . 6.7. Congenic Strains (CS) . . . . . . . . . . . . . . . . . . . . . 6.8. Gene Discovery and Validation . . . . . . . . . . . . . . . The Genetic Tools for Analysis of Host Response in Humans. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Association Studies . . . . . . . . . . . . . . . . . . . . . . . 7.2. Linkage Analysis . . . . . . . . . . . . . . . . . . . . . . . . . 7.3. Affected Family Member Studies. . . . . . . . . . . . . . 7.4. Future Approaches to Mapping Host Response Genes in Human Populations . . . . . . . . . . . . . . . . The Genetics of Host Response to Leishmaniasis . . . . . 8.1. Genetics of Susceptibility to Leishmaniasis in Mice. 8.2. Genetics of Susceptibility to Leishmaniasis in Humans. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Environmental Factors Influencing Leishmaniasis . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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ABSTRACT Leishmania are digenetic protozoa which inhabit two highly specific hosts, the sandfly where they grow as motile, flagellated promastigotes in the gut, and the mammalian macrophage where they grow intracellularly as non-flagellated amastigotes. Leishmaniasis is the outcome of an evolutionary ‘arms race’ between the host’s immune system and the parasite’s evasion mechanisms which ensure survival and transmission in the population. The spectrum of disease manifestations and severity reflects the interaction between the genome of the host and that of the parasite, and the pathology is caused by a combination of host and parasite molecules. This chapter examines the genetic basis of host susceptibility to disease in humans and animal models. It describes the genetic tools used to map and identify susceptibility genes, and the lessons learned from murine and human cutaneous leishmaniasis.

GENES AND SUSCEPTIBILITY TO LEISHMANIASIS

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1. INTRODUCTION ‘‘From moments after birth, we constantly breathe, eat, and come into physical contact with millions of microorganisms until we are consumed by them at death (Jones and Falkow, 1996).’’

Not surprisingly, the interaction of humans with the microbial world has, to a large extent shaped human as well as microbial evolution. However, there is no clear consensus on what constitutes a pathogen, and why some, but not all individuals get sick. The ‘Damage–Response’ framework of microbial pathogenesis proposed by Casadevall and Pirofski (2003) provides a model within which to consider the outcome of the inevitable encounters that we have with the microbial world around us. The model is based on three tenets that (i) pathogenesis is the consequence of the interaction between the genome of the host and that of the microbe and is attributable neither to the host alone nor to the microbe alone, (ii) the outcome of this interaction is defined by the degree of damage caused to the host, (iii) the damage to the host is caused by a combination of host and microbe molecules. Since the host and the microbe are inextricably bound in a war of their genomes, it is no wonder that it has been so difficult to reach consensus on the host genes responsible for susceptibility to most infections. In the ‘Damage–Response’ framework, a pathogen is defined as an organism capable of causing damage to the host. The microbemediated damage can result from production of virulence factors, such as toxins or from subversion of host homeostatic or immune mechanisms. Host-mediated damage can result from the host response to the microbes or their products. Although the outcomes of the interaction between the host and microbe is a continuum and damage can occur throughout the interaction, it is most prominent at the extremes, when the host either mounts an ineffectual response or mounts a response that is very strong qualitatively or quantitatively (Figure 1 is Plate 1.1 in the Separate Color Plate section) that it can damage tissues and lead to organ dysfunction (Casadevall and Pirofski, 2003). This model focuses on the immune system as the major player in the ‘Damage–Response’ because of the perception

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of the critical role of the immune system as ‘‘a weapon of mass destruction invented by evolution to even the odds during the war of the DNAs’’ (Cohn, 2002). This is the framework in which we will examine the data on the genetics of host response to Leishmania infection.

2. LEISHMANIA AND LEISHMANIASIS Leishmaniasis is the umbrella name for a group of parasitic diseases affecting around 12 million people, mostly in the developing world, with an estimated incidence of 1.5–2 million new cases each year (WHO, 2000). The type and severity of disease depends on the species and strain of parasite, environmental factors and the genetic predisposition to disease of the infected host. Even in the case of a single disease entity, such as cutaneous leishmaniasis, where there is a good animal model of disease, the genetics of the host response to infection is complex. This complexity has caused difficulty in the identification of genes underlying the observed naturally occurring differences in disease susceptibility. With the availability of the human and mouse genome sequences and better tools for gene mapping and cloning, there is hope that progress will be made more rapidly. We hope that the identification of disease susceptibility genes will make a contribution to the prevention and treatment of leishmaniasis. Leishmania species are protozoan parasites transmitted by the phlebotomine sandfly. They infect a range of animal hosts including humans, rodents and dogs. The parasite exists as a motile, flagellated ‘promastigote’ in the sandfly and a non-motile ‘amastigote’ within macrophages of the infected host (Figure 2). The amastigote form divides by binary fission, eventually escaping from the macrophage to infect surrounding macrophages. Amastigotes are taken up by the female phlebotomine sandfly when it takes a blood meal from an infected host. The parasites emerge from the macrophages in the gut of the sandfly and undergo a series of developmental changes leading to the mature or infectious form, the metacyclic promastigote (Sacks,


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Sandfly

Attachment

Macrophage

Proliferation

Intracellular amastigote Lysis

Transformation

Mammalian host

Figure 2 The Leishmania life cycle. Leishmania are transmitted by the bite of infected female sandflies, which regurgitate a small number of infectious metacyclic promastigotes into a pool of blood in the skin. These forms are phagocytosed by blood monocytes or macrophages and targeted to phagolysosomes where they transform into the non-motile amastigotes. Amastigotes are taken up by sandflies with their blood meal into the gut where they transform into the flagellated promastigotes. The promastigotes undergo a maturation program culminating with the infectious metacyclic forms in the mouthparts ready for transmission to the vertebrate hosts.

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1988, 1989). These metacyclic promastigotes are delivered to the mammalian host when a subsequent blood meal is taken, and they infect macrophages, transform into the amastigote form, thus completing the cycle. Most stages of the life cycle can be mimicked in the laboratory. Amastigotes can be isolated from an infected animal or human and the transformation to the promastigote form takes place in tissue culture. Depending on the species of Leishmania that is studied, animals can be infected with promastigotes by intradermal or intravenous injection. Depending on the species studied, amastigotes can be generated in cell-free cultures under conditions that simulate those present in the macrophage phagolysosome, i.e. 33–371C and pH 5.0. In humans, infection with parasites causing cutaneous leishmaniasis in the Old World, such as Leishmania major or L. tropica usually causes a cutaneous ulcer of 6–12 months duration. However, there is evidence that disease severity varies considerably; in some individuals lesions caused by infection with L. tropica are not curable. This may be due to an inability to mount an appropriate immune response. In these individuals, most of the tissue damage is caused by the host immune response and it occurs in the presence of very few parasites in the lesions (Jacobson, 2003). Similarly, New World cutaneous leishmaniasis caused by the L. braziliensis complex can lead to metastasis to the mucosal membranes resulting in severe facial disfigurement (Tapia et al., 1994; Bosque et al., 2000). L. donovani and L. infantum cause a visceral form of the disease where the parasite targets the internal organs and is often fatal without treatment (Herwaldt, 1999). Treatment involves use of highly toxic antimonial drugs, which often have severe side effects, but efforts are underway to identify new therapies (Croft and Coombs, 2003; Davis et al., 2004). To date, there is no vaccine against any form of leishmaniasis. However, in the case of cutaneous leishmaniasis caused by L. major, controlled inoculation with live parasites (carried out in an inconspicuous spot to hide scarring) has been used for many years in Iran, Russia and Israel in order to provide protection against subsequent infection (Handman et al., 1974; Greenblatt, 1980). This process carries the risk that


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parasites remain latent in the host after cure of the initial lesion with the possibility of recrudescence when the host sustains some trauma or immunosuppression (Aebischer et al., 1993; Mendonca et al., 2004). HIV/AIDS patients are at increased clinical risk of leishmaniasis as latent infections reactivate and new infections cannot be eliminated (WHO, 2000).

3. ANIMAL MODELS OF LEISHMANIASIS Generally the search for genetic determinants of response to parasites in humans has met with little success. As discussed in the Introduction, this is due in part to the heterogeneous nature of the populations of hosts and parasites being studied. This heterogeneity refers not only to the genetics of the population, but also the different exposure of individuals to environmental variables ranging from nutrition and health status, to rates of exposure to the vector and infection with parasites of varying virulence. In the case of leishmaniasis, since the disease is primarily a zoonosis, susceptible animals have been used to establish animal models of the various types observed in the human disease. By doing so, many of the environmental variables can be controlled. Using clonal parasite lines also removes variation in parasite virulence (Handman et al., 1983). Thus, animal models for leishmaniasis include several rodent species such as mouse, gerbil, guinea pig and hamster, as well as dogs and monkeys (Sacks and Melby, 1998). The use of the mouse has advantages for studies of the genetics of susceptibility to disease. One of the important observations that made the mouse the preferred animal model for cutaneous leishmaniasis has been the fact that inbred strains of mice have been shown to differ significantly in their susceptibility to infection with L. major (Preston and Dumonde, 1976; Handman et al., 1979). In addition, mice have a relatively short generation time and their immune system is well studied. Most importantly, a large number of tools have been developed which aid in their genetic analysis, including the availability of many inbred strains of mice, genetic and physical maps, the

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availability of the complete genome sequence, YAC and bacterial artificial chromosome (BAC) libraries and many protocols for genetic manipulation such as specific gene deletion by homologous recombination. Mice also show many similarities to humans in their anatomy, physiology, immunology and genome, allowing the potential transfer of knowledge gained in the murine system to the human disease (Copeland and Jenkins, 1999).

4. THE IMMUNOLOGY OF MURINE CUTANEOUS LEISHMANIASIS AND ITS ROLE IN HOST RESISTANCE TO DISEASE Leishmaniasis in the mouse has been used as a model to study T cell responses to intracellular microbial pathogens (Locksley, 1997). To a large extent, the interest in this model stemmed from the early studies showing that cell-mediated immune responses are necessary and sufficient to explain susceptibility to L. major infection (Preston et al., 1972; Preston and Dumonde, 1976; Handman et al., 1979). Later studies showed that resistance or susceptibility to leishmaniasis was associated with the T helper 1 (Th1) and Th2 type immune responses, respectively (see below). The current understanding of the immune response to infection by L. major is described in Figure 3 (Figure 3 is Plate 1.3 in the Separate Color Plate section). Initially, cells of the innate immune response, such as dendritic cells (DCs), neutrophils and natural killer (NK) cells are responsible for detection and initial containment of the pathogen. Their activation leads, on the one hand, to recruitment of additional cells of the inflammatory response, and on the other to antigen presentation to naı¨ ve T cells and to priming of the adaptive immune response. Through these interactions, T cells become primed to develop into either Th1 or Th2 type cells, thought to promote healing or disease, respectively, through their effect on the activation of macrophages to kill the intracellular parasites, or subversion of these processes and exacerbation of disease (Sacks and Noben-Trauth, 2002). Each of the cells interacts with others at the site of inflammation by secretion of, and response to,


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cytokines and chemokines. It is possible that genes controlling response to infection with L. major could be acting at any of these points in the detection and clearance of the parasites. Therefore, it is valuable to consider the role of each component of the immune response with respect to susceptibility and resistance to L. major infection in more detail.

4.1. The Innate Immune Response in Murine Leishmaniasis The innate, as opposed to the adaptive immune response is initiated instantaneously when a ‘danger signal’ in the form of a foreign microbe is detected. It is evolutionarily conserved throughout most multicellular organisms and initiates a protective response against a broad range of pathogens (Matzinger, 1998; Kimbrell and Beutler, 2001). No previous exposure to the antigen is required to ‘prime’ the innate response, as cells are activated by recognition of ‘microbial patterns’ by receptors such as lectins and Toll-like receptors (Medzhitov and Janeway, 2000; Kaisho and Akira, 2003). The inflammatory response initiated by the innate immune response is thought to limit the spread of infectious agents and to prime and regulate the adaptive immune response (Kimbrell and Beutler, 2001). Cells of the innate immune system are particularly important in infection with L. major as it is these cells, mainly macrophages but also DCs and neutrophils, which are both the host cells of the parasite and the effector cells responsible for parasite clearance (Prina et al., 2004; Ribeiro-Gomes et al., 2004). 4.1.1. The role of macrophages in murine leishmaniasis Macrophages play a major role in the mammalian response to L. major, being both the host cell for the parasite, required for parasite survival and replication in the mammalian host, and the effector cell, responsible for killing of parasites to clear infection. Many mechanisms are involved in the fine balance between death and survival.

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The promastigote form of Leishmania has been shown to subvert macrophages by inhibiting their production of IL-12 after infection. IL-12 is required for the optimal production of IFN-g from T cells, which then results in activation of the macrophage. In vitro studies have shown that this occurs in macrophages from both resistant and susceptible mice and the inhibition is restricted to IL-12, with other inflammatory cytokines only slightly affected (Reiner, 1994; Carrera et al., 1996). Infection with L. major, L. amazonensis or L. braziliensis promastigotes has also been shown to lead to the secretion of the anti-inflammatory cytokines IL-10 and TGF-b (Sacks and Noben-Trauth, 2002). Both the invading promastigotes and the obligatory intracellular amastigotes enter the macrophage by receptor-mediated uptake after attachment to macrophage receptors, which initiate phagocytosis. In the case of promastigotes, the complement receptors 1 and 3 (CR1, CR3), the fibronectin receptor and mannose receptor have been shown to play a role. In the case of amastigotes, the Fc receptors have been implicated in uptake as well as persistence of infection (Kima et al., 2000). On the parasite side, at least in L. major, the surface lipophosphoglycan (LPG) and proteophosphoglycans (PPG) have been implicated, as well as the glycoproteins leishmanolysin and gp46/ parasite surface antigen 2 (PSA-2) (Ilgoutz and McConville, 2001; Turco et al., 2001). Much less is known about the parasite molecules involved in the uptake of other species of Leishmania (Turco et al., 2001) or of amastigotes. It has been shown that phagocytosis of metacyclic promastigotes is increased by C3b and C3bi complement opsonisation (Mosser and Edelson, 1987). Opsonised promastigotes are taken up via CR1 and CR3 (da Silva et al., 1988, 1989). In addition to facilitating entry into the cell, use of these ‘silent’ receptors does not result in the release of inflammatory cytokines and thus results in a much lower respiratory burst compared with either non-opsonised parasites or uptake via different receptors. This is one system the parasite exploits to evade the host innate immune response allowing it to establish and survive in the phagolysosome of the macrophage. Parasite establishment may


11

also be facilitated by down-regulation of several signalling pathways leading to macrophage deactivation. This may be due, at least in part, to the activation by Leishmania molecules of several negative regulators such as the phosphotyrosine phosphatase SHP-1 (Nandan et al., 2002) and suppressor of cytokine signalling 3 (SOCS3) (Bertholet et al., 2003; Kubo et al., 2003). As noted above, the macrophage is also the final effector cell, and the mechanism used by macrophages to kill parasites involves activation of the macrophage by IFN-g to produce inducible nitric oxide synthase (iNOS), an enzyme which catalyses the production of nitric oxide (NO) from L-arginine (Liew et al., 1990b). It has been suggested that the difference in the ability of the Th1, but not Th2-type immune responses to clear infection is due to the ability of macrophages to be activated to produce NO, a molecule toxic to many microbes. According to this model, in a response dominated by Th1 cells, IFN-g will be produced, activating the macrophage to produce iNOS and therefore NO and facilitate killing of parasites. If Th2 cells are present, IFN-g-induced activation of macrophages will not occur and, in the absence of NO, parasites will survive and replicate. The importance of iNOS was confirmed in an iNOS knockout mouse, which despite being on a genetically resistant background was susceptible to L. major infection in the presence of a strong Th1-type cytokine response (Wei et al., 1995). Also, it has been shown that mice recovered from infection require continuous iNOS activity in order to prevent recurrence of disease caused by the persistent parasites present even in otherwise immune hosts (Stenger et al., 1996). Interestingly, the role of iNOS is not limited to the adaptive immune response. The expression and activity of iNOS could be detected in the first 24 h of infection and was dependent on the presence of type I interferons (IFNa/b), suggesting that these are critical regulators of the innate immune response to L. major (Diefenbach et al., 1998). We have evidence that the plasmacytoid DCs are a major producer of type I IFN, but their role in leishmaniasis has just started to be studied (Baldwin et al., 2004). In macrophages, iNOS mRNA and protein are undetectable unless the cells are activated by IFN-g or by microbial products. Attempts

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have been made to identify the individual components of L. major that induce or inhibit iNOS production in macrophages (reviewed in Bogdan (1997) and Liew et al. (1997)). The membrane-bound glycoinositolphospholipids (GIPLs) as well as PPG and LPG, present in both membrane-bound and secreted forms in L. major promastigotes are capable of down-regulating iNOS expression. However, amastigotes or LPG in the presence of IFN-g can up-regulate its expression. The context in which the parasite is seen by the macrophage is undoubtedly important for the effect on iNOS production and subsequently the effect on the balance between killing of parasites and evasion of this process. The Fas/FasL system has also been reported to play a role in the ability of macrophages to control parasite replication. It is thought to be involved both in apoptosis and in stimulating macrophages to their full microbicidal function (Chakour et al., 2003). Recently, it has been recognised that macrophages exposed to different cytokine environments can differentiate into distinct subsets that perform specific immunological functions. The classically activated macrophages, described above, have been studied in detail. On the other hand, it has become apparent that macrophages exposed to Th2-type cytokines turn on an alternative activation programme. An examination of the nature and function of these cells in leishmaniasis is very recent (Noe¨l et al., 2004). 4.1.2. The role of dendritic cells in murine leishmaniasis Over the past decade, DCs have come to prominence as an important link between the innate and adaptive arms of the immune system (Banchereau et al., 2000). Early studies by Moll (1993) led to the concept that Langerhans cells, the specialised DCs found in the skin where the parasites are deposited by the sand fly, and where infection is most likely to occur, transport L. major to the draining lymph nodes where they present antigen to T cells. Although all DCs can be infected with Leishmania both in vitro and in vivo (Henri et al., 2002; Baldwin et al., 2004), their role in the initiation of infection and in the transport of parasites to the lymph nodes has been questioned in


13

studies showing that it is macrophages in the skin that harbour the first parasites and that they are the cells that migrate out of the infected skin and most likely transport the parasites to the draining lymph node (Belkaid et al., 2000; von Stebut et al., 2000; Baldwin et al., 2004). Moreover, in real life infections, it is likely that bloodderived monocytes may play a much more important role as the primary infected cell than originally considered because the parasites are deposited into the pool of blood created by the sandfly bite at the site. More recently, the focus has shifted from the DCs role in antigen capture and presentation to the way they can influence the ensuing adaptive immune response. It was determined that some populations of DCs could produce IL-12 in response to the uptake of antigen (Macatonia et al., 1995; von Stebut et al., 1998) and were therefore thought to be able to induce a Type 1 T cell response. This led to the proposal that distinct DC populations, ‘DC1’ or ‘DC2’ subsets are capable of inducing a Type 1 or Type 2 T cell response, respectively (Rissoan et al., 1999). Currently, it is thought that the type and dose of microbe, the local tissue environment and extra regulatory signals, for example CD40 (Marovich et al., 2000; MacDonald et al., 2002), may all play a role in the response of DCs to antigen (Kelsall et al., 2002; Scott and Hunter, 2002). TLR4 and its associated adapter protein MyD88 have been shown to be important in clearance of L. major infection. TLR4 detection of L. major associated molecular pattern molecules and subsequent downstream signalling is thought to result in DC secretion of IL-12 (Kropf et al., 2004; Muraille et al., 2003). Further confusing the potential role of DCs with respect to L. major infection has been the discovery that DCs can themselves be infected by L. major as well as L. mexicana parasites. Different subsets of DCs are differentially permissive to infection and this seems to be inversely related to their ability to produce IL-12p70 (Henri et al., 2002). The mechanism controlling the induction of IL-12 in these cells and the functional differences between the IL-12-producing cells and the non-producers are still to be determined. To date, several studies have investigated differences between BALB/c and C57BL/6 DCs

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with respect to L. major infection. von Stebut et al. (2000), using foetal skin-derived DCs, found no difference between the ability of BALB/c and C57BL/6 DCs to be infected, up-regulate activation markers or produce cytokines, including IL-12p70. Moll et al. (2002), on the other hand, investigated the cytokine receptors found on the surface of Langerhans cells from the ear epidermis. They found that BALB/c DCs up-regulate IL-4 receptor when infected with L. major and down-regulate the production of IL-12p40. In studies in vivo, using the metacyclic promastigotes-ear infection model of cutaneous leishmaniasis Baldwin et al. (2004) found that the main difference between the BALB/c and C57BL/6 mice was an increased number of plasmacytoid DCs in the lymph nodes of the susceptible mice. Similarly, there was a 10-fold larger number in the BALB/c skin early in infection compared to C57BL/6J. The relevance of these findings to the pathogenesis of disease remains to be established. 4.1.3. The role of additional mechanisms of innate immunity in murine leishmaniasis In addition to macrophages and DCs, several other cellular and noncellular components of the innate immune system play a role in the response to infection with L. major. The cells of the innate immune system attracted to the site of infection with L. major have been studied. A mixture of cells including neutrophils, macrophages, eosinophils and lymphocytes is recruited very rapidly. Resistant and susceptible strains have been shown to recruit these cells in different proportions, with neutrophils constituting a much higher proportion of the cells recruited by BALB/c mice compared with C57BL/6. BALB/c mice have high numbers until at least day 8 post-infection. In C57BL/6 mice the neutrophils are replaced with high numbers of monocyte/macrophages (Beil et al., 1992; Tacchini-Cottier et al., 2000). Several groups have investigated the role of the neutrophil in infection with L. major with contradictory results. Lima et al. (1998) showed that depletion of neutrophils before infection results in an increase in disease features and parasite load in both resistant and susceptible mice during the first weeks after infection, whereas


15

Tacchini-Cottier et al. (2000) report that depletion of neutrophils in BALB/c mice leads to abrogation of early IL-4 production and alters the T cell response in these mice to a curative Type 1, rather than the usual disease exacerbating Type 2 response. They found no difference in infection of C57BL/6 mice depleted of neutrophils (Tacchini-Cottier et al., 2000). The difference between these two studies may be explained by the use of a different L. major strain and dose of injected organisms. Both groups however agree that there is an effect due to neutrophils early in infection. L. major promastigotes have been shown to produce a chemoattractant for neutrophils and to use the neutrophils as an initial host before the appearance of macrophages. There are little data on the transformation of promastigotes to amastigotes or replication of the parasites within neutrophils. However, infected neutrophils are induced to produce chemokines conducive to parasite survival (Van Zandbergen et al., 2002). This agrees with earlier work indicating that neutrophils are inefficient at killing parasites (Beil et al., 1992) and, together with the abundant and prolonged neutrophil infiltration seen in BALB/c mice, may be in part responsible for the susceptibility of BALB/c mice. Interestingly, it has also been shown that dead neutrophils ingested by macrophages may contain factors able to modulate the parasite/macrophage relationship (Ribeiro-Gomes et al., 2004). NK cells have also been shown to be important early in the innate response to L. major infection. Several groups have shown, through depletion experiments, that NK cells are responsible for early containment of the parasites at the site of infection and the draining lymph nodes (Laskay et al., 1993, 1995; Scharton and Scott, 1993). This occurs in the resistant mice, but not in the susceptible BALB/c mice, that show dissemination of parasites within 10–24 h postinfection to organs throughout the body (Laskay et al., 1995). Depletion of NK cells in C57BL/6 mice results in BALB/c-like dissemination, while activation of NK cells in BALB/c results in decreased spread and ameliorated disease. IFN-g has been shown to be important in this process. One study, using transplanted T cells into NK cell-deficient mice where almost no NK cell development was detected, indicated that NK cells were not important in the

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IL-12-mediated production of a Th1 immune response and subsequent cure in resistant mice (Satoskar et al., 1999). However, the majority of studies support a host-protective role for NK cells early in infection. Two recent studies focussing on the NK-activating chemokines have shown differences in the expression of these chemokines in resistant and susceptible mice (Vester et al., 1999; Robertson, 2002), indicating that despite the presence of similar numbers of NK cells in the lymph nodes of BALB/c and C57BL/6 mice, BALB/c NK cells are not being activated. Treatment of BALB/c mice with the NK activating chemokine INF-g-inducible protein 10 (IP-10) resulted in increased NK activation indicating that these cells are potentially able to be activated if they receive the correct stimulus. The role of chemokines in the innate immune response to infection is one of co-ordinating the trafficking and activation of the many and varied cell types involved in the response. Chemokines are produced in instances of both inflammation and tissue wounding by a variety of cell types including infiltrating lymphocytes, neutrophils, macrophages and mast cells, as well as resident fibroblasts, epithelial and endothelial cells (Gillitzer and Goebeler, 2001; Matte and Olivier, 2002). Not surprisingly, since chemokines regulate cell traffic and tissue localisation of effector cells, many chemokines and chemokine receptors have been implicated in the response to L. major. For example, monocyte chemotactic protein 1 (MCP-1) has been shown in vitro to activate human monocytes to kill parasites and IL-4 can inhibit this process (Ritter and Moll, 2000). Also in humans, the CCL2/MCP1 chemokine profile is detected in localised cutaneous leishmaniasis lesions in contrast to diffuse cutaneous leishmaniasis where MIP-1a/CCL3 dominate (Ritter and Korner, 2002). In the mouse model, the impact of CCL2/MCP1 has been demonstrated in mice where deletion of the gene encoding the receptor for CCL2/ MCP1 leads to susceptibility to infection (Sato et al., 2000). In another study, the chemokines MIP-1a; -1b; RANTES (regulated on activation normal T cell-expressed and -secreted protein) and lymphotactin have been shown to be co-expressed with IFN-g in individual cells. As well as being chemoattractants for cells such as monocytes, basophils, eosinophils and lymphocytes, they are able to co-activate,


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with IFN-g; macrophages to up-regulate production of IL-12, TNF-a and CD40, which could be important in the response to L. major (Dorner et al., 2002). The innate immune response to infection, therefore, has the complex role of containing infection and attracting and activating effector cells from both the innate and adaptive arms of the immune system. Differences in the innate response between resistant and susceptible strains of mice may be crucial in determining whether a successful, host protective, or deleterious immune response is induced by infection.

4.2. The Role of the Adaptive Immune Response in Leishmaniasis 4.2.1. The role of T cells in murine cutaneous leishmaniasis Different strains of inbred mice show different levels of susceptibility to infection with L. major. BALB/c mice are highly susceptible while CBA/H and C57BL/6 are resistant. However, all three strains, when containing the nu/nu mutation rendering them unable to produce T cells, were highly susceptible, indicating that T cells are required for the rapid cure seen in intact C57BL/6 and CBA/H mice (Handman et al., 1979). Further work using irradiated BALB/c mice reconstituted with various cell types confirmed a role for CD4+ T cells and not B cells or CD8+ T cells (Mitchell et al., 1980; Howard et al., 1981). Also, no protective effect was elicited after antibody transfer, indicating that there is no role for B cells in resistance to infection with L. major (Mitchell and Handman, 1983). Reconstitution with different numbers of normal spleen cells indicated that a resistant or susceptible phenotype could be elicited in BALB/c mice depending on the transfer of low or high numbers of cells, respectively (Mitchell et al., 1981b). When spleen cells from chronically infected BALB/c mice were transferred into irradiated mice, disease progression was observed after infection. When a similar experiment was done with cells from healed mice, the mice receiving the cells remained resistant. This indicated that a very fine balance between disease promoting

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and suppressing cells existed, that some sort of threshold of disease promotion was acting and that this balance was shifted depending on the state of the transferred T cells (Mitchell et al., 1981b). Confusingly, both the susceptibility and resistance effects seemed to be mediated by CD4+ T cells. However, light was soon to be thrown on this strange situation with the discovery of functionally different subsets of CD4+ or ‘helper’ T cells. 4.2.2. T helper cell polarisation In 1986, Mosmann et al. (1986) reported the identification of two distinct mouse T helper cell subsets, based on the pattern of cytokines they produced in culture. They defined Th1 cells as producing IL-2, IFN-g; GM-CSF and IL-3 when stimulated, and Th2 cells as producing IL-3, BSF-1 and two other growth factors not identified at that time (later IL-4, IL-5 and IL-13 were identified as Th2 type cytokines). Soon after, adoptive transfer experiments using polarised T cell lines demonstrated that in the mouse model for leishmaniasis, host ‘protective’ T cells were of the Th1 phenotype and ‘non-protective’ T cells were of the Th2 type (Scott et al., 1988; Holaday et al., 1991). At the same time, it was shown that in L. major-infected C57BL/6 mice, healing was associated with an increase in IFN-g production in spleen cells, whereas susceptible BALB/c mice showed an increase in IL-4 after infection (Heinzel et al., 1989). This was also shown in the draining lymph nodes (Heinzel et al., 1991). In addition, BALB/c mice treated with antibodies to CD4, a treatment previously shown to efficiently remove CD4+ T cells and render BALB/c mice resistant to infection (Titus et al., 1985), were shown to be repopulated with IFN-g- and IL-2-producing T cells. Treatment with antibodies to IL-4 also resulted in an increase in IFN-g-producing cells and a decrease in IL-4-producing cells (Heinzel et al., 1991). These data supported the involvement of the two subsets of T helper cells in resistance and susceptibility to disease. Investigation of the cytokine profile of single T cell clones soon after infection with L. major also supports a role for these subsets. Two groups have reported that T cells from draining lymph nodes of both BALB/c and C57BL/6


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mice produce IL-4 or IFN-g as early as 1 week after infection. However, later in infection, when the disease pattern becomes polarised, i.e. the C57BL/6 mice cure and the BALB/c progress towards severe disease, the predominant population of T cells from C57BL/6 mice produces IFN-g without IL-4, whereas BALB/c T cells predominantly produce IL-4 (Morris et al., 1992; Sommer et al., 1998). 4.2.3. The early T cell response to L: major infection In addition to the innate immune response acting very rapidly in response to L. major infection, it has been reported that a subset of T cells also contributes to the immediate response. Launois et al. (1995) showed that in the susceptible strains BALB/c and DBA/2, a burst of IL-4 mRNA was detected in the draining lymph nodes within 16 h of infection in the footpad. C57BL/6, C3 H and CBA on the other hand produced no IL-4 during the 48 h post-infection and maintained a low level of production throughout infection. BALB/c mice down-regulated this burst after 48 h, but a second increase was detected 96 h after infection to a very high level of IL-4 which was maintained throughout infection. No IFN-g was detected in any of the strains this early in infection (Launois et al., 1995). Manipulation of several Th1/Th2 cytokines before the burst of IL-4 was shown to be effective in reversing both the disease phenotype and the elicited T helper-type response. For example, treatment of BALB/c mice with recombinant IL-12 suppressed the early IL-4 burst and converted mice into resistance with a Th1-type response. Concomitant treatment of the mice with antibodies against IFN-g resulted in abrogation of this effect, thus indicating that the switch to the Th1 response was acting via an IFN-g-mediated pathway. The cells responsible for the IL-4 burst were shown to be CD4+ T cells (Launois et al., 1995), which are Vb4; Va8-restricted (Launois et al., 1997). Earlier work had determined that this subset of T cells was expanded in draining lymph nodes (Reiner et al., 1993) and responded to an immunodominant antigen from L. major called LACK (Leishmania homologue of mammalian RACK1 (receptors of activated C kinases), Mougneau et al., 1995). Tolerance to this antigen had

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previously been shown to induce resistance in BALB/c mice, supporting the involvement of these cells in susceptibility (Julia et al., 1996). The Vb4; Va8 CD4+ T cell subset was proposed to be of a memory/effector cell phenotype, primed by cross-reactive microbial antigens in the gut (Julia et al., 2000). However, the ability of this subset to be manipulated to a Th1-type response argues for a less-differentiated population of T cells. It is possible that these manipulations caused a new, previously unidentified, Th1 population to arise (Maillard et al., 2001). It has been suggested that the presence of the IL-4 burst in susceptible, but not resistant mice, may be due to an imbalance in the numbers of this T cell subset or an inherent property of BALB/c T cells to produce IL-4. However, recent work has shown that C57BL/6 mice have similar numbers of Vb4; Va8 CD4+ T cells as BALB/c mice and that, in C57BL/6 mice treated with antibodies against either IL-12 or IFN-g; a similar burst of IL-4 from this subset of T cells is observed (Launois et al., 2002; Stetson et al., 2002). It is now proposed that resistant mice have an even earlier (10–12 h post-infection) burst of IL-12 and IFN-g that results in inhibition of the IL-4 burst seen in the susceptible strains. Surprisingly, other studies including ours have not been able to detect this early burst of IL-4 in the susceptible BALB/c mice, and this may be due to the use of a different site of infection and a different strain of L. major (reviewed in Sacks and Noben-Trauth, 2002; Elso et al., 2004a). 4.2.4. Regulatory T cells Very recently, a subset of T cells identified by the surface markers CD4+CD25+ have been found to play an important regulatory role in many immune processes in the mouse. Initially identified through their role in suppressing T cell-mediated autoimmune diseases (Sakaguchi et al., 1995), these regulatory T cells (T reg) have now been implicated in the response to infectious disease (Aseffa et al., 2002; Belkaid et al., 2002; Xu et al., 2003; Mendez et al., 2004). T reg cells are found in increased numbers at the site of infection in mice infected with L. major and, through a series of adoptive transfer studies, have been shown to reduce the magnitude of both the Th2


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response in susceptible BALB/c mice and the Th1 response in resistant C57BL/6 mice. In BALB/c mice, removal of T reg cells results in an increase in disease severity (Belkaid et al., 2002). However, the presence of T reg cells is important for the persistence of concomitant immunity to reinfection. This immunity is dependent on the persistence of parasites in the immune animals, and their survival is maintained through the production of IL-10, which in turn is regulated by the T reg cells (Belkaid et al., 2002). In further studies it has been shown that superinfection can alter the extremely fine balance between T reg and effector T cells with implications for recrudescence (Mendez et al., 2004). While T reg cells have not yet been characterised in humans, it is tempting to speculate that they are a key factor in establishing and maintaining the latent infection seen in humans that can result in recurrence of the disease when changes in the host immune system occur, whether due to disease such as HIV/ AIDS, immunosuppressive treatment or due to ageing.

4.3. The Role of Cytokines and Other Immunomodulatory Molecules in Murine Leishmaniasis Many cytokines and other molecules have been implicated in the response to infection with L. major. It is beyond the scope of this review to fully document the role of them all, but we will touch on some of the cytokines and accessory molecules and pathways that may be responsible for the dichotomy in response to infection in resistant and susceptible inbred strains of mice. 4.3.1. Interleukin-4 and -10 As discussed in the previous section, IL-4 has been shown to be produced during the first 24 h post-infection in susceptible BALB/c mice. Data supporting the importance of IL-4 early in infection show that treatment of BALB/c mice with antibodies against IL-4 at the time of infection leads to a Th1 response and healing in an

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IFN-g-dependent manner. However, treatment of the resistant C3 H mice with recombinant IL-4 did not result in disease susceptibility, but rather a transient Th2 response which reverted to a Th1 response over time (Chatelain et al., 1992). This indicated that IL-4 was necessary but not sufficient for susceptibility. A later report argued that IL-4 was both necessary and sufficient to initiate the maturation of Th2 cells and susceptibility to infection with L. major (Himmelrich et al., 2000). This group used BALB/c mice rendered resistant by a deficiency in Vb4 T cells, and thus loss of the subset of T cells shown to produce the early burst of IL-4 observed in BALB/c mice. By treating the mice with IL-4 the phenotype reverted to susceptibility and a concomitant Th2 response. Depletion of CD4+ T cells in addition to treatment with IL-4 abrogated this result indicating that the action of IL-4 on T cells was necessary for susceptibility (Himmelrich et al., 2000). These data indicate that the genetic background of affected mice play a role in whether IL-4 is sufficient to induce susceptibility. An attempt to correlate levels of IL-4 at 1 week postinfection with disease outcome failed to do so, finding rather that early IL-12 was a more reliable predictor of disease outcome, as IL-4 was detected in C57BL/6 as well as susceptible strains at this stage (Scott et al., 1996). However, IL-4 levels late in an established infection were found to be a reliable indicator of disease, whereas IFNg levels were not (Morris et al., 1993). IL-10 was discovered due to its ability to inhibit cytokine production by Th1 cell clones, thereby earning classification as a Type 2 cytokine. It acts on macrophages to suppress IFN-g production by Th1 cells (Fiorentino et al., 1991). Treatment of BALB/c mice rendered resistant by depletion of CD4+ T cells with IL-4, IL-10 or both can inhibit Th1 function in vivo (Powrie et al., 1993) as evidenced by a suppression of IFN-g production. Treatment of resistant or susceptible mice with antibodies against IL-10 resulted in no change in the phenotype showing that it is not necessary for normal resistance or susceptibility (Chatelain et al., 1999), but knocking out the IL-10 gene in BALB/c mice resulted in intermediate resistance (Kane and Mosser, 2001). Infection with L. mexicana and L. amazonensis is also less severe in BALB/c mice lacking IL-10, with an associated increase


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in Th1 cytokines (Padigel et al., 2003). However, knocking out IL-10 on a C57BL/6 background produced some startling results (Belkaid et al., 2001). Contrary to the typical persistence of L. major parasites after cure of overt disease even in these resistant mice (Aebischer et al., 1993), the lack of IL-10 resulted in sterile cure of the mice. This was also seen in mice lacking both IL-10 and IL-4 but not in those lacking only IL-4. Treatment of C57BL/6 mice with antibodies to IL-10 after natural healing of the lesions also resulted in sterile cure. This result gives IL-10 a very clear and important role in the long-term persistence of parasites after cure from disease. Recently, the source of this IL-10 may have been discovered. As discussed previously, the production of IL-10 by T reg cells is required for a persistent chronic infection to remain (Belkaid et al., 2002). Observations from IL-4 knockout mice have been less clear, with one report showing that the disruption of IL-4 on a BALB/c background conferred susceptibility (Kopf et al., 1996) but another that it resulted in no or only partial cure, depending on the strain of L. major used (Noben-Trauth et al., 1999). The response of a BALB/c IL-4 receptor knockout was also shown to be parasite-straindependent. Both groups, however, reported that no increase in IFN-g or other known inflammatory cytokines was observed in these mice. A second group investigating the response of BALB/c mice lacking the IL-4 receptor found that no impairment of the Th2 response was detected, indicating that development of the Type 2 response is independent of signalling through this receptor (Mohrs et al., 2000). Further work using IL-4R- or IL-10-deficient mice has confirmed that both cytokines are important for susceptibility and that while disruption of one or the other gene confers only partial resistance on normally susceptible mice, disruption of both genes confers significantly more resistance (Noben-Trauth et al., 2003). In contrast to the data discussed thus far, some research has shown that IL-4 does not always induce a Th2-type response (Carter et al., 1989; Biedermann et al., 2001). If resistant Vb4-deficient BALB/c mice are treated with IL-4 at infection and 8 h post-infection, resistance is maintained. However, treatment extending over 64 h postinfection results in a susceptible phenotype. In vitro studies indicated

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that resistance was due to the effect of IL-4 on DCs, resulting in the stimulation of IL-12 and a Th1-type response. IL-4 present later, when T cells were being primed, resulted in T cells being driven towards a Th2 phenotype (Biedermann et al., 2001). Therefore, the timing of the production of IL-4 may have an effect on the resultant T helper cell response and the outcome of infection. Although most of the data points to IL-4 as necessary for the initiation of a Th2 response, whether it is sufficient to cause disease susceptibility depends on the strain of mouse and the timing of its presence. Production of IL-4 as early as 16 h after infection with L. major has been shown to elicit a Th2 response and a susceptible phenotype. IL-10 plays a lesser role in the establishment of the Th2 response but has a crucial role for the long-term persistence of parasites in the immune host after recovery from disease. 4.3.2. Interferon-g and IL-12 IFN-g and IL-12 are the two cytokines involved in the initiation of the Th1 response. IFN-g or IL-12 act on macrophages to produce more IL-12, which then allows CD4+ T cells to differentiate into Th1 cells, themselves capable of producing IFN-g: Positive feedback allows the amplification of the inflammatory response. IFN-g acting on macrophages can then induce the production of NO and oxygen radicals required for parasite killing. Moreover, cross-talk between these cytokines and the Th2-type cytokines is thought to downregulate the production of the Th2 cytokines, thus driving the immune response towards a sustained Th1 response. Even before the description of the two T helper cell subsets, IFN-g production by cells from the lymph nodes draining the infection site was found to correlate with resistance. Namely, T cells from C57BL/6 mice stimulated in vitro with L. major antigens produced IFN-g; whereas cells from BALB/c mice did not (Sadick et al., 1986). Surprisingly, treatment of the relatively resistant C3H/He mice with antibodies to IFN-g did not convert these mice into a completely susceptible phenotype. The mice initially became susceptible but eventually cured (Belosevic et al., 1989). However, more evidence for


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the importance of IFN-g came when it was shown that 3 days postinfection with L. major, lymph node cells from BALB/c produced high levels of IL-4 but low IFN-g in contrast to C3H/He mice, which produced high levels of IFN-g and no IL-4 (Scott, 1991). Treatment of C3H/He with antibodies to IFN-g resulted in up-regulation of Th2 cytokines and disease, while treatment of BALB/c mice with IFN-g resulted in delayed disease with a transient switch to a Th1 response (Scott, 1991). The conclusion was that one or more cytokine(s) in addition to IFN-g must have been acting to promote the Th1 response. IL-12 was discovered to have properties, which recommended it for this role. It was found to be produced by macrophages in response to inflammatory stimuli and to promote cell proliferation and IFN-g release by T and NK cells. It was also found to be inhibited by IL-10, TGF-b and IL-4 (Heinzel, 1994). Treatment of BALB/c mice with recombinant IL-12 during the first week post-infection resulted in cure with a decrease in IL-4 and increase in IFN-g production. This cure was dependent on IFN-g (Heinzel et al., 1993). Further work on this model revealed that IL-4 was required for disease even in the absence of IFN-g or IL-12, and that IL-12 was required for the stimulation of IFN-g-producing cells (Heinzel et al., 1995). Knocking out either the IFN-g gene or its receptor in resistant mice resulted in completely uncontrolled growth of the parasite and rapid death of the host from systemic disease (Wang et al., 1994; Swihart et al., 1995). The IFN-g-deficient mice produced a Th2 response, whereas mice lacking the IFN-g receptor maintained a Th1 response, presumably through a sustained inflammatory response due to prolonged antigenic stimulation. Although IFN-g was present, it was unable to activate macrophages to produce NO to kill parasites in the absence of its receptor. Having established that IFN-g and IL-12 were important for both host resistance and the development of a Th1 response, it remained to elucidate how these cytokines could affect the Th1/Th2 balance. A series of studies addressed this issue, identifying differential expression of the IL-12Rb2 chain on Th1 and Th2 cells as a possible mechanism (Guler et al., 1996; Szabo et al., 1997; Himmelrich et al.,

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1998). It was shown that IL-12 signalling was lost very early in Th2 cells due to the loss of expression of the receptor. While present in the cells of both resistant and susceptible mice up to 2 days after infection (and activation through the T cell receptor), after 48 h, T cells from BALB/c mice could no longer respond to IL-12. IFN-g treatment of early Th2 cells allowed them to maintain expression of the receptor and IL-4 was shown to inhibit its expression. This indicated that IL4-induced down-regulation of the IL-12Rb2 chain may be responsible for the susceptibility of BALB/c mice as their T cells were not able to receive the IL-12 stimulus required to differentiate into Th1 cells (Szabo et al., 1997; Himmelrich et al., 1998). Work on a transgenic model with constant expression of the IL-12Rb2 chain has called into question this hypothesis as cells expressing the receptor are still able to differentiate into Th2 cells (Heath et al., 2000) and BALB/c mice containing the transgene exhibit a non-healing phenotype with a strong Th2 response, even when treated with exogenous IL-12. This suggests that down-regulation of IL-12Rb2 on Th2 cells is not required for Th2 development but may be necessary for Th1 development by depriving Th2 cells of the proliferative stimulus that IL-12 confers (Nishikomori et al., 2001). A recent study showed that deletion of the IL-12Rb2 gene in C57BL/6 mice resulted in the production of a Th2-dominated response to infection with L. major, lending weight to this hypothesis (Chakir et al., 2003). Recently, both IL-12p35 and p40 have been shown to play a role late in infection and in maintenance of immunity to secondary infection (Park et al., 2000; Stobie et al., 2000). It is thought that they may be required to sustain memory/effector Th1 cells and to prevent their loss due to activation-induced apoptosis. This was demonstrated using IL-12p35 and p40 knockout mice. In contrast to untreated mice that were unable to control infection, mice treated with IL-12 during the first 2 weeks after infection were able to cure the disease but did not develop immunity to secondary infection. This was apparent even in the absence of a Th2 response (Park et al., 2000, 2002). IFN-g and IL-12 therefore have important roles in the initiation and maintenance of a Th1-type response. The mechanisms for the selective development of Th1 or Th2 cells are still not clear but are


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likely to be affected by the cytokines produced in the early hours after infection. The resultant down-regulation of IL-12Rb2 in Th2 cells may still play an important role in the development of Th1 cells by depriving Th2 cells of the IL-12 proliferative signal. 4.3.3. TNF-a; TGF-b and accessory molecules TNF-a is a pro-inflammatory cytokine, which induces NO-mediated parasite killing by macrophages (Liew et al., 1990b). It has been shown to play a role in resistance to L. major. Treatment of resistant mice with antibodies against TNF-a led to increased susceptibility, while intra-lesion injection of recombinant TNF-a resulted in less severe disease (Liew et al., 1990a). Disruption of the gene encoding TNF-a resulted in uncontrolled infection in C57BL/6 mice, but interestingly without any skin ulceration (Wilhelm et al., 2001). TNF-a can signal through two different receptors, TNFRp55 (TNFR1) and TNFRp75 (TNFR2). Disruption of the TNFR2 was shown to have no effect on infection. Also, the loss of TNFR1 or both receptors together did not alter the normal Th1 response and clearance of the parasites, but a role for signalling through these receptors was indicated in resolution of the lesion, possibly due to involvement in the down-regulation of the inflammatory response (Vieira et al., 1996; Nashleanas et al., 1998). Not surprisingly, due to the complexity of the immune system, many other molecules have been shown to play a part in the inflammatory response resulting in clearance of the Leishmania parasites. Among these are membrane-bound molecules, which act as accessory signals on binding of the T cell receptor to the MHC–peptide complex. The B7 molecules and their interaction with CTLA-4, which inhibits T cell activation, or CD28, which activates the T cell response, are examples of such a system. CTLA-4 engagement has been shown to induce expression of TGF-b: TGF-b is thought to have a down-regulatory effect on macrophages and has been shown to modulate T cell subsets. These effects differ depending on the stage of maturation of the T cell, promoting Th1 cells early in differentiation but favouring Th2 cells at more mature stages of development (Gomes and DosReis, 2001). The depletion of TGF-b can increase

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resistance to established L. major infection, while treatment with TGF-b can make mice more susceptible to visceral leishmaniasis (Wilson et al., 1998; Li et al., 1999). Knocking out the gene encoding CTLA-4 resulted in the production of a Th2 response, which led to susceptibility to L. major (Masteller et al., 2000). This could be explained by the resultant loss of stimulation of TGF-b; which promotes early Th1 differentiation. Another interaction shown to be important in inflammatory responses is the CD40–CD40L (CD154) interaction. This interaction has been shown to be involved in the activation and differentiation of a number of cell types in the immune system. As well as influencing T cells, it has been shown to up-regulate DCs to produce IL-12 and up-regulate macrophages to produce inflammatory mediators (Grewal and Flavell, 1998). Disruption of either CD40 or CD40L has indicated that the interaction is required for successful resistance to infection with L. major (Campbell et al., 1996; Grewal and Flavell, 1998). Similarly, BALB/c mice stimulated through CD40 become more resistant to infection (Ferlin et al., 1998).

4.4. Transcription Factors Involved in the Th1/Th2 Response The signalling events underlying the development and cross inhibition of the different effector subsets of T helper cells is becoming better understood. Naı¨ ve T cells are activated upon binding of peptide–MHC to the appropriate T cell receptor. In addition to this, the cross-linking of various accessory receptors and co-stimulatory molecules leads to a cascade of signalling events in the cytoplasm of the T cell that results in transcription of genes required for effector function. For induction of a Th1 phenotype, interaction of IL-12 and IL-12 receptor activates the JAK-STAT pathway through STAT-4, which results in an increase in IFN-g transcription. Signalling of IL-18 through the IL-18 receptor can also result in this up-regulation of IFN-g: Several transcription factors have been found to bind to the IFN-g promoter and other regulatory elements and affect its


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expression. These include T-bet (Szabo et al., 2000), WSX-1 (Yoshida et al., 2001), NFAT, NF-kB, and members of the IRF1 family (Murphy et al., 2000). T-bet is a T-box transcription factor found in Th1 but not Th2 cells. T-bet up-regulates expression of IFN-g and can revert Th2 effector cells to a Th1-type phenotype if inappropriately expressed (Szabo et al., 2000). WSX-1 has been shown to be important in the initial production of IFN-g (Yoshida et al., 2001). NF-kB1 has also recently been shown to be required for development of type 1 T cells and disruption of this gene in C57BL/6 mice results in susceptibility to infection with L. major (Artis et al., 2003). A Th2 phenotype is induced upon signalling through IL-4 and the IL-4 receptor. Once again the JAK-STAT pathway is utilised, but in this case STAT-6 is activated (Murphy et al., 2000). The transcription factor GATA-3 has been shown to up-regulate IL-4, -5 and -13 and also to inhibit expression of the IL-12 receptor, making it a very important factor in the establishment of a Th2 response (Zheng and Flavell, 1997). STAT-4 and BCL-6 have been shown to inhibit this pathway. When transcription of IFN-g in Th1 cells or IL-4 in Th2 cells is established, complex regulatory feedback loops ensure that the established T helper phenotype is promoted and the alternative phenotype is inhibited. Also playing a role in persistence of the functional polarisation of T cells is silencing of cytokine genes from the opposing response by their repositioning into the heterochromatin (Grogan et al., 2001). However, there is accumulating data suggesting that these processes must be quite leaky since single clones of T cells have been shown to produce both IL-4 and IFN-g; simultaneously (Morris et al., 1992). This is not so surprising in view of the complexity of the immune system and the requirements made on it by various complex pathogens (Parham, 1990).

5. THE IMMUNOLOGY OF HUMAN CUTANEOUS LEISHMANIASIS AND ITS ROLE IN HOST RESISTANCE TO DISEASE The elucidation of the immune response to infection of humans with Leishmania species is more difficult than that of mice. First, the

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immune response early in infection is not easily studied, as people presenting to clinics are generally at an advanced stage of the disease. Second, the availability of tissues for examination and for experimental long-term studies is limited. Despite this, there have been several studies investigating the cytokines produced by peripheral blood mononuclear cells (PBMC) or present in the lesion in patients with cutaneous leishmaniasis. Very early studies showed that recovery from infection correlated with the induction of delayed-type hypersensitivity to Leishmania antigens, pointing to the importance of the cell-mediated immune response (Bryceson, 1970). Measurement of the proliferative response of PBMC induced by stimulation with L. major preparations has indicated that a higher level of proliferation is associated with milder disease and healing, presumably due to a more robust T cell response to infection (Kemp et al., 1994; Gaafar et al., 1995a, b; Ajdary et al., 2000). Several groups have measured the cytokine profile of PBMC from patients infected with L. major and showed that in patients who had cured their infection, had sub-clinical infection or had milder disease, high levels of IFN-g and low levels of IL-4 were detected. Those with more severe or non-healing disease had low levels of IFN-g and most reported high levels of IL-4 (Kemp et al., 1994; Gaafar et al., 1995b; Ajdary et al., 2000; Habibi et al., 2001). When IL-10 and IL-12 were measured in addition to these two cytokines, IL-12 was found to correlate with IFN-g: IL-10 was found in one study to correlate with IL-4, but it was not detected in the second study (Ajdary et al., 2000; Habibi et al., 2001). Louzir et al. (1998) investigated the cytokine response in the lesion itself using a semi-quantitative RT-PCR on RNA prepared from skin biopsies. They reported that IL-12p40 and IFN-g were positively associated with the presence of parasites in the lesion and that elevated levels of IL-12p40, IFN-g and IL-10 were associated with an unfavourable evolution of disease. They suggested that IL-10 was inhibiting the macrophage activating effects of IFN-g in this situation. Similar investigations in patients infected with L. guyanensis found an association between high levels of IL-10 and poor response to treatment and a possible role for IL-13 in the down-regulation of


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IL-12Rb2; an indicator of a predominantly Th2 response (Bourreau et al., 2001a, b). A report on the cytokines detected in biopsies of patients infected with L. braziliensis or L. mexicana has indicated a difference in response in patients with varying severity of the disease. Patients with diffuse cutaneous leishmaniasis, or non-healing disease, exhibited a Th2 cytokine profile, those who cured their lesions produced Th1-type cytokines and those with an intermediate form of the disease produced a mixed response (Diaz et al., 2002). Each of these studies then suggests that a Th1/Th2 dichotomy may also be acting in humans. Until recently, the role of NO in human leishmaniasis has been controversial. However, it has now been reported that iNOS can be detected in cells from cutaneous lesions, indicating that NO is likely to be involved in macrophage-mediated parasite killing in humans (Arevalo et al., 2002). This is supported by in vitro data, which demonstrated that human macrophages killed parasites in a NOdependent manner (Vouldoukis et al., 1995). Therefore, a strong T cell response, with the production of IFN-g to induce iNOS expression in macrophages seems also to be required for the clearance of the parasite in humans. It seems likely then that the immune response we detect in mice in response to experimental infection with L. major is, at least in part, an appropriate model for the response in the natural infection in humans.

6. THE GENETIC TOOLS FOR ANALYSIS OF HOST RESPONSE TO INFECTION IN THE MOUSE While host response to L. major has been studied for several decades, we still are unsure as to the important mechanisms which differentiate between a successful host response which results in the elimination of parasites with minimal host damage and a response which is slow to eliminate parasites and leaves the host with extended periods of substantive pathology. Classical immunology has uncovered a number of mechanisms important or even crucial for the host response to infection (as described above), but their relationship to the clinical

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manifestation of disease is not easy to ascertain. This is where the genetic study of disease is important. Genetics allows the direct connection of a genetic change to an observed phenotype. In lower organisms, the study of genetic mutations has led to the understanding of such complex mechanisms as the cell cycle and development. This has been done using saturation mutagenesis and by the careful sifting through millions of embryos/organisms searching for mutants. While this is becoming possible for mammals using mutagenesis screens in mice, the phenotypes are usually too complex for rapid screens and one is therefore left to analyse those genetic polymorphisms thrown up by nature. Naturally occurring functional polymorphisms are common. Indeed, the response of almost all inbred laboratory mouse strains to infection differs in one phenotype or another. This has led to the use of inter-strain comparisons to tease out the biologic consequences of host response. While this has led to some fundamental insights into the biology of disease, it is not without risk. These animals probably differ at thousands of genes across their genomes and an association between a host response to disease and another phenotype in a small number of strains (often only two, as has been the case for leishmaniasis) can lead to incorrect assumptions about the underlying biology. Often these associations are beguiling as they fit nicely into a model of how the host responds to disease and this drives the ascertainment of the associated phenotype in the first place. This approach is usually chosen to validate a hypothesis about host response and when the ‘resistant’ strain behaves in one direction and the ‘susceptible’ strain behaves in the other direction, then this is often reported as evidence for the two phenotypes being causally linked. Obviously, publication is more likely for a polarised response between the two strains, and so the literature is full of these observations, of which many are undoubtedly spurious. Notwithstanding these concerns, strain differences do hold the key to the study of host response in infectious disease. How, therefore, are the nuggets distinguished from the mullock using strain differences?


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6.1. Multiple Strain Comparisons To increase the chances that the inter-strain association of two phenotypes is biologically relevant, the experiments could be repeated in a number of different inbred strains. For example if one were looking to associate the CD4+ T cell response to L. major and the ability of the strain to heal lesions, then comparisons between BALB/c and C57BL/6 animals would associate a Th2 response with susceptibility and a Th1 response with rapid lesion resolution. If this observation could be repeated in many other strains then there would be increasing support for the hypothesis that the Th response is linked to outcome following L. major infection. The difficulty with this approach is that the strains that are normally used as either ‘resistant’ or ‘susceptible’ are usually those strains with the most extreme phenotype and when other strains are tested, they fall somewhere between these two in response to infection, or in their allied phenotypes, in this example, their Th response. This is probably indicative of both phenotypes being genetically complex, with a number of factors influencing outcome. Therefore, a more sophisticated approach is needed and this involves the genetic isolation (and mapping) of the host response loci.

6.2. Mapping Host Response Loci in the Mouse There are many approaches for mapping loci involved in a given trait in the mouse. Several genes have been identified which govern the response of strains of mice to infection. To date, almost all of these are for single-gene Mendelian traits. The most relevant example to leishmaniasis is the Lsh gene (now Slc11a1) (Blackwell, 1982), which was cloned by a straightforward positional cloning exercise. However, most other strain differences are complex genetic events, governed by multiple genes and probably with multiple allelic variants at these loci. The phenotypes are often quantitative, the sum of effects from each locus giving rise to a fraction of the total phenotype. Often loci will interact

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epistatically. These effects are difficult to identify. The strategy used to identify the loci controlling these quantitative phenotypes is quantitative trait loci (QTL) mapping. There are several strategies available.

6.3. Intercrosses or Backcrosses The production of either intercross progeny or backcross progeny generates mice which are genetically unique yet comprise only the grand-parental genotypes, and because normal laboratory mice are inbred, will exhibit only a maximum of two different alleles at any one locus. Large numbers of such progeny can be produced allowing the mapping of loci contributing to only a small fraction of the total genetic variance. The initial step in the generation of a F2 intercross is the mating of the two parental lines to produce the F1 generation. These lines will be chosen based on functional differences in the traits of interest. These F1 animals are then intercrossed producing the F2 generation. The N2 backcross generation is produced by crossing F1 animals back to one (or both) of the parental lines. Phenotypes and genotypes are measured in the F2 and N2 animals. At this point, disease response and the associated phenotype are measured in these animals and an association between the two can now be made with a statistically meaningful significance. If there is a causal relationship between the disease phenotypes and the response, then they will occur together in the same animal more frequently than would otherwise be expected by chance alone. The addition of the genotypes of these animals can now link phenotype to chromosomal interval using one of the number of available statistical methods (Lander et al., 1987; Broman et al., 2003). Basically, the contribution of each genotype at each locus to the phenotype is calculated and a probability of linkage is assigned. With the exception of the ability to link phenotypes, this information is not really very useful as a standalone observation. This is due to the difficulty of identifying a causative gene from a QTL scan. The peaks are often broad, encompassing many centimorgans (cM) and many hundreds of genes. However, it can be put to much use by


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breeding congenic animals (see below). There are a number of permutations to this approach, one useful tactic has been to take the inbreeding to advanced stages to produce advanced intercross lines (AILs) . These AILs represent many more meioses and therefore the length of the parental haplotypes in each of these individual animals is much smaller than for the F1 and N2 animals, allowing for more fine-mapping of the interval. This approach, when used with a large number of progeny, has the ability to give narrower QTL peaks.

6.4. Recombinant Inbred Strains (RIS) RIS are produced by inbreeding F1 animals from two strains of interest (Bailey, 1971; Swank and Bailey, 1973). A number of lines are produced and are maintained as completely inbred lines, homozygous at all loci. They represent a random mix of the two parental genotypes with each animals possessing, on average, 50% contribution from each of the parental lines as long stretches of homozygous parental haplotype. Each of the lines has random assortments of these haplotypes varying in their ancestry, length and start and finish positions. Most of the RIS have been extensively genotyped and this information is available in databases. The lines can be phenotyped and the phenotype is associated with the known genotypes. Again, if a particular genotype at a locus is associated with a given phenotype more often than would be expected by chance alone, then it may be contributing towards the phenotype. The advantage of RIS is that the genotypes are already known, and therefore all that is needed is to phenotype the animals. They are particularly useful for testing the association of phenotypes. However, there are limited lines available and in some of these panels, there are limited numbers of strains. The ability to test a large number of animals with identical genetic make up decreases environmental variance considerably, and often loci conferring a small effect, which are not seen in larger intercross experiments, can be found in RIS (Hasegawa et al., 2000; Roberts et al., 2000). Unfortunately, as the genetic complexity increases, the small number of lines becomes limiting.

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6.5. Recombinant Congenic Strains (RCS) The production of RCS with a mosaic of homozygous parental haplotypes has been of extreme use for the dissection of complex diseases (Groot et al., 1992; Demant et al., 1996). RCS lines are similar to RIS in that strains are homozygous for random segments of parental haplotypes. However, RCS are produced by inbreeding the second backcross generation and therefore each strain has only, on average, 12% contribution from the donor strain. This serves to isolate loci from one strain away from any other confounding loci. The effects of complex genetic phenotypes are therefore broken down into single effect loci. Several groups have produced RCS (Groot et al., 1992; Fortin et al., 2001) and the genotype data for these strains are available.

6.6. Common Haplotype Mapping The availability of the mouse genome sequence has refined the positional cloning approach, but has also added a new mapping approach to the mapping arsenal. Single nucleotide polymorphism (SNP) analysis on numerous inbred mouse strains has demonstrated that there are only about three different haplotypes present at most loci across the murine genome. This is possibly an historical artefact of the production of laboratory mice. However, this can be used to advantage to map genes for complex traits. If the locus-specific phenotype is known for a number of different strains of inbred mice and the haplotype structure of the locus is known in these particular strains, then a haplotype association study can be performed. Haplotypes giving rise to a given phenotype will necessarily harbour the causative gene. The small size of these haplotypes will accurately pinpoint the causative interval (Park et al., 2003). The advent of dense SNP mapping and the eventual publication of the SNP haplotype of the mouse genome will make this the technique of choice for future mapping studies (Wade et al., 2002). However, initial mapping studies must be carried out to determine the effect on the phenotype of the locus under study in a number of inbred strains.


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6.7. Congenic Strains (CS) As stated above, the discovery of linkage of a genetically complex phenotype to a chromosomal segment is not by itself particularly useful. To verify the linkage and to generate reagents useful for the study of the biology of the locus, congenic animals need to be bred. CS are produced by backcrossing one strain onto another and keeping the chromosomal region of interest heterozygous until all the background genome is fixed. This usually takes about 10 backcross generations, but can be accelerated using strategies to select animals with a greater percent of the background fixed for breeding the next generation (Estill and Garcia, 2000; Jeffs et al., 2000; Burt et al., 2002; Elso et al., 2004c). The net result is a mouse strain with a small interval from one chromosome of the donor strain isolated on the genome of the recipient strain. This allows for the study of the biology of the linked locus on an otherwise isogenic strain. Direct comparisons can be made between the recipient strain and the CS, and any differences will be due to the presence of the congenic interval from the donor strain. As this region can be narrowed down by the selection of further recombinations across the interval and the breeding of sub-congenic lines, the certainty that the coincidence of phenotypes and genotype is causal increases along with an increase in the resolution of the map location of the causative gene. Another advantage of congenic lines is the ability to design useful DNA microarray experiments to compare gene expression between the congenic and the parental lines. Changes in transcription in the relevant tissues or cells between the congenic and its parental line (which are isogenic at all other loci) will be conferred by the genes present in the donor interval. It may even be possible to map transcriptional changes to this interval, thereby aiding in the identification of the causative gene/genes.

6.8. Gene Discovery and Validation Gene discovery in the mouse is still difficult for QTLs. This is in part due to the inaccuracy in localisation of the gene under a linkage peak,

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but is also due to the difficulty in assigning importance to sequence differences observed in candidate genes. There is a great deal of sequence polymorphism between inbred strains of mice. This confounds the association of sequence polymorphisms to phenotype. In humans, one can examine the association between sequence change and phenotype in thousands of individuals and be reasonably certain of the association. Unfortunately, there are insufficient lines of inbred mice for a similar strategy. Therefore, confirmation of candidature must be done by other means. The absolute proof is a knock-in of the mutation from one strain to the other along with a change in the phenotype similar to that seen in the congenic. Other means to the same end involve the knockout of the candidate gene and its re-introduction by BAC transgenesis, looking for phenotypic complementation. There are complexities to this technique. For example, one would normally make BACs from the strain of mouse displaying the dominantly acting locus and inject them into the pronucleus of cells from the strain displaying the recessive phenotype. This will work if the locus is operating through a dominant gain of function, but the dominance could also be due to a haplo-insufficiency. In this case, the complementation would only work in the opposite direction. Insufficient QTLs have been identified so far to accurately predict which direction would be the most prudent to try. Obviously, BACs may carry more than one gene and, therefore, care must be taken to ensure that the candidate is indeed the gene mediating the change in phenotype.

7. THE GENETIC TOOLS FOR ANALYSIS OF HOST RESPONSE IN HUMANS The genetic analysis of the human response to infectious disease falls behind that of the mouse for several reasons. The genetics of host response is, for the most part, a complex genetic trait and these are still very difficult to analyse in human populations. Secondly, the genetic analysis of host response is the epitome of the study of environment–genetic interactions. The infection is the environmental


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component and the host response is largely driven by genetics (including the development of immunity, both innate and adaptive). Therefore, in order to use some of the methods available for genetic analysis, one needs families, or at least related individuals who have seen the same infectious agent and have had a similar opportunity to become infected. This set of circumstances has been used in some diseases, which will be mentioned below, but is uncommon. Therefore, the most common method to study the genetics of complex host response to infection is at the population level.

7.1. Association Studies Association studies provide the easiest tools for implicating genes in the host response. Sequence polymorphisms from around a candidate gene are tested in a number of unrelated individuals from a population with the disease phenotype of interest (cases) and without the phenotype (controls). A skewing of allele frequencies into either one of these groups associates the skewed allele with the phenotype, and therefore implicates the chosen gene as being functionally relevant to the phenotype chosen for study. While this approach has implicated a number of genes in response to infection (McGuire et al., 1994), there are many difficulties. The control population must be very closely matched to the cases otherwise spurious associations will occur due to ethnic differences in allele frequency distributions. This is especially important in the study of infectious diseases where it is known that there are major differences in susceptibility genes based on ethnicity. One way around this problem is to use the non-transmitted chromosome as a control. In this design, parents are genotyped along with children, the nontransmitted alleles, or even better, haplotypes are used as the control. This supplies a better match between the test and control cohorts than population controls and also allows transmission disequilibrium testing as part of the analysis. Secondly, there is the major problem of multiple testing. Many laboratories test numerous loci for association and each locus may be

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tested for multiple sequence polymorphisms. These polymorphisms may not be in linkage disequilibrium and, therefore, will be independent tests. Very few results are reported in the literature with penalties attached for multiple testing and, as more and more tests are done, these penalties become great. The way around this is to repeat the experiment with a replication set that will need to be greater than the original set. An association replicated in another set of cases and controls will lend more credibility to the original observation. It is of interest that few associations to host response in infectious disease have been replicated. Finally, the greatest difficulty of association studies is the choice of candidate genes. It is unlikely that new paradigms will be unearthed by choosing a candidate based on a current hypothesis. It will, at best, confirm the hypothesis, which is often based on similar findings in mice, or in in vitro studies. The paradigm shifts in host response will come from hypothesis-free, genome-wide scans, as described above for studies in mice.

7.2. Linkage Analysis While most host responses to infection will be complex genetic events, this will not always be the case. There are some rare disorders, which are manifest by an increased susceptibility to infectious disease. Note that due to points raised in the discussion above, susceptibility to infection will be an unlikely familial occurrence. In some rare Mendelian disorders where susceptibility to infection does occur in families, classical linkage and positional cloning strategies are powerful approaches. They have resulted in the identification of particular cytokines being responsible for atypical mycobacterial infections (Reichenbach et al., 2001; Casanova and Abel, 2002, 2004; StaretzHaham et al., 2003). Similarly, significant linkage has been found examining large families with a high risk of severe schistosomiasis (Marquet et al., 1996, 1999). In the case of leishmaniasis, a locus for susceptibility to visceral leishmaniasis (Kala Azar) has been described using genome-wide scans in families (Bucheton et al., 2003).


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Therefore, the classical familial linkage approach looking for a single major locus has been successful.

7.3. Affected Family Member Studies Until recently, the only method for tackling complex genetic diseases was to look for linkage using large affected sib-pair methods. Affected sibs are genotyped and a skewing of the normal segregation ratio of marker alleles indicates linkage. As these sib pairs are not related to the other sib pairs investigated in the study, and as there is often genetic heterogeneity, in the absence of a major contributory locus, many thousands of such sib pairs may be needed to find statistically significant linkage. While such studies with significant statistical power have been carried out for diabetes, multiple sclerosis and other Western diseases, there has been insufficient funding to perform similar studies for diseases of the third world.

7.4. Future Approaches to Mapping Host Response Genes in Human Populations With the advent of the human genome sequence, there have been several proposals to increase our ability to identify genes for complex genetic diseases. There is a project in progress describing many of the SNPs and aiming to use this information to generate haplotype maps of the human population (www.hapmap.org). This information, (especially the identification of large numbers of SNPs) could allow genome-wide SNP association studies. There are DNA-carrying chips being produced with hundreds of thousands of SNPs, which could provide one-assay genome-wide SNP genotypes. There are many issues to be resolved with this technique, especially as most of the SNPs have been described in populations, which do not have those diseases that most infectious disease researchers are studying. The analysis of these data is still in its infancy but shows considerable promise.

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A more specialised use of the SNP data is in admixture studies. As mentioned above, populations of different ethnicity show different responses to infectious agents. There are also subsets of the SNPs, which are different between ethnicities. In populations with ethnic admixture, it may be possible to take advantage of these observations to map loci harbouring either resistant or susceptible alleles (Zhu et al., 2004).

8. THE GENETICS OF HOST RESPONSE TO LEISHMANIASIS Genes responsible for the natural differences in host response to leishmaniasis have been difficult to identify both in humans and in animal models. Studies carried out in mice have implicated several loci in progression of disease and have shown that response to Leishmania species is multigenic and interactions between loci may exist, confounding attempts to identify individual genes. So far, only one gene involved in mouse resistance to infection with the viscerotropic L. donovani species (Blackwell, 1989) has been identified. The gene Slc11a1, previously known as Nramp1, and originally designated Lsh, encodes a membrane transport protein. Slc11a1 is the principal gene involved in resistance to not only L. donovani, but also Mycobacterium bovis, BCG and Salmonella typhimurium in mice. All these organisms are either obligatory or facultative intracellular pathogens in macrophages. More recently, Slc11a1 has been associated with a number of other infectious and autoimmune diseases in humans (Blackwell, 2001). This provides one example of a gene relevant to human disease, identified through genetic studies in mice.

8.1. Genetics of Susceptibility to Leishmaniasis in Mice In the late 1970s, several groups investigated the response of a series of inbred strains of mice to infection with L. major. Reports showed that C57BL/6 and NZB mice were resistant to infection with rapid


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cure of disease. CBA/H, A/J and C3H/He were of intermediate phenotype, exhibiting a delayed cure and BALB/c and DBA/2 were susceptible, exhibiting signs of progression to the visceral form of the disease and death (Preston and Dumonde, 1976; Handman et al., 1979). This difference in inbred strains of mice infected under exactly the same conditions argued for a genetic factor predisposing the mice to disease or cure. These initial observations were confirmed by others, with C57BL/6 mice consistently resistant and BALB/c always extremely susceptible (De Tolla et al., 1980; Howard et al., 1980). Armed with the knowledge that inbred strains of mice exhibit different responses to infection with L. major, several groups set out to find the genes underlying this phenomenon. It was shown that F1 mice from BALB/c C57BL/6 intercross experiments exhibited an intermediate phenotype (Howard et al., 1980; De Tolla et al., 1981). Analysis of the progress of infection in progeny from an F1 intercross of these strains and a BALB/c F1 (BALB/c C57BL/6) backcross was claimed to support the involvement of one major gene responsible for the observed difference in susceptibility between these two strains. However, contrary to the conclusion of these studies that susceptibility was mediated by a single gene, it was noted by Howard et al. (1980) that in the F2 generation mice, the phenotype of the intermediate and resistant strains could not be clearly distinguished. This ‘‘was not surprising in view of the presumed polygenic nature of immune intervention’’ (Howard et al., 1980). In addition, De Tolla et al. (1981) reported that in the BALB/c F1 (BALB/c C57BL/6) backcross, over 90% of the mice exhibited a BALB/c-like phenotype, whereas only 50% would be expected to do so. It was suggested that other BALB/c genes could be modifying susceptibility to L. major, but these genes were ‘‘irrelevant to the eventual outcome of the infection’’ (De Tolla et al., 1981). These genes would be now classed as disease modifiers, thus performing an important role in the pattern of disease. Therefore, contrary to the reported conclusion of both groups that a single gene was responsible for disease due to L. major, these studies, in fact, support a polygenic model of response to infection.

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A number of early studies assessed the contribution of several loci initially identified for their role in L. donovani infection, to infection with L. major (De Tolla et al., 1981). H-11 and Ir-2, as well as the Lsh gene (now Slc11a1, Howard et al., 1980) were all excluded from having an effect in the L. major mouse model. A number of minor histocompatibility loci, the immunoglobulin heavy chain, lipopolysaccharide, as well as loci linked to markers in a genetic linkage test strain (Rop/Le) were also excluded (Mock et al., 1993). Roberts et al. (1993) and Mock et al. (1993) both reported that susceptibility to L. major showed high concordance rates with BALB/c alleles on chromosome11 (Scl-1) in two different sets of recombinant inbred mice (C B and C S) and in two infection systems, infection with amastigotes in the footpad and promastigotes in the rump. Seven strains of mice obtained from the C B (BALB/c C57BL/6) set were used and either 13 or 14 strains from the C S (BALB/c STS/A) set. While achieving concordant results from these studies lends confidence to the observations, these recombinant inbred sets do not have the statistical power to unequivocally confirm or exclude linkage to a particular chromosome. This group also found a locus on chromosome 4, which they named Scl-2, that appeared to be important in susceptibility to L. mexicana infection (Roberts et al., 1990), and linkage to the T lymphocyte ‘T helper 2’ cluster of genes on a more proximal region of chromosome 11 considered important, late in infection with L. major (Roberts et al., 1993). Howard et al. (1980) assessed the effect of the MHC on the course of infection using sets of mice congenic for various H-2 loci. A series of congenic strains on the C57BL/10 and BALB backgrounds were infected and their disease progression followed. All strains on the C57BL/10 background showed early arrest of lesion growth. However, mice carrying H-2s, H-2a, or H-2k on this background completely healed their lesion, whereas mice with H-2b, H-2d or H-2q all showed some mild residual disease. BALB/c, BALB/b and BALB/k mice all showed progressive disease but, in contrast to the other two strains, BALB/k showed slower progression of disease when given lower infectious doses. The result with the BALB series of congenic mice was confirmed by Mitchell et al. (1981a). These results indicated


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that while not responsible for a major effect on disease progression, the MHC or genes contained within the congenic interval were affecting in some way the response to infection. It is notable that no difference was observed on either background between H-2b and H-2d, the haplotypes carried by C57BL/6 and BALB/c mice, respectively. When whole genome linkage analysis was made feasible with the availability of new reagents and high throughput techniques, it was used by a number of groups to search for the genes underlying response to infection with L. major. In 1997, two linkage studies were published. The first, from this laboratory, was carried out on a cohort of 199 mice from an intercross between BALB/c and C57BL/6 mice (Roberts et al., 1997). The mice were infected intradermally–subcutaneously at the base of the tail and the progression of disease followed for 23 weeks. The average lesion score for each mouse from weeks 3 to 14 was used as the criterion for disease severity and was used for linkage analysis. The mice were genotyped at approximately 12 cM density. Regions suggestive of linkage were confirmed in a further 271 F2 mice. Two loci named lmr for L. major response were confirmed: proximal chromosome 17 (lmr1) and proximal chromosome 9 (lmr2, Roberts et al., 1997). On subsequent analysis it was found that a locus on the X chromosome (lmr3) was exerting an epistatic effect in combination with lmr1, such that the C57BL/6 allele at lmr3 conferred susceptibility on the mouse in the presence of BALB/c homozygosity at lmr1 (Roberts et al., 1999). The contribution of each of these alleles has been validated since in a series of mice congenic for each of these loci, as well as combinations of the loci on both the C57BL/6 and BALB/c genetic background (Elso et al., 2004b). Beebe et al. (1997) approached the problem in a different way, attempting to map resistance alleles using a serial backcrossing method. Susceptible BALB/c mice were crossed with resistant B10.D2 mice to create F1 generation mice, which were fully resistant. F1 mice were backcrossed onto BALB/c and infected in the footpad with L. major. Resistant or intermediate male progeny based on footpad swelling were retained as breeders if 420% of their progeny were resistant. By breeding only from mice with a resistant or intermediate

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phenotype, it was hoped to enrich for B10.D2 alleles encoding resistance while halving the frequency of B10.D2 alleles at each generation. Genotyping was carried out on N5 mice and loci where B10.D2 alleles were found at greater than three times the frequency expected by random segregation were considered as candidates. Loci on chromosomes 6, 11 and 15 had values suggestive of linkage, but loci on chromosomes 7, 10 and 16 were not found to be linked. Pairwise analysis indicated linkage of disease severity to pairs including all of these six loci. One advantage of this experimental design is that mice congenic for the loci mapped are produced during the experiment, thus speeding up the process of gene detection. The design of this experiment, however, also had major problems. A percentage (5–10% reported by Beebe et al., 1997) of male BALB/c mice exhibited a resistant phenocopy. This phenomenon has been previously reported (Roberts et al., 1993, 1997) but its mechanism is still not understood. In addition, mice with resistant or intermediate phenotypes were designated ‘resistant’ for the purpose of breeding. The presence of the resistant phenocopy combined with the somewhat ‘soft’ definition of the phenotype of animals used for breeding and analysis could confound the results. Mice with a ‘susceptible’ genotype but ‘resistant’ phenotype could be bred, therefore losing the ‘resistant’ alleles from this particular line. Breeding from a limited number of mice could also introduce a significant founder effect into the distribution of alleles observed, not necessarily being linked to resistance. Further work was carried out by this group on mice congenic for the proximal portion of chromosome 11 (2–40 cM). The male congenic mice also contained the Y chromosome from B10.D2. In this study, male congenics were significantly protected by the presence of the B10.D2 allele at the chromosome 11 locus, whereas females were not. These data suggest that a resistance gene, which also mediates differential effects on the sexes, is found on chromosome 11. However, it may be prudent to repeat the challenge with mice containing the BALB/c Y chromosome to ensure that the B10.D2 Y chromosome was not having any effect on the infection. It is unlikely that


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this locus is the Scl1 locus described previously, since Scl1 was reported to be on distal rather than proximal chromosome 11. The most recent studies on the genetics of the immunological response in L. major-infected mice involve the use of resistant and susceptible recombinant congenic (RC) strain CcS-5/Dem and its susceptible BALB/c and resistant STS parental strains (Lipoldova et al., 2000; Badalova et al., 2002; Vladimirov et al., 2003). The RC strains contain 12.5% of genes from the STS and 87.5% of genes from the BALB/c parents. Mice from F2 intercrosses between parental and RC strains were genotyped at the regions, in which the STS alleles were segregating and analysed for a wide variety of responses to infection with L. major found to be different between the BALB/c and STS strains. These included hepatomegaly, splenomegaly, levels of IgE, IFN-g; IL-12 and IL-4 in serum, parasite load and lesion development. Linkage to 13 different regions, called lmr3– 15, has now been reported by this group. It is unfortunate that despite the fact that this report was published after Roberts and colleagues designated the X chromosome locus as lmr3 (Roberts et al., 1999), the authors used the same nomenclature. Many of the loci showed linkage not to actual disease phenomena but to immunological traits. Therefore, this would seem to argue against the possibility of these loci having a major role in controlling the infection, and thus naming them L. major response loci is somewhat of a misnomer. The authors suggest that these loci may affect pathology on a different genetic background, citing the observed protection elicited by the presence of the B10.D2 allele at a locus coinciding with lmr6 in the study by Beebe et al. (1997). It is also interesting to note that linkage was found in the F2 cohort to phenotypes for serum IL-12 levels and hepatomegaly, which were shown in the same study not to be different between the parental BALB/c and CcS-5/ Dem strains. Although this issue was not discussed in the paper, it could be explained by interactions that are not observed in the parental strains. Lipoldova et al. (2000) point out that the loci identified in their studies do not explain the entire difference between the BALB/c and STS strains, as other strains from the CcS series carrying STS alleles at different loci also have varying responses to L. major infection.

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Chr.5

Chr.6

Chr.9

Chr.10

Chr.11

Chr.15

Chr.17

Chr.X

telomere

centromere

Combined, these studies suggest that a very large pool of genes could be influencing the host response to L. major infection and that combinations of resistance and susceptibility alleles at each of those genes are contributing, to varying degrees, to the disease outcome. Already, various phenotypes in L. major-infected mice have been linked to mouse chromosomes 5, 6, 9, 10, 11, 13, 14, 15, 17, 18 and X (Figure 4). An additional layer of complexity can be added to this because, in each study discussed, only the alleles from two inbred strains were considered. Other inbred strains will, no doubt, have different alleles at many of these loci, so there is likely to be a spectrum in the contribution of different alleles of each gene to resistance or susceptibility. This situation is observed in outbred populations

Figure 4 Genetic loci implicated in susceptibility to infection with L. major. Several loci have been reported to be linked to either the pathology or the immune responses to infection with L. major. Loci described by Lipoldova et al. (2000) are in black and include genes linked to splenomegaly (chromosome 5), lesion size (chromosomes 4 and 10), IL-4 (chromosome 11) and lymphocyte proliferation (chromosome 17). Studies from Roberts et al. (1993) and Mock et al. (1993) identified loci involved in lesion size and in the production of Th2-type cytokines (shown cross-hatched on chromosome 11). Additional loci linked to lesion size have been described by Beebe et al. (1997) and are depicted in dark grey on chromosomes 6, 11 and 15. Other loci linked to lesion size have been identified on chromosome 9, 17 and X and are shown as light grey (Roberts et al., 1997, 1999).


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such as humans and contributes to the difficulties in identifying causative genes.

8.2. Genetics of Susceptibility to Leishmaniasis in Humans Several groups have attempted to find association of severity of human leishmaniasis with candidate genes. These studies have largely centred on specific alleles of the HLA class I, II and III regions. Cabrera et al. (1995) found association of mucocutaneous leishmaniasis (MCL) to alleles of the HLA class III genes, TNF-a and TNF-b in a group of individuals in Venezuela. However, Meddeb-Garnaoui et al. (2001) did not find association to these alleles in Tunisians with visceral leishmaniasis (VL). A further study found association of TNF locus polymorphisms with severity of disease in people infected with L. chagasi (Karplus et al., 2002). Alleles of HLA-A and -B were found to be associated with disease in a study of cutaneous leishmaniasis (CL) in Venezuela (Lara et al., 1991), but not in a South American study of MCL (Petzl-Erler et al., 1991) or VL in India (Singh et al., 1997). HLA-DR and DQ were also found to be variably associated with disease in these studies. Differences that could explain the apparently contradictory results between these studies include the clinical form of leishmaniasis being studied, the species and strain of the infecting parasite and the human population under study. It is likely that different genes and different alleles exert different influences in each of the above situations. Another possibility is that the HLA alleles themselves are not predisposing for disease, but show association with leishmaniasis because they are in linkage-disequilibrium with genes in the region which are involved in the response to the disease, i.e. ‘at a greater frequency together than predicted by the individual frequencies of the single alleles’ (Beck and Trowsdale, 2000). Linkage disequilibrium is very strong at the MHC locus (Beck and Trowsdale, 2000). This could explain those associations that were weak and variable in the human studies discussed above. A similar explanation could then be envisaged for the weak

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differences seen between mice congenic for different H-2 haplotypes if congenic intervals for some haplotypes contained a gene involved in the host response to leishmaniasis and others did not. Blackwell et al. (1997) conducted a study on families in Brazil in an attempt to find linkage and/or association of VL, leprosy and tuberculosis to genes and loci previously implicated in response to infection with various Leishmania species in the mouse model of these diseases. These genes or loci were the MHC, NRAMP1 (now SLC11A1), two loci syntenic with regions on mouse chromosome 11 (Scl1 and the ‘T helper 2’ cytokine gene cluster) and a locus syntenic with chromosome 4 (Scl2). The MHC was found to have weak allelic association with VL, but none of the other loci showed any linkage or association to disease (Blackwell, 1998). The failure to find in human disease a corresponding effect of loci identified in murine leishmaniasis could be explained in a number of ways. First, keeping in mind that the mouse is not the natural host of L. major or L. donovani, the response to infection may differ between human and mouse. Second, the loci identified in the mouse may themselves not be important in the human disease, but the biological pathway in which they act may be important. Third, these genes may not be implicated in host response to disease in the particular families studied, but may be important in other populations or in individuals infected with different Leishmania species. For example, recent studies in the Sudan examining the Masalit tribe who are extremely susceptible to VL, with high rates of severe disease and low rates of positive skin test, and their neighbouring Hawsa tribe who have low rates of severe disease and high skin test positivity, have demonstrated linkage between clinical VL and SLC11A1 as well as IL-4 in Masalit families (Mohamed et al., 2003, 2004; Blackwell et al., 2004). These studies also showed that IFN-g receptor 1 (IFNGR1) is associated specifically with the post-kala-azar dermal leishmaniasis syndrome (PKDL). A model of the pathways that may be regulated by these and other genes involved in innate and adaptive immunity has been proposed by Blackwell and colleagues (Figure 5 is Plate 1.5 in the Separate Color Plate section).


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9. ENVIRONMENTAL FACTORS INFLUENCING LEISHMANIASIS A range of factors have been identified which modify the response to infection with L. major and may contribute to the severity of disease. These include the dose and site of infection, the presence of modulatory factors in the sandfly saliva and co-infection with other organisms. There is no doubt that in the case of VL, the state of nutrition of the host has a major influence on disease severity (Anstead et al., 2001, 2003). It is conceivable that a genetic modulator of disease may be involved in the response to these types of factors, which in turn would result in different outcomes of leishmaniasis in the infected host. In natural infections, the parasite is introduced into the dermis via the bite of a sandfly. A range of sandfly and parasite-derived molecules are co-injected with the parasites. Among the sandfly bioactive molecules are some designed to aid the sandfly in obtaining its blood meal. These include vasodilators and anti-clotting agents (Locksley, 2000). Some of these molecules have immunomodulatory actions, which favour establishment of infection with the parasites. Thus, co-injection of saliva with parasites led to disease exacerbation (Kamhawi et al., 2000; Morris et al., 2001; Norsworthy et al., 2004). Paradoxically, vaccination with salivary gland preparations or specific proteins from the preparation could induce protection against subsequent infection with a small dose of parasites introduced with sandfly saliva (mimicking natural conditions) (Kamhawi et al., 2000; Morris et al., 2001). The dose of parasite used in most experimental infections is undoubtedly very much higher than that delivered in natural infection. In fact, although the exact numbers are not known, it has been shown that about 1000 parasites may be introduced into the host by an infectious bite (Rogers et al., 2004). Variation of the dose in experimental infections of inbred mice has resulted in different responses to infection. For example, the susceptibility of BALB/c mice to infection with L. major has been well established. However, after infection with

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a very low dose of L. major, BALB/c mice were able to resolve the infection and develop immunity to re-infection (Bretscher et al., 1992). This was associated with the development of a Th1 response indicative of the presence of a dosage threshold for the development of disease (Doherty and Coffman, 1996). Experiments with low-dose infections of C57BL/6 mice demonstrate that a ‘silent’ phase of parasite growth lasting several weeks precedes pathological change and parasite clearance. This silent period is characterised by no overt signs of disease or inflammatory infiltrate (Belkaid et al., 2000; Lira et al., 2000). This is thought to mimic the normal progression in natural infection (albeit without contribution from the sandfly). An important consideration overlooked in most studies is the potential difference between parasites grown in the laboratory and those developing in the natural habitat of the sandfly. Not only these are grown in vitro, but also they have been selected for these conditions from a larger population. In natural infections, promastigotes undergo a maturation process in the sandfly gut, termed metacyclogenesis, which results in characteristic changes in surface and secreted glycoconjugates. These changes are thought to allow the release of the mature metacyclic promastigotes from the gut wall to be regurgitated into the new host when the sandfly feeds (Rogers et al., 2004). These are the most infectious form of the promastigote. When promastigotes are grown in culture to a stationary phase of growth, only approximately 10% are metacyclic with the remaining 90% remaining in the immature procyclic form. Moreover, in the sand fly gut, the parasites are surrounded by a thick gel of PPG, which has been shown to contribute significantly to the parasites’ ability to establish infection in the mammalian host (Rogers et al., 2004). This material is absent in the thoroughly washed parasites that are usually injected into mice. The site of infection has also been shown to result in different disease outcomes even when metacyclic promastigotes derived from a cloned line of L. major were used. For example, a recent comparison of mice infected at the base of the tail with mice infected in the ear dermis has shown that different responses to infection are observed in individual inbred strains. For example, C3H/He mice that show


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intermediate susceptibility to infection at the base of the tail show neither lesion pathology nor the presence of parasites in the draining lymph nodes when infected in the ear dermis (Baldwin et al., 2003). A similar effect has also been shown in hamsters with differences in the course of infection seen after inoculation of L. panamensis in the foot or the snout (Osorio et al., 2003). Co-infection with other infectious agents, which modulate the immune system, plays a role in the severity of disease due to infection with Leishmania species. This could include other parasitic infections as well as bacterial or viral infections. For example, schistosomiasis has been shown to reduce the ability of macrophages to kill L. major parasites (La Flamme et al., 2002). Due to the persistence of parasites after cure in otherwise immune individuals, leishmaniasis is increasingly affecting people infected with HIV who are exhibiting the immunosuppressive features of AIDS (Ashford, 2000; Dedet and Pratlong, 2000).

10. CONCLUSIONS The response to infection with Leishmania is clearly influenced by a large number of factors from the host, the parasite and the environment. Despite this complexity, work in the murine model of disease, as well as data obtained in humans, indicate that there is a clear host genetic component in this response. In experimental CL, different inbred strains of mice are differentially resistant to disease, but association and linkage studies have not, to date, been successful in determining the underlying cause of this differential response. A number of genes mediating host response to infectious diseases have been identified, including genes involved in the development of T cell responses through the IL-12/IFN-g pathway mediating resistance to mycobacterial diseases (Doffinger et al., 2001), and Slc11a1, which plays a role in the response to L. donovani, S. typhimurium and M. bovis BCG (Blackwell, 1982; Vidal et al., 1995; Blackwell et al., 2004). These genes act in a monogenic Mendelian fashion, which has facilitated their identification. However, host response to infectious

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diseases more often presents as a complex trait, and studies of the response to L. major infection have indicated that this phenotype is indeed mediated by multiple genes. The difference between resistant and susceptible mice has encouraged detailed examination of the immunological response to L. major and has resulted in the elucidation of much that is known about the polarization of T helper cell responses. It is possible that some aspect of this dichotomy or the mechanism initiating the bias of the T helper response may be the basis of the genetic difference between the inbred strains. However, while the T cell response is clearly necessary for the elimination of L. major parasites, and a difference in T cell response between the resistant and susceptible strains of mice is evident, it is still not clear that the T cell response is the causative difference in the response to infection with L. major. A very strong influence on outcome of infection from components of the innate arm of the immune response is also apparent, demonstrated by the difference in cellular infiltrate early in infection and the different abilities to restrict spread of parasite from the site of infection. With the increase in valuable resources such as the availability of the human and mouse genome sequences, identification of large numbers of SNPs and the promise of haplotype mapping, we are entering a time where the genes involved in complex systems, such as the host response to infection will be increasingly obtainable. This will allow us further insight into the age-old DNA war between host and pathogen.

ACKNOWLEDGEMENTS The work from the authors’ laboratories has been supported by the Australian National Health and Medical Research Council, the National Institutes of Health, USA and the World Health Organization TDR Program. We apologise to our many valued colleagues whose work has not been cited due to space limitations. We are grateful to Fiona Mitchell for excellent editorial support.


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REFERENCES Aebischer, T., Moody, S.F. and Handman, E. (1993). Persistence of virulent Leishmania major in murine cutaneous leishmaniasis: a possible hazard for the host. Infection and Immunity 61, 220–226. Ajdary, S., Alimohammadian, M.H., Eslami, M.B., Kemp, K. and Kharazmi, A. (2000). Comparison of the immune profile of nonhealing cutaneous Leishmaniasis patients with those with active lesions and those who have recovered from infection. Infection and Immunity 68, 1760–1764. Anstead, G.M., Chandrasekar, B., Zhang, Q. and Melby, P.C. (2003). Multinutrient undernutrition dysregulates the resident macrophage proinflammatory cytokine network, nuclear factor-kappaB activation, and nitric oxide production. Journal of Leukocyte Biology 74, 982–991. Anstead, G.M., Chandrasekar, B., Zhao, W., Yang, J., Perez, L.E. and Melby, P.C. (2001). Malnutrition alters the innate immune response and increases early visceralization following Leishmania donovani infection. Infection and Immunity 69, 4709–4718. Arevalo, I., Ward, B. and Matlashewski, G. (2002). Detection of iNOS gene expression in cutaneous leishmaniasis biopsy tissue. Molecular and Biochemical Parasitology 121, 145–147. Artis, D., Speirs, K., Joyce, K., Goldschmidt, M., Caamano, J., Hunter, C.A. and Scott, P. (2003). NF-kappa B1 is required for optimal CD4+ Th1 cell development and resistance to Leishmania major. Journal of Immunology 170, 1995–2003. Aseffa, A., Gumy, A., Launois, P., MacDonald, A.S., Louis, J.A. and Tacchini-Cottier, F. (2002). The early IL-4 response to Leishmania major and the resulting Th2 cell maturation steering progressive disease in BALB/c mice are subject to the control of regulatory CD4+CD25+ T cells. Journal of Immunology 169, 3232–3241. Ashford, R.W. (2000). The leishmaniases as emerging and reemerging zoonoses. International Journal for Parasitology 30, 1269–1281. Badalova, J., Svobodova, M., Havelkova, H., Vladimirov, V., Vojtiskova, J., Engova, J., Pilcik, T., Volf, P., Demant, P. and Lipoldova, M. (2002). Separation and mapping of multiple genes that control IgE level in Leishmania major infected mice. Genes and Immunity 3, 187–195. Bailey, D.W. (1971). Recombinant-inbred strains. An aid to finding identity, linkage, and function of histocompatibility and other genes. Transplantation 11, 325–327. Baldwin, T., Henri, S., Curtis, J., O’Keeffe, M., Vremec, D., Shortman, K. and Handman, E. (2004). Dendritic cell populations in the Leishmania major infected skin and draining lymph nodes. Infection and Immunity 72, 1991–2001.

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Baldwin, T., Elso, C., Curtis, J., Buckingham, L. and Handman, E. (2003). The site of Leishmania major infection determines the disease severity and immune responses. Infection and Immunity 71, 6830–6834. Baldwin, T., Henri, S., Curtis, J., O’Keeffe, M., Vremec, D., Shortman, K. and Handman, E. (2004). Dendritic cell populations in Leishmania majorinfected skin and draining lymph nodes. Infection and Immunity 72, 1991–2001. Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y.J., Pulendran, B. and Palucka, K. (2000). Immunobiology of dendritic cells. Annual Review of Immunology 18, 767–811. Beck, S. and Trowsdale, J. (2000). The human major histocompatibility complex: lessons from the DNA sequence. Annual Review of Genomics Human Genetics 1, 117–137. Beebe, A.M., Mauze, S., Schork, N.J. and Coffman, R.L. (1997). Serial backcross mapping of multiple loci associated with resistance to Leishmania major in mice. Immunity 6, 551–557. Beil, W.J., Meinardus-Hager, G., Neugebauer, D.C. and Sorg, C. (1992). Differences in the onset of the inflammatory response to cutaneous leishmaniasis in resistant and susceptible mice. Journal of Leukocyte Biology 52, 135–142. Belkaid, Y., Hoffmann, K.F., Mendez, S., Kamhawi, S., Udey, M.C., Wynn, T.A. and Sacks, D.L. (2001). The role of interleukin (IL)-10 in the persistence of Leishmania major in the skin after healing and the therapeutic potential of anti-IL-10 receptor antibody for sterile cure. Journal of Experimental Medicine 194, 1497–1506. Belkaid, Y., Mendez, S., Lira, R., Kadambi, N., Milon, G. and Sacks, D. (2000). A natural model of Leishmania major infection reveals a prolonged ‘‘silent’’ phase of parasite amplification in the skin before the onset of lesion formation and immunity. Journal of Immunology 165, 969–977. Belkaid, Y., Piccirillo, C.A., Mendez, S., Shevach, E.M. and Sacks, D. (2002). CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature 420, 502–507. Belosevic, M., Finbloom, D.S., Van Der Meide, P.H., Slayter, M.V. and Nacy, C.A. (1989). Administration of monoclonal anti-IFNgamma antibodies in vivo abrogates natural resistance of C3H/HeN mice to infection with Leishmania major. Journal of Immunology 143, 266–274. Bertholet, S., Dickensheets, H.L., Sheikh, F., Gam, A.A., Donnelly, R.P. and Kenney, R.T. (2003). Leishmania donovani-induced expression of suppressor of cytokine signaling 3 in human macrophages: a novel mechanism for intracellular parasite suppression of activation. Infection and Immunity 71, 2095–2101.


57

Biedermann, T., Zimmermann, S., Himmelrich, H., Gumy, A., Egeter, O., Sakrauski, A.K., Seegmuller, I., Voigt, H., Launois, P., Levine, A.D., Wagner, H., Heeg, K., Louis, J.A. and Rocken, M. (2001). IL-4 instructs TH1 responses and resistance to Leishmania major in susceptible BALB/c mice. Nature Immunology 2, 1054–1060. Blackwell, J.M. (1982). Genetic control of recovery from visceral leishmaniasis. Transactions of the Royal Society of Tropical Medicine and Hygiene 76, 147–151. Blackwell, J.M. (1989). The macrophage resistance gene Lsh/Ity/Bcg. Research in Immunology 140, 767–769. Blackwell, J.M. (1998). Genetics of host resistance and susceptibility to intramacrophage pathogens: a study of multicase families of tuberculosis, leprosy and leishmaniasis in north-eastern Brazil. International Journal for Parasitology 28, 21–28. Blackwell, J. (2001). Genetics and genomics in infectious disease susceptibility. Trends in Molecular Medicine 7, 521–526. Blackwell, J.M., Black, G.F., Peacock, C.S., Miller, E.N., Sibthorpe, D., Gnananandha, D., Shaw, J.J., Silveira, F., Lins-Lainson, Z., Ramos, F., Collins, A. and Shaw, M.A. (1997). Immunogenetics of leishmanial and mycobacterial infections: the Belem Family Study. Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 352, 1331–1345. Blackwell, J., Mohamed, H.S. and Ibrahim, M. (2004). Genetics and visceral leishmaniasis in the Sudan: seeking a link. Trends in Parasitology 20, 268–274. Bogdan, C. (1997). Of microbes, macrophages and nitric oxide. Behring Institute Mitteilungen 99, 58–72. Bosque, F., Saravia, N.G., Valderrama, L. and Milon, G. (2000). Distinct innate and acquired immune responses to Leishmania in putative susceptible and resistant human populations endemically exposed to L. (Viannia) panamensis infection. Scandinavian Journal of Immunology 51, 533–541. Bourreau, E., Prevot, G., Gardon, J., Pradinaud, R. and Launois, P. (2001a). High intralesional interleukin-10 messenger RNA expression in localized cutaneous leishmaniasis is associated with unresponsiveness to treatment. Journal of Infectious Diseases 184, 1628–1630. Bourreau, E., Prevot, G., Pradinaud, R. and Launois, P. (2001b). Interleukin (IL)-13 is the predominant Th2 cytokine in localized cutaneous leishmaniasis lesions and renders specific CD4+ T cells unresponsive to IL-12. Journal of Infectious Diseases 183, 953–959. Bretscher, P.A., Wei, G., Menon, J.N. and Bielefeldt-Ohmann, H. (1992). Establishment of stable, cell-mediated immunity that makes ‘‘susceptible’’ mice resistant to Leishmania major. Science 257, 539–542.

58

E. HANDMAN ET AL.

Broman, K.W., Wu, H., Sen, S. and Churchill, G.A. (2003). R/qtl: QTL mapping in experimental crosses. Bioinformatics 19, 889–890. Bryceson, A.D.M. (1970). Immunological aspects of clinical leishmaniasis. Proceedings of the Royal Society of Medicine 63, 1056–1060. Bucheton, B., Abel, L., El-Safi, S., Kheir, M.M., Pavek, S., Lemainque, A. and Dessein, A.J. (2003). A major susceptibility locus on chromosome 22q12 plays a critical role in the control of kala-azar. American Journal of Human Genetics 73, 1052–1060. Burt, R.A., Marshall, V.M., Wagglen, J., Rodda, F.R., Senyschen, D., Baldwin, T.M., Buckingham, L.A. and Foote, S.J. (2002). Mice that are congenic for the char2 locus are susceptible to malaria. Infection and Immunity 70, 4750–4753. Cabrera, M., Shaw, M.A., Sharples, C., Williams, H., Castes, M., Convit, J. and Blackwell, J.M. (1995). Polymorphism in tumor necrosis factor genes associated with mucocutaneous leishmaniasis. Journal of Experimental Medicine 182, 1259–1264. Campbell, K.A., Ovendale, P.J., Kennedy, M.K., Fanslow, W.C., Reed, S.G. and Maliszewski, C.R. (1996). CD40 ligand is required for protective cell-mediated immunity to Leishmania major. Immunity 4, 283–289. Carrera, L., Gazzinelli, R.T., Badolato, R., Hieny, S., Muller, W., Kuhn, R. and Sacks, D.L. (1996). Leishmania promastigotes selectively inhibit interleukin 12 induction in bone marrow-derived macrophages from susceptible and resistant mice. Journal of Experimental Medicine 183, 515–526. Carter, K.C., Gallagher, G., Baillie, A.J. and Alexander, J. (1989). The induction of protective immunity to Leishmania major in the BALB/c mouse by interleukin 4 treatment. European Journal of Immunology 19, 779–782. Casadevall, A. and Pirofski, L.A. (2003). The damage-response framework of microbial pathogenesis. Nature Reviews Microbiology 1, 17–24. Casanova, J.L. and Abel, L. (2002). Genetic dissection of immunity to mycobacteria: the human model. Annual Review of Immunology 20, 581–620. Casanova, J.L. and Abel, L. (2004). The human model: a genetic dissection of immunity to infection in natural conditions. Nature Reviews Immunology 4, 55–66. Chakir, H., Campos-Neto, A., Mojibian, M. and Webb, J.R. (2003). IL12Rbeta2-deficient mice of a genetically resistant background are susceptible to Leishmania major infection and develop a parasite-specific Th2 immune response. Microbes and Infection 5, 241–249. Chakour, R., Guler, R., Bugnon, M., Allenbach, C., Garcia, I., Mauel, J., Louis, J. and Tacchini-Cottier, F. (2003). Both the Fas ligand and inducible nitric oxide synthase are needed for control of parasite replication within lesions in mice infected with Leishmania major whereas the


59

contribution of tumor necrosis factor is minimal. Infection and Immunity 71, 5287–5295. Chatelain, R., Mauze, S. and Coffman, R.L. (1999). Experimental Leishmania major infection in mice: role of IL-10. Parasite Immunology 21, 211–218. Chatelain, R., Varkila, K. and Coffman, R.L. (1992). IL-4 induces a Th2 response in Leishmania major-infected mice. Journal of Immunology 148, 1182–1187. Cohn, M. (2002). The immune system: a weapon of mass destruction invented by evolution to even the odds during the war of the DNAs. Immunological Reviews 185, 24–38. Copeland, N.G. and Jenkins, N.A. (1999). The mouse as a gene discovery tool in the modern genome era. Harvey Lectures 95, 137–155. Croft, S.L. and Coombs, G.H. (2003). Leishmaniasis—current chemotherapy and recent advances in the search for novel drugs. Trends in Parasitology 19, 502–508. da Silva, R., Hall, B.F., Joiner, K.A. and Sacks, D.L. (1988). CR1 mediates binding of L. major metacyclic promastigotes to human macrophages. Memorias do Instituto Oswaldo Cruz 1, 459–463. da Silva, R.P., Hall, B.F., Joiner, K.A. and Sacks, D.L. (1989). CR1, the C3b receptor, mediates binding of infective Leishmania major metacyclic promastigotes to human macrophages. Journal of Immunology 143, 617–622. Davis, A., Murray, H.W. and Handman, E. (2004). Drugs against leishmaniasis: new approaches and new targets for drug development. Trends in Parasitology 20, 73–76. De Tolla, L.J., Scott, P.A. and Farrell, J.P. (1981). Single gene control of resistance to cutaneous leishmaniasis in mice. Immunogenetics 14, 29–39. De Tolla, L.J., Semprevivo, L.H., Palczuk, N.C. and Passmore, H.J. (1980). Genetic control of acquired resistance to visceral leishmaniasis in mice. Immunogenetics 10, 353–361. Dedet, J.P. and Pratlong, F. (2000). Leishmania, Trypanosoma and monoxenous trypanosomatids as emerging opportunistic agents. The Journal of Eukaryotic Microbiology 47, 37–39. Demant, P., Lipoldova, M. and Svobodova, M. (1996). Resistance to Leishmania major in mice. Science 274, 1392–1393. Diaz, N.L., Zerpa, O., Ponce, L.V., Convit, J., Rondon, A.J. and Tapia, F.J. (2002). Intermediate or chronic cutaneous leishmaniasis: leukocyte immunophenotypes and cytokine characterisation of the lesion. Experimental Dermatology 11, 34–41. Diefenbach, A., Schindler, H., Donhauser, N., Lorenz, E., Laskay, T., MacMicking, J., Rollinghoff, M., Gresser, I. and Bogdan, C. (1998). Type 1 interferon (IFNa=b) and type 2 nitric oxide synthase regulate the innate immune response to a protozoan parasite. Immunity 8, 77–87.

60

E. HANDMAN ET AL.

Doffinger, R., Dupuis, S., Picard, C., Fieschi, C., Feinberg, J., BarcenasMorales, G. and Casanova, J.-L. (2001). Inherited disorders of IL-12and IFN-gamma-mediated immunity: a molecular genetics update. Molecular Immunology 38, 903–909. Doherty, T.M. and Coffman, R.L. (1996). Leishmania major: effect of infectious dose on T cell subset development in BALB/c mice. Experimental Parasitology 84, 124–135. Dorner, B.G., Scheffold, A., Rolph, M.S., Huser, M.B., Kaufmann, S.H., Radbruch, A., Flesch, I.E. and Kroczek, R.A. (2002). MIP-1alpha, MIP-1beta, RANTES, and ATAC/lymphotactin function together with IFN-gamma as type 1 cytokines. Proceedings of the National Academy of Sciences USA 99, 6181–6186. Elso, C., Kumar, B., Smith, G.K., Foote, S.J. and Handman, E. (2004a). Dissociation of disease susceptibility, inflammation and cytokine profile in lmr1/2 congenic mice infected with Leishmania major. Genes and Immunity 5, 188–196. Elso, C., Roberts, L.J., Smith, G.K., Thomson, R.J., Baldwin, T.M., Foote, S.J. and Handman, E. (2004b). Leishmaniasis host response loci (lmr1,2 and 3) modify disease severity through a Th1/Th2 independent pathway. Genes and Immunity 5, 93–100. Elso, C.M., Roberts, L.J., Smyth, G.K., Thomson, R.J., Baldwin, T.M., Foote, S.J. and Handman, E. (2004c). Leishmaniasis host response loci (lmr1-3) modify disease severity through a Th1/Th2-independent pathway. Genes and Immunity 5, 93–100. Estill, S.J. and Garcia, J.A. (2000). A marker assisted selection protocol (MASP) to generate C57BL/6J or 129S6/SvEvTac speed congenic or consomic strains. Genesis 28, 164–166. Ferlin, W.G., von der Weid, T., Cottrez, F., Ferrick, D.A., Coffman, R.L. and Howard, M.C. (1998). The induction of a protective response in Leishmania major-infected BALB/c mice with anti-CD40 mAb. European Journal of Immunology 28, 525–531. Fiorentino, D.F., Zlotnik, A., Vieira, P., Mosmann, T.R., Howard, M., Moore, K.W. and O’Garra, A. (1991). IL-10 acts on the antigenpresenting cell to inhibit cytokine production by Th1 cells. Journal of Immunology 146, 3444–3451. Fortin, A., Diez, E., Rochefort, D., Laroche, L., Malo, D., Rouleau, G.A., Gros, P. and Skamene, E. (2001). Recombinant congenic strains derived from A/J and C57BL/6J: a tool for genetic dissection of complex traits. Genomics 74, 21–35. Gaafar, A., el Kadaro, A.Y., Theander, T.G., Permin, H., Ismail, A., Kharazmi, A. and el Hassan, A.M. (1995a). The pathology of cutaneous leishmaniasis due to Leishmania major in Sudan. American Journal of Tropical Medicine and Hygiene 52, 438–442.


61

Gaafar, A., Kharazmi, A., Ismail, A., Kemp, M., Hey, A., Christensen, C.B., Dafalla, M., el Kadaro, A.Y., el Hassan, A.M. and Theander, T.G. (1995b). Dichotomy of the T cell response to Leishmania antigens in patients suffering from cutaneous leishmaniasis; absence or scarcity of Th1 activity is associated with severe infections. Clinical and Experimental Immunology 100, 239–245. Gillitzer, R. and Goebeler, M. (2001). Chemokines in cutaneous wound healing. Journal of Leukocyte Biology 69, 513–521. Gomes, N.A. and DosReis, G.A. (2001). The dual role of CTLA-4 in Leishmania infection. Trends in Parasitology 17, 487–491. Greenblatt, C.L. (1980). The present and future of vaccination for cutaneous leishmaniasis. Progress in Clinical and Biological Research 47, 259–285. Grewal, I.S. and Flavell, R.A. (1998). CD40 and CD154 in cell-mediated immunity. Annual Review of Immunology 16, 111–135. Grogan, J.L., Mohrs, M., Harmon, B., Lacy, D.A., Sedat, J.W. and Locksley, R.M. (2001). Early transcription and silencing of cytokine genes underlie polarization of T helper cell subsets. Immunity 14, 205–215. Groot, P.C., Moen, C.J., Dietrich, W., Stoye, J.P., Lander, E.S. and Demant, P. (1992). The recombinant congenic strains for analysis of multigenic traits: genetic composition. Faseb Journal 6, 2826–2835. Guler, M.L., Gorham, J.D., Hsieh, C.S., Mackey, A.J., Steen, R.G., Dietrich, W.F. and Murphy, K.M. (1996). Genetic susceptibility to Leishmania: IL-12 responsiveness in TH1 cell development. Science 271, 984–987. Habibi, G.R., Khamesipour, A., McMaster, W.R. and Mahboudi, F. (2001). Cytokine gene expression in healing and non-healing cases of cutaneous leishmaniasis in response to in vitro stimulation with recombinant gp63 using semi-quantitative RT-PCR. Scandinavian Journal of Immunology 54, 414–420. Handman, E., Ceredig, R. and Mitchell, G.F. (1979). Murine cutaneous leishmaniasis: disease patterns in intact and nude mice of various genotypes and examination of differences between normal and infected macrophages. Australian Journal of Experimental Biology and Medical Science 57, 9–29. Handman, E., Hocking, R.E., Mitchell, G.F. and Spithill, T.W. (1983). Isolation and characterization of infective and non-infective clones of Leishmania tropica. Molecular and Biochemical Parasitology 7, 111–126. Handman, E., Spira, D.T., Zuckerman, A. and Montilio, B. (1974). Standardization and quality control of Leishmania tropica vaccine. Journal of Biological Standards 2, 11–22. Hasegawa, M., Baldwin, T.M., Metcalf, D. and Foote, S.J. (2000). Progenitor cell mobilization by granulocyte colony-stimulating factor controlled by loci on chromosomes 2 and 11. Blood 95, 1872–1874.

62

E. HANDMAN ET AL.

Heath, V.L., Showe, L., Crain, C., Barrat, F.J., Trinchieri, G. and O’Garra, A. (2000). Cutting edge: ectopic expression of the IL-12 receptor-beta 2 in developing and committed Th2 cells does not affect the production of IL- 4 or induce the production of IFN-gamma. Journal of Immunology 164, 2861–2865. Heinzel, F.P. (1994). Interleukin 12 and the regulation of CD4+ T-cell subset responses during murine leishmaniasis. Parasitology Today 10, 190–192. Heinzel, F.P., Rerko, R.M., Ahmed, F. and Pearlman, E. (1995). Endogenous IL-12 is required for control of Th2 cytokine responses capable of exacerbating leishmaniasis in normally resistant mice. Journal of Immunology 155, 730–739. Heinzel, F.P., Sadick, M.D., Holaday, B.J., Coffman, R.L. and Locksley, R.M. (1989). Reciprocal expression of interferon-g or interleukin-4 during the resolution or progression of murine leishmaniasis. Evidence for expansion of distinct helper T cell subsets. Journal of Experimental Medicine 169, 59–72. Heinzel, F.P., Sadick, M.D., Mutha, S.S. and Locksley, R.M. (1991). Production of interferon g; interleukin 2, interleukin 4, and interleukin 10 by CD4+ lymphocytes in vivo during healing and progressive murine leishmaniasis. Proceedings of the National Academy of Sciences USA 88, 7011–7015. Heinzel, F.P., Schoenhaut, D.S., Rerko, R.M., Rosser, L.E. and Gately, M.K. (1993). Recombinant interleukin 12 cures mice infected with Leishmania major. Journal of Experimental Medicine 177, 1505–1509. Henri, S., Curtis, J., Hochrein, H., Vremec, D., Shortman, K. and Handman, E. (2002). Hierarchy of susceptibility of dendritic cell subsets to infection by Leishmania major: inverse relationship to interleukin-12 production. Infection and Immunity 70, 3874–3880. Herwaldt, B.L. (1999). Leishmaniasis. The Lancet 354, 1191–1199. Himmelrich, H., Launois, P., Maillard, I., Biedermann, T., TacchiniCottier, F., Locksley, R.M., Rocken, M. and Louis, J.A. (2000). In BALB/c mice, IL-4 production during the initial phase of infection with Leishmania major is necessary and sufficient to instruct Th2 cell development resulting in progressive disease. Journal of Immunology 164, 4819–4825. Himmelrich, H., Parra-Lopez, C., Tacchini-Cottier, F., Louis, J.A. and Launois, P. (1998). The IL-4 rapidly produced in BALB/c mice after infection with Leishmania major down-regulates IL-12 receptor B2-chain expression on CD4+ T cells resulting in a state of unresponsiveness to IL-12. Journal of Immunology 161, 6156–6163. Holaday, B.J., Sadick, M.D., Wang, Z.E., Reiner, S.L., Heinzel, F.P., Parslow, T.G. and Locksley, R.M. (1991). Reconstitution of Leishmania


63

immunity in severe combined immunodeficient mice using Th1- and Th2like cell lines. Journal of Immunology 147, 1653–1658. Howard, J.G., Hale, C. and Chan-Liew, W.L. (1980). Immunological regulation of experimental cutaneous leishmaniasis. 1. Immunogenetic aspects of susceptibility to Leishmania tropica in mice. Parasite Immunology 2, 214–303. Howard, J.G., Hale, C. and Liew, F.Y. (1981). Immunological regulation of experimental cutaneous leishmaniasis. IV. Prophylactic effect of sublethal irradiation as a result of abrogation of suppressor T cell generation in mice genetically susceptible to Leishmania tropica. Journal of Experimental Medicine 153, 557–568. Ilgoutz, S.C. and McConville, M.J. (2001). Function and assembly of the Leishmania surface coat. International Journal for Parasitology 21, 899–908. Jacobson, R.L. (2003). Leishmania tropica (Kinetoplastida: Trypanosomatidae)—a perplexing parasite. Folia Parasitologica 50, 241–250. Jeffs, B., Negrin, C.D., Graham, D., Clark, J.S., Anderson, N.H., Gauguier, D. and Dominiczak, A.F. (2000). Applicability of a ‘‘speed’’ congenic strategy to dissect blood pressure quantitative trait loci on rat chromosome 2. Hypertension 35, 179–187. Jones, B.D. and Falkow, S. (1996). Salmonellosis: host immune responses and bacterial virulence determinants. Annual Review of Immunology 14, 533–561. Julia, V., McSorley, S.S., Malherbe, L., Breittmayer, J.P., Girard-Pipau, F., Beck, A. and Glaichenhaus, N. (2000). Priming by microbial antigens from the intestinal flora determines the ability of CD4+ T cells to rapidly secrete IL-4 in BALB/c mice infected with Leishmania major. Journal of Immunology 165, 5637–5645. Julia, V., Rassoulzadegan, M. and Glaichenhaus, N. (1996). Resistance to Leishmania major induced by tolerance to a single antigen. Science 274, 421–423. Kaisho, T. and Akira, S. (2003). Regulation of dendritic cell function through Toll-like receptors. Current Molecular Medicine 3, 759–771. Kamhawi, S., Belkaid, Y., Modi, G., Rowton, E. and Sacks, D. (2000). Protection against cutaneous leishmaniasis resulting from bites of uninfected sand flies. Science 290, 1351–1354. Kane, M.M. and Mosser, D.M. (2001). The role of IL-10 in promoting disease progression in leishmaniasis. Journal of Immunology 166, 1141–1147. Karplus, T.M., Jeronimo, S.M., Chang, H., Helms, B.K., Burns, T.L., Murray, J.C., Mitchell, A.A., Pugh, E.W., Braz, R.F., Bezerra, F.L. and Wilson, M.E. (2002). Association between the tumor necrosis factor locus and the clinical outcome of Leishmania chagasi infection. Infection and Immunity 70, 6919–6925.

64

E. HANDMAN ET AL.

Kelsall, B.L., Biron, C.A., Sharma, O. and Kaye, P.M. (2002). Dendritic cells at the host-pathogen interface. Nature Immunology 3, 699–702. Kemp, M., Hey, A.S., Kurtzhals, J.A., Christensen, C.B., Gaafar, A., Mustafa, M.D., Kordofani, A.A., Ismail, A., Kharazmi, A. and Theander, T.G. (1994). Dichotomy of the human T cell response to Leishmania antigens. I. Th1-like response to Leishmania major promastigote antigens in individuals recovered from cutaneous leishmaniasis. Clinical and Experimental Immunology 96, 410–415. Kima, P.E., Constant, S.L., Hannum, L., Colmenares, M., Lee, K.S., Haberman, A.M., Schlomchik, M.J. and McMahon-Pratt, D. (2000). Internalization of Leishmania mexicana complex amastigotes via the Fc receptor is required to sustain infection in murine cutaneous leishmaniasis. Journal of Experimental Medicine 191, 1063–1067. Kimbrell, D.A. and Beutler, B. (2001). The evolution and genetics of innate immunity. Nature Reviews. Genetics 2, 256–267. Kopf, M., Brombacher, F., Kohler, G., Kienzle, G., Widmann, K.H., Lefrang, K., Humborg, C., Ledermann, B. and Solbach, W. (1996). IL-4deficient Balb/c mice resist infection with Leishmania major. Journal of Experimental Medicine 184, 1127–1136. Kropf, P., Freudenberg, M.A., Modolell, M., Price, H.P., Herath, S., Antoniazi, S., Galanos, C., Smith, D.F. and Muller, I. (2004). Toll-like receptor 4 contributes to efficient control of infection with the protozoan parasite Leishmania major. Infection and Immunity 72, 1920–1928. Kubo, M., Hanada, T. and Yoshimura, A. (2003). Suppressors of cytokine signalling and immunity. Nature Immunology 4, 1169–1176. La Flamme, A.C., Scott, P. and Pearce, E.J. (2002). Schistosomiasis delays lesion resolution during Leishmania major infection by impairing parasite killing by macrophages. Parasite Immunology 24, 339–345. Lander, E.S., Green, P., Abrahamson, J., Barlow, A., Daly, M.J., Lincoln, S.E. and Newburg, L. (1987). MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1, 174–181. Lara, M.L., Layrisse, Z., Scorza, J.V., Garcia, E., Stoikow, Z., Granados, J. and Bias, W. (1991). Immunogenetics of human American cutaneous leishmaniasis. Study of HLA haplotypes in 24 families from Venezuela. Human Immunology 30, 129–135. Laskay, T., Diefenbach, A., Rollinghoff, M. and Solbach, W. (1995). Early parasite containment is decisive for resistance to Leishmania major infection. European Journal of Immunology 25, 2220–2227. Laskay, T., Rollinghoff, M. and Solbach, W. (1993). Natural killer cells participate in the early defense against Leishmania major infection in mice. European Journal of Immunology 23, 2237–2241.


65

Launois, P., Gumy, A., Himmelrich, H., Locksley, R.M., Rocken, M. and Louis, J.A. (2002). Rapid IL-4 production by Leishmania homolog of mammalian RACK1-reactive CD4(+) T cells in resistant mice treated once with anti-IL-12 or -IFN- gamma antibodies at the onset of infection with Leishmania major instructs Th2 cell development, resulting in nonhealing lesions. Journal of Immunology 168, 4628–4635. Launois, P., Maillard, I., Pingel, S., Swihart, K.G., Xeńarios, I., Acha-Orbea, H., Diggelmann, H., Locksley, R.M., McDonald, H.R. and Louis, J.A. (1997). IL-4 rapidly produced by Vb4 Va8 CD4+ T cells instructs Th2 development and susceptibility to Leishmania major in BALB/c mice. Immunity 6, 541–549. Launois, P., Ohteki, T., Swihart, K., MacDonald, H.R. and Louis, J.A. (1995). In susceptible mice, Leishmania major induce very rapid interleukin-4 production by CD4+ T cells which are NK11. European Journal of Immunology 25, 3298–3307. Li, J., Hunter, C.A. and Farrell, J.P. (1999). Anti-TGF-beta treatment promotes rapid healing of Leishmania major infection in mice by enhancing in vivo nitric oxide production. Journal of Immunology 162, 974–979. Liew, F.Y., Li, Y. and Millott, S. (1990a). Tumor necrosis factor-a synergizes with IFN-g in mediating killing of Leishmania major through the induction of nitric oxide. Journal of Immunology 145, 4306–4310. Liew, F.Y., Li, Y. and Millott, S. (1990b). Tumour necrosis factor (TNFalpha) in leishmaniasis. II. TNF-alpha-induced macrophage leishmanicidal activity is mediated by nitric oxide from L-arginine. Immunology 71, 556–559. Liew, F.Y., Wei, X.Q. and Proudfoot, L. (1997). Cytokines and nitric oxide as effector molecules against parasitic infections. Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 352, 1311–1315. Lima, G.M., Vallochi, A.L., Silva, U.R., Bevilacqua, E.M., Kiffer, M.M. and Abrahamsohn, I.A. (1998). The role of polymorphonuclear leukocytes in the resistance to cutaneous Leishmaniasis. Immunology Letters 64, 145–151. Lipoldova, M., Svobodova, M., Krulova, M., Havelkova, H., Badalova, J., Nohynkova, E., Holan, V., Hart, A.A., Volf, P. and Demant, P. (2000). Susceptibility to Leishmania major infection in mice: multiple loci and heterogeneity of immunopathological phenotypes. Genes and Immunity 1, 200–206. Lira, R., Doherty, M., Modi, G. and Sacks, D. (2000). Evolution of lesion formation, parasitic load, immune response, and reservoir potential in C57BL/6 mice following high- and low-dose challenge with Leishmania major. Infection and Immunity 68, 5176–5182.

66

E. HANDMAN ET AL.

Locksley, R.M. (1997). Exploitation of immune and other defence mechanisms by parasites: an overview. Parasitology 115, S5–S7. Locksley, R.M. (2000). Flying doctors. Nature Immunology 1, 457–458. Louzir, H., Melby, P.C., Ben Salah, A., Marrakchi, H., Aoun, K., Ben Ismail, R. and Dellagi, K. (1998). Immunologic determinants of disease evolution in localized cutaneous leishmaniasis due to Leishmania major. Journal of Infectious Diseases 177, 1687–1695. Macatonia, S.E., Hosken, N.A., Litton, M., Vieira, P., Hsieh, C.S., Culpepper, J.A., Wysocka, M., Trinchieri, G., Murphy, K.M. and O’Garra, A. (1995). Dendritic cells produce IL-12 and direct the development of Th1 cells from naive CD4+ T cells. Journal of Immunology 154, 5071–5079. MacDonald, A.S., Straw, A.D., Dalton, N.M. and Pearce, E.J. (2002). Cutting edge: Th2 response induction by dendritic cells: a role for CD40. Journal of Immunology 168, 537–540. Maillard, I., Launois, P., Himmelrich, H., Acha-Orbea, H., Diggelmann, H., Locksley, R.M. and Louis, J.A. (2001). Functional plasticity of the LACKreactive Vbeta4-Valpha8 CD4(+) T cells normally producing the early IL4 instructing Th2 cell development and susceptibility to Leishmania major in BALB/ c mice. European Journal of Immunology 31, 1288–1296. Marovich, M.A., McDowell, M.A., Thomas, E.K. and Nutman, T.B. (2000). IL-12p70 production by Leishmania major-harboring human dendritic cells is a CD40/CD40 ligand-dependent process. Journal of Immunology 164, 5858–5865. Marquet, S., Abel, L., Hillaire, D. and Dessein, A. (1999). Full results of the genome-wide scan which localises a locus controlling the intensity of infection by Schistosoma mansoni on chromosome 5q31-q33. European Journal of Human Genetics 7, 88–97. Marquet, S., Abel, L., Hillaire, D., Dessein, H., Kalil, J., Feingold, J., Weissenbach, J. and Dessein, A.J. (1996). Genetic localization of a locus controlling the intensity of infection by Schistosoma mansoni on chromosome 5q31-q33. Nature Genetics 14, 181–184. Masteller, E.L., Chuang, E., Mullen, A.C., Reiner, S.L. and Thompson, C.B. (2000). Structural analysis of CTLA-4 function in vivo. Journal of Immunology 164, 5319–5327. Matte, C. and Olivier, M. (2002). Leishmania-induced cellular recruitment during the early inflammatory response: modulation of proinflammatory mediators. Journal of Infectious Diseases 185, 673–681. Matzinger, P. (1998). An innate sense of danger. Seminars in Immunology 10, 399–415. McGuire, W., Hill, A.V., Allsopp, C.E., Greenwood, B.M. and Kwiatkowski, D. (1994). Variation in the TNF-alpha promoter region associated with susceptibility to cerebral malaria. Nature 371, 508–510.


67

Meddeb-Garnaoui, A., Gritli, S., Garbouj, S., Ben Fadhel, M., El Kares, R., Mansour, L., Kaabi, B., Chouchane, L., Ben Salah, A. and Dellagi, K. (2001). Association analysis of HLA-class II and class III gene polymorphisms in the susceptibility to mediterranean visceral leishmaniasis. Human Immunology 62, 509–517. Medzhitov, R. and Janeway, C.J. (2000). The toll receptor family and microbial recognition. Trends in Microbiology 8, 452–456. Mendez, S., Reckling, S.K., Piccirillo, C.A., Sacks, D. and Belkaid, Y. (2004). Role for CD4(+) CD25(+) regulatory T cells in reactivation of persistent leishmaniasis and control of concomitant immunity. Journal of Experimental Medicine 200, 201–210. Mendonca, M.G., de Brito, M.E., Rodrigues, E.H., Bandeira, V., Jardim, M.L. and Abath, F.G. (2004). Persistence of leishmania parasites in scars after clinical cure of American cutaneous leishmaniasis: is there a sterile cure? Journal of Infectious Diseases 189, 1018–1023. Mitchell, G.F., Curtis, J.M. and Handman, E. (1981a). Resistance to cutaneous leishmaniasis in genetically susceptible BALB/c mice. Australian Journal of Experimental Biology and Medical Science 59, 555–565. Mitchell, G.F., Curtis, J.M., Handman, E. and McKenzie, I.F.C. (1980). Cutaneous leishmaniasis in mice: disease patterns in reconstituted nude mice of several genotypes infected with Leishmania tropica. Australian Journal of Experimental Biology and Medical Science 58, 521–532. Mitchell, G.F., Curtis, J.M., Scollay, R.G. and Handman, E. (1981b). Resistance and abrogation of resistance to cutaneous leishmaniasis in reconstituted BALB/c nude mice. Australian Journal of Experimental Biology and Medical Science 59, 539–554. Mitchell, G.F. and Handman, E. (1983). Leishmania tropica major in mice: vaccination against cutaneous leishmaniasis in mice of high genetic susceptibility. Australian Journal of Experimental Biology and Medical Science 61, 11–25. Mock, B., Blackwell, J., Hilgers, J., Potter, M. and Nacy, C. (1993). Genetic control of Leishmania major infection in congenic, recombinant inbred and F2 populations of mice. European Journal of Immunogenetics 20, 335–348. Mohamed, H.S., Ibrahim, M.E., Miller, E.N., Peacock, C.S., Khalil, E.A., Cordell, H.J., Howson, J.M., El Hassan, A.M., Bereir, R.E. and Blackwell, J.M. (2003). Genetic susceptibility to visceral leishmaniasis in the Sudan: linkage and association with IL4 and IFNGR1. Genes and Immunity 4, 351–355. Mohamed, H.S., Ibrahim, M.E., Miller, E.N., White, J.K., Cordell, H.J., Howson, J.M., Peacock, C.S., Khalil, E.A., El Hassan, A.M. and Blackwell, J.M. (2004). SLC11A1 (formerly NRAMP1) and susceptibility to

68

E. HANDMAN ET AL.

visceral leishmaniasis in the Sudan. European Journal of Human Genetics 12, 66–74. Mohrs, M., Holscher, C. and Brombacher, F. (2000). Interleukin-4 receptor alpha-deficient BALB/c mice show an unimpaired T helper 2 polarization in response to Leishmania major infection. Infection and Immunity 68, 1773–1780. Moll, H. (1993). Experimental cutaneous leishmaniasis: langerhans cells internalize Leishmania major and induce an antigen-specific T-cell response. Advances in Experimental Medicine and Biology 329, 587–592. Moll, H., Scharner, A. and Kampgen, E. (2002). Increased interleukin 4 (IL-4) receptor expression and IL-4-induced decrease in IL-12 production by Langerhans cells infected with Leishmania major. Infection and Immunity 70, 1627–1630. Morris, L., Troutt, A.B., Handman, E. and Kelso, A. (1992). Changes in the precursor frequencies of IL-4 and IFN-g secreting CD4+ cells correlate with resolution of lesions in murine cutaneous leishmaniasis. Journal of Immunology 149, 2715–2721. Morris, L., Troutt, A.B., McLeod, K.S., Kelso, A., Handman, E. and Aebischer, T. (1993). Interleukin-4 but not gamma interferon production correlates with the severity of murine cutaneous leishmaniasis. Infection and Immunity 61, 3459–3465. Morris, R.V., Shoemaker, C.B., David, J.R., Lanzaro, G.C. and Titus, R.G. (2001). Sandfly maxadilan exacerbates infection with Leishmania major and vaccinating against it protects against L. major infection. Journal of Immunology 167, 5226–5230. Mosmann, T.R., Cherwinski, H., Bond, M.W., Giedlin, M.A. and Coffman, R.L. (1986). Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. Journal of Immunology 136, 2348–2356. Mosser, D.M. and Edelson, P.J. (1987). The third component of complement (C3) is responsible for the intracellular survival of Leishmania major. Nature 327, 329–331. Mougneau, E., Altare, F., Wakil, A.E., Zheng, S., Coppola, T., Wang, Z.-E., Waldmann, R., Locksley, R.M. and Glaichenhaus, N. (1995). Expression cloning of a protective Leishmania antigen. Science 268, 563–566. Muraille, E., De Trez, C., Brait, M., De Baetselier, P., Leo, O. and Carlier, Y. (2003). Genetically resistant mice lacking MyD88-adapter protein display a high susceptibility to Leishmania major infection associated with a polarized Th2 response. Journal of Immunology 170, 4237–4241. Murphy, K.M., Ouyang, W., Farrar, J.D., Yang, J., Ranganath, S., Asnagli, H., Afkarian, M. and Murphy, T.L. (2000). Signaling and transcription in T helper development. Annual Review of Immunology 18, 451–494.


69

Nandan, D., Yi, T., Lopez, M., Lai, C. and Reiner, N.E. (2002). Leishmania EF-1alpha activates the Src homology 2 domain containing tyrosine phosphatase SHP-1 leading to macrophage deactivation. The Journal for Biological Chemistry 277, 50190–50197. Nashleanas, M., Kanaly, S. and Scott, P. (1998). Control of Leishmania major infection in mice lacking TNF receptors. Journal of Immunology 160, 5506–5513. Nishikomori, R., Gurunathan, S., Nishikomori, K. and Strober, W. (2001). BALB/c mice bearing a transgenic IL-12 receptor beta 2 gene exhibit a nonhealing phenotype to Leishmania major infection despite intact IL-12 signaling. Journal of Immunology 166, 6776–6783. Noben-Trauth, N., Lira, R., Nagase, H., Paul, W.E. and Sacks, D.L. (2003). The relative contribution of IL-4 receptor signaling and IL-10 to susceptibility to Leishmania major. Journal of Immunology 170, 5152–5158. Noben-Trauth, N., Paul, W.E. and Sacks, D.L. (1999). IL-4- and IL-4 receptor-deficient BALB/c mice reveal differences in susceptibility to Leishmania major parasite substrains. Journal of Immunology 162, 6132–6140. Noe¨l, W., Raes, G., Ghassabeh, H.G. and De Baetselier, P. (2004). Alternatively activated macrophages during parasite infections. Trends in Parasitology 20, 126–133. Norsworthy, N.B., Sun, J., Elnaiem, D., Lanzaro, G. and Soong, L. (2004). Sand fly saliva enhances Leishmania amazonensis infection by modulating interleukin-10 production. Infection and Immunity 72, 1240–1247. Osorio, Y., Melby, P.C., Pirmez, C., Chandrasekar, B., Guarin, N. and Travi, B.L. (2003). The site of cutaneous infection influences the immunological response and clinical outcome of hamsters infected with Leishmania panamensis. Parasite Immunology 25, 139–148. Padigel, U.M., Alexander, J. and Farrell, J.P. (2003). The role of interleukin-10 in susceptibility of BALB/c mice to infection with Leishmania mexicana and Leishmania amazonensis. Journal of Immunology 171, 3705–3710. Parham, P. (1990). Immunology. Some savage cuts in defence. Nature 344, 709–711. Park, Y.G., Clifford, R., Buetow, K.H. and Hunter, K.W. (2003). Multiple cross and inbred strain haplotype mapping of complex-trait candidate genes. Genome Research 13, 118–121. Park, A.Y., Hondowicz, B., Kopf, M. and Scott, P. (2002). The role of IL-12 in maintaining resistance to Leishmania major. Journal of Immunology 168, 5771–5777. Park, A.Y., Hondowicz, B.D. and Scott, P. (2000). IL-12 is required to maintain a Th1 response during Leishmania major infection. Journal of Immunology 165, 896–902.

70

E. HANDMAN ET AL.

Petzl-Erler, M.L., Belich, M.P. and Queiroz-Telles, F. (1991). Association of mucosal leishmaniasis with HLA. Human Immunology 32, 254–260. Powrie, F., Menon, S. and Coffman, R.L. (1993). Interleukin-4 and interleukin-10 synergize to inhibit cell-mediated immunity in vivo. European Journal of Immunology 23, 3043–3049. Preston, P.M. and Dumonde, D.C. (1976). Experimental cutaneous leishmaniasis. V. Protective immunity in subclinical and selfhealing infection in the mouse. Clinical and Experimental Immunology 23, 126–138. Preston, P.M., Carter, R.L., Leuchars, E., Davies, A.J.S. and Dumonde, D.C. (1972). Experimental cutaneous leishmaniasis. III. Effects of thymectomy on the course of infection of CBA mice with Leishmania tropica. Clinical and Experimental Immunology 10, 337–357. Prina, E., Abdi, S.Z., Lebastard, M., Perret, E., Winter, N. and Antoine, J.C. (2004). Dendritic cells as host cells for the promastigote and amastigote stages of Leishmania amazonensis: the role of opsonins in parasite uptake and dendritic cell maturation. Journal of Cell Science 117, 315–325. Reichenbach, J., Rosenzweig, S., Doffinger, R., Dupuis, S., Holland, S.M. and Casanova, J.L. (2001). Mycobacterial diseases in primary immunodeficiencies. Current Opinion in Allergy and Clinical Immunology 1, 503–511. Reiner, N.E. (1994). Altered cell signaling and mononuclear phagocyte deactivation during intracellular infection. Immunology Today 15, 374–381. Reiner, S.L., Wang, Z.-E., Hatam, F., Scott, P. and Locksley, R.M. (1993). Th1 and Th2 cell antigen receptors in experimental leishmaniasis. Science 259, 1457–1460. Ribeiro-Gomes, F.L., Otero, A.C., Gomes, N.A., Moniz-De-Souza, M.C., Cysne-Finkelstein, L., Arnholdt, A.C., Calich, V.L., Coutinho, S.G., Lopes, M.F. and DosReis, G.A. (2004). Macrophage interactions with neutrophils regulate Leishmania major infection. Journal of Immunology 172, 4454–4462. Rissoan, M.C., Soumelis, V., Kadowaki, N., Grouard, G., Briere, F., de Waal Malefyt, R. and Liu, Y.J. (1999). Reciprocal control of T helper cell and dendritic cell differentiation. Science 283, 1183–1186. Ritter, U. and Korner, H. (2002). Divergent expression of inflammatory dermal chemokines in cutaneous leishmaniasis. Parasite Immunology 24, 295–301. Ritter, U. and Moll, H. (2000). Monocyte chemotactic protein-1 stimulates the killing of Leishmania major by human monocytes, acts synergistically with IFN-gamma and is antagonized by IL-4. European Journal of Immunology 30, 3111–3120.


71

Roberts, A.W., Hasegawa, M., Metcalf, D. and Foote, S.J. (2000). Identification of a genetic locus modulating splenomegaly induced by granulocyte colony-stimulating factor in mice. Leukemia 14, 657–661. Roberts, L.J., Baldwin, T.M., Curtis, J.M., Handman, E. and Foote, S.J. (1997). Resistance to Leishmania major is linked to the H2 region on chromosome 17 and to chromosome 9. Journal of Experimental Medicine 185, 1–6. Roberts, L.J., Baldwin, T.M., Speed, T.P., Handman, E. and Foote, S.J. (1999). Chromosomes X, 9, and the H2 locus interact epistatically to control Leishmania major infection. European Journal of Immunology 29, 3047–3050. Roberts, M., Alexander, J. and Blackwell, J.M. (1990). Genetic analysis of Leishmania mexicana infection in mice: single gene (Scl-2) controlled predisposition to cutaneous lesion development. Journal of Immunogenetics 17, 89–100. Roberts, M., Mock, B.A. and Blackwell, J.M. (1993). Mapping of genes controlling Leishmania major infection in CXS recombinant inbred mice. European Journal of Immunogenetics 20, 349–362. Robertson, M.J. (2002). Role of chemokines in the biology of natural killer cells. Journal of Leukocyte Biology 71, 173–183. Rogers, M.E., Ilg, T., Nikolaev, A.V., Ferguson, M.A.J. and Bates, P.A. (2004). Transmission of cutaneous leishmaniasis by sand flies is enhanced by regurgitation of fPPG. Nature 430, 463–467. Sacks, D.L. (1988). Developmental biology of Leishmania promastigotes. In: The Biology of Parasitism (P. Englund and A. Sher, eds), pp. 93–103. New York: Alan R. Liss, Inc. Sacks, D.L. (1989). Metacyclogenesis in Leishmania promastigotes. Experimental Parasitology 69, 100–103. Sacks, D. and Melby, P.C. (1998). Animal models for the analysis of immune responses to leishmaniasis. In: Current Protocols in Immunology (R. Coico, series editor), pp. 19.2.1–19.2.20. New York: John Wiley & Sons. Sacks, D. and Noben-Trauth, N. (2002). The immunology of susceptibility and resistance to Leishmania major in mice. Nature Reviews. Immunology 2, 845–858. Sadick, M.D., Locksley, R.M., Tubbs, C. and Raff, H.V. (1986). Murine cutaneous leishmaniasis: resistance correlates with the capacity to generate interferon-gamma in response to Leishmania antigens in vitro. Journal of Immunology 136, 655–661. Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M. and Toda, M. (1995). Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. Journal of Immunology 155, 1151–1164.

72

E. HANDMAN ET AL.

Sato, N., Ahuja, S.K., Quinones, M., Kostecki, V., Reddick, R.L., Melby, P.C., Kuziel, W.A. and Ahuja, S.S. (2000). CC chemokine receptor (CCR)2 is required for langerhans cell migration and localization of T helper cell type 1 (Th1)-inducing dendritic cells. Absence of CCR2 shifts the Leishmania major-resistant phenotype to a susceptible state dominated by Th2 cytokines, b cell outgrowth, and sustained neutrophilic inflammation. Journal of Experimental Medicine 192, 205–218. Satoskar, A.R., Stamm, L.M., Zhang, X., Satoskar, A.A., Okano, M., Terhorst, C., David, J.R. and Wang, B. (1999). Mice lacking NK cells develop an efficient Th1 response and control cutaneous Leishmania major infection. Journal of Immunology 162, 6747–6754. Scharton, T.M. and Scott, P. (1993). Natural killer cells are a source of interferon gamma that drives differentiation of CD4+ T cell subsets and induces early resistance to Leishmania major in mice. Journal of Experimental Medicine 178, 567–577. Scott, P. (1991). IFN-gamma modulates the early development of Th1 and Th2 responses in a murine model of cutaneous leishmaniasis. Journal of Immunology 147, 3149–3155. Scott, P. and Hunter, C.A. (2002). Dendritic cells and immunity to leishmaniasis and toxoplasmosis. Current Opinion in Immunology 14, 466–470. Scott, P., Eaton, A., Gause, W.C., Zhou, X.-D. and Hondowicz, B. (1996). Early IL-4 production does not predict susceptibility to Leishmania major. Experimental Parasitology 84, 178–187. Scott, P., Natovitz, P., Coffman, R.L., Pearce, E. and Sher, A. (1988). Immunoregulation of cutaneous leishmaniasis. T cell lines that transfer protective immunity or exacerbation belong to different T helper subsets and respond to distinct parasite antigens. Journal of Experimental Medicine 168, 1675–1684. Singh, N., Sundar, S., Williams, F., Curran, M.D., Rastogi, A., Agrawal, S. and Middleton, D. (1997). Molecular typing of HLA class I and class II antigens in Indian kala-azar patients. Tropical Medicine and International Health 2, 468–471. Sommer, F., Meixner, M., Mannherz, M., Ogilvie, A.L., Rollinghoff, M. and Lohoff, M. (1998). Analysis of cytokine patterns produced by individual CD4+ lymph node cells during experimental murine leishmaniasis in resistant and susceptible mice. International Immunology 10, 1853–1861. Staretz-Haham, O., Melamed, R., Lifshitz, M., Porat, N., Fieschi, C., Casanova, J.L. and Levy, J. (2003). Interleukin-12 receptor beta1 deficiency presenting as recurrent Salmonella infection. Clinical Infectious Diseases 37, 137–140.


73

Stenger, S., Donhauser, N., Thuring, H., Rollinghoff, M. and Bogdam, C. (1996). Reactivation of latent leishmaniasis by inhibition of inducible nitric oxide synthase. Journal of Experimental Medicine 183, 1501–1514. Stetson, D.B., Mohrs, M., Mallet-Designe, V., Teyton, L. and Locksley, R.M. (2002). Rapid expansion and IL-4 expression by Leishmaniaspecific naive helper T cells in vivo. Immunity 17, 191–200. Stobie, L., Gurunathan, S., Prussin, C., Sacks, D.L., Glaichenhaus, N., Wu, C.Y. and Seder, R.A. (2000). The role of antigen and IL-12 in sustaining Th1 memory cells in vivo: IL-12 is required to maintain memory/effector Th1 cells sufficient to mediate protection to an infectious parasite challenge. Proceedings of the National Academy of Sciences USA 97, 8427–8432. Swank, R.T. and Bailey, D.W. (1973). Recombinant inbred lines: value in the genetic analysis of biochemical variants. Science 181, 1249–1252. Swihart, K., Fruth, U., Messmer, N., Hug, K., Behin, R., Huang, S., Del Giudice, G., Aguet, M. and Louis, J.A. (1995). Mice from a genetically resistant background lacking the interferon gamma receptor are susceptible to infection with Leishmania major but mount a polarized T helper cell 1-type CD4+ T cell response. Journal of Experimental Medicine 181, 961–971. Szabo, S.J., Dighe, A.S., Gubler, U. and Murphy, K.M. (1997). Regulation of the interleukin (IL)-12R b2 subunit expression in developing T helper 1 (Th1) and Th2 cells. Journal of Experimental Medicine 185, 817–824. Szabo, S.J., Kim, S.T., Costa, G.L., Zhang, X., Fathman, C.G. and Glimcher, L.H. (2000). A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell 1000, 655–669. Tacchini-Cottier, F., Zweifel, C., Belkaid, Y., Mukankundiye, C., Vasei, M., Launois, P., Milon, G. and Louis, J.A. (2000). An immunomodulatory function for neutrophils during the induction of a CD4+ Th2 response in BALB/c mice infected with Leishmania major. Journal of Immunology 165, 2628–2636. Tapia, F.J., Caceres-Dittmar, G. and Sanchez, M.A. (1994). Inadequate epidermal homing leads to tissue damage in human cutaneous leishmaniasis. Immunology Today 15, 160–165. Titus, R.G., Ceredig, R., Cerottini, J.C. and Louis, J.A. (1985). Therapeutic effect of anti-L3T4 monoclonal antibody GK1.5 on cutaneous leishmaniasis in genetically-susceptible BALB/c mice. Journal of Immunology 135, 2108–2114. Turco, S.J., Spa¨th, G.F. and Beverley, S.M. (2001). Is lipophosphoglycan a virulence factor? A surprising diversity between Leishmania species. Trends in Parasitology 17, 223–226. Van Zandbergen, G., Hermann, N., Laufs, H., Solbach, W. and Laskay, T. (2002). Leishmania promastigotes release a granulocyte chemotactic

74

E. HANDMAN ET AL.

factor and induce interleukin-8 release but inhibit gamma interferoninducible protein 10 production by neutrophil granulocytes. Infection and Immunity 70, 4177–4184. Vester, B., Muller, K., Solbach, W. and Laskay, T. (1999). Early gene expression of NK cell-activating chemokines in mice resistant to Leishmania major. Infection and Immunity 67, 3155–3159. Vidal, S., Tremblay, M.L., Govoni, G., Gauthier, S., Sebastiani, G., Malo, D., Skamene, E., Olivier, M., Jothy, S. and Gros, P. (1995). The Ity/Lsh/ Bcg locus: natural resistance to infection with intracellular parasites is abrogated by disruption of the Nramp1 gene. Journal of Experimental Medicine 182, 655–666. Vieira, L.Q., Goldschmidt, M., Nashleanas, M., Pfeffer, K., Mak, T. and Scott, P. (1996). Mice lacking the TNF receptor p55 fail to resolve lesions caused by infection with Leishmania major, but control parasite replication. Journal of Immunology 157, 827–835. Vladimirov, V., Badalova, J., Svobodova, M., Havelkova, H., Hart, A.A., Blazkova, H., Demant, P. and Lipoldova, M. (2003). Different genetic control of cutaneous and visceral disease after Leishmania major infection in mice. Infection and Immunity 71, 2041–2046. von Stebut, E., Belkaid, Y., Jakob, T., Sacks, D.L. and Udey, M.C. (1998). Uptake of Leishmania major amastigotes results in activation and interleukin 12 release from murine skin-derived dendritic cells: implications for the initiation of anti-Leishmania immunity. Journal of Experimental Medicine 188, 1547–1552. von Stebut, E., Belkaid, Y., Nguyen, B.V., Cushing, M., Sacks, D.L. and Udey, M.C. (2000). Leishmania major-infected murine langerhans celllike dendritic cells from susceptible mice release IL-12 after infection and vaccinate against experimental cutaneous Leishmaniasis. European Journal of Immunology 30, 3498–3506. Vouldoukis, I., Riveros-Moreno, V., Dugas, B., Ouaaz, F., Becherel, P., Debre, P., Moncada, S. and Mossalayi, M.D. (1995). The killing of Leishmania major by human macrophages is mediated by nitric oxide induced after ligation of the Fc epsilon RII/CD23 surface antigen. Proceedings of the National Academy of Sciences USA 92, 7804–7808. Wade, C.M., Kulbokas, E.J., III, Kirby, A.W., Zody, M.C., Mullikin, J.C., Lander, E.S., Lindblad-Toh, K. and Daly, M.J. (2002). The mosaic structure of variation in the laboratory mouse genome. Nature 420, 574–578. Wang, Z.E., Reiner, S.L., Zheng, S., Dalton, D.K. and Locksley, R.M. (1994). CD4+ effector cells default to the Th2 pathway in interferon gamma-deficient mice infected with Leishmania major. Journal of Experimental Medicine 179, 1367–1371.


75

Wei, X.Q., Charles, I.G., Smith, A., Ure, J., Feng, G.J., Huang, F.P., Xu, D., Muller, W., Moncada, S. and Liew, F.Y. (1995). Altered immune responses in mice lacking inducible nitric oxide synthase. Nature 375, 408–411. WHO. (2000). The Leishmaniases and Leishmania/HIV Co-infections. Geneva: World Health Organisation. Wilhelm, P., Ritter, U., Labbow, S., Donhauser, N., Rollinghoff, M., Bogdan, C. and Korner, H. (2001). Rapidly fatal leishmaniasis in resistant C57BL/6 mice lacking TNF. Journal of Immunology 166, 4012–4019. Wilson, M.E., Young, B.M., Davidson, B.L., Mente, K.A. and McGowan, S.E. (1998). The importance of TGF-beta in murine visceral leishmaniasis. Journal of Immunology 161, 6148–6155. Xu, D., Liu, H., Komai-Koma, M., Campbell, C., McSharry, C., Alexander, J. and Liew, F.Y. (2003). CD4+CD25+ regulatory T cells suppress differentiation and functions of Th1 and Th2 cells, Leishmania major infection, and colitis in mice. Journal of Immunology 170, 394–399. Yoshida, H., Hamano, S., Senaldi, G., Covey, T., Faggioni, R., Mu, S., Xia, M., Wakeham, A.C., Nishina, H., Potter, J., Saris, C.J. and Mak, T.W. (2001). WSX-1 is required for the initiation of Th1 responses and resistance to L. major infection. Immunity 15, 569–578. Zheng, W. and Flavell, R.A. (1997). The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 89, 587–596. Zhu, X., Cooper, R.S. and Elston, R.C. (2004). Linkage analysis of a complex disease through use of admixed populations. American Journal of Human Genetics 74, 1136–1153.

Death

Host damage

Disease

Persistence

Infection

Exposure Weak

Host response

Strong

Plate 1.1 The ‘damage– response’ framework (adapted from Casadevall and Pirofski, 2003). The diagram shows the damage to the host caused by infection as a function of the amplitude of the host response. In this model, infection can lead to no symptoms, to the persistence of the microbe in a latent state or to disease. Damage to the host is most severe at the two ends of the spectrum of host responses, when they are weak or inappropriate, or when they are exaggerated or inappropriate.

CD4+ T cells Dendritic cell

IL-12 Macrophage

IFN-γ CD8+ T cells

i NOS

M

? NK cells

Plate 1.3 The immune response to L. major infection. Early events after infection with L. major result in a cytokine environment, which promotes production of IFN-g or IL-4 from T cells and other cells. Macrophages harbouring parasites are activated to produce NO and kill the parasites if IFN-g is present. IL-4 inhibits IFN-g production, thus allowing the parasites to survive in the macrophage.

Plate 1.5 Immune responses that affect susceptibility to visceral leishmaniasis in Sudan (reproduced with permission from Blackwell et al., 2004). SLC11A1 regulates macrophage activation directly and also contributes to the regulation of Th cell responses. Polymorphism at the IL-4 locus contributes to susceptibility to clinical disease, whereas polymorphism at the IFNGR1 only affects development of the severe form of disease, postkala-azar dermal leishmaniasis (PKDL). T reg cells may also contribute to the development of PKDL through their effect on IFNGR1 and increased levels of IL-10.

Cryptosporidium and Cryptosporidiosis R.C.A. Thompson1,, M.E. Olson2, G. Zhu3,4, S. Enomoto4,5, Mitchell S. Abrahamsen5,6 and N.S. Hijjawi1 1

Division of Veterinary and Biomedical Sciences, Murdoch University, Murdoch WA 6150, Australia 2 Microbiology and Infectious Diseases, University of Calgary, Alberta, Canada 3 Department of Veterinary Pathobiology, Texas AM University, College Station, TX 77843-4467, USA 4 Faculty of Genetics Program, Texas AM University, College Station, TX 77843-4467, USA 5 Department of Veterinary and Biomedical Science, College of Veterinary Medicine, University of Minnesota, 55108, St. Paul, MN, USA 6 Biomedical Genomics Center, University of Minnesota, 55108, St. Paul, MN, USA

Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Phylogenetic Relationships and Taxonomy . . . . . . . . . . . 2.1. Biological and Molecular Characteristics . . . . . . . . 2.2. Current Taxonomy . . . . . . . . . . . . . . . . . . . . . . . . 3. Life Cycle and Development . . . . . . . . . . . . . . . . . . . . . 3.1. Establishment . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Life Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Developmental Stages in the Life Cycle. . . . . . . . . 3.4. Maintenance and Amplification of Cryptosporidium . 4. Host–Parasite Relationship . . . . . . . . . . . . . . . . . . . . . . 4.1. Pathogenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Immunobiology . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Infections in Humans . . . . . . . . . . . . . . . . . . . . . .

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. 78 . 79 . 79 . 79 . 85 . 88 . 88 . 89 . 91 . 95 . 99 . 99 . 101 . 104

Author for correspondence. E-mail: [email protected]

ADVANCES IN PARASITOLOGY VOL 59 ISSN: 0065-308X DOI: 10.1016/S0065-308X(05)59002-X


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5.

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7.

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4.4. Infections in Cattle . . . . . . . . . . . . . . . . . . . . . 4.5. Infections in Sheep and Goats . . . . . . . . . . . . 4.6. Infections in Pigs . . . . . . . . . . . . . . . . . . . . . . 4.7. Infections in Poultry . . . . . . . . . . . . . . . . . . . . 4.8. Infection in Horses . . . . . . . . . . . . . . . . . . . . . 4.9. Infection in Dogs and Cats . . . . . . . . . . . . . . . 4.10. Infections in Reptiles . . . . . . . . . . . . . . . . . . . 4.11. Infections in Fish . . . . . . . . . . . . . . . . . . . . . . The Regulation of Biochemical Processes . . . . . . . . 5.1. Cryptosporidium parvum Genome . . . . . . . . . . 5.2. Core Energy Metabolism . . . . . . . . . . . . . . . . 5.3. Fatty Acid and Polyketide Synthesis . . . . . . . . 5.4. Nucleic Acid Metabolism. . . . . . . . . . . . . . . . . 5.5. Amino Acid Metabolism . . . . . . . . . . . . . . . . . 5.6. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . Epidemiology and Transmission . . . . . . . . . . . . . . . 6.1. Cycles of Transmission and Zoonotic Potential. 6.2. Epidemiology of Infections in Humans . . . . . . . Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Detection. . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Chemotherapy . . . . . . . . . . . . . . . . . . . . . . . . 7.3. Immunotherapy and Immunoprophylaxis . . . . . Perspectives for the Future . . . . . . . . . . . . . . . . . . . Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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105 107 108 109 110 110 110 111 111 112 115 118 120 121 122 123 123 127 130 130 132 136 138 138 139

ABSTRACT Cryptosporidium is one of the most common enteric protozoan parasites of vertebrates with a wide host range that includes humans and domestic animals. It is a significant cause of diarrhoeal disease and an ubiquitous contaminant of water which serves as an excellent vehicle for transmission. A better understanding of the development and life cycle of Cryptosporidium, and new insights into its phylogenetic relationships, have illustrated the need to re-evaluate many aspects of the biology of Cryptosporidium. This has been reinforced by information obtained from the recent successful Cryptosporidium genome sequencing project, which has emphasised the uniqueness of this organism in terms of its parasite life style and evolutionary biology.

CRYPTOSPORIDIUM AND CRYPTOSPORIDIOSIS

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This chapter provides an up to date review of the biology, biochemistry and host parasite relationships of Cryptosporidium.

1. INTRODUCTION Cryptosporidium is a significant cause of diarrhoeal disease in humans, livestock and other animals throughout the world, and a major economic burden to the water industry. During recent years, research into our understanding of Cryptosporidium and cryptosporidiosis has become increasingly diversified yet reviews have tended to concentrate on taxonomy and nomenclature, or waterborne transmission. This undoubtedly reflects the relatively recent development of molecular epidemiological tools, and the pressure from the water industry for adequate methodologies for surveillance and control. As a consequence, the biology and host–parasite relationship of Cryptosporidium have not received the attention they should have, given the uniqueness of this organism. Recent developments in vitro cultivation and life cycle propagation, and elucidation of the complete genome sequence of C. parvum have served to illustrate the need to re-evaluate many aspects of the biology and ecology of Cryptosporidium. Our aim in this chapter is thus to redress the balance and provide an up to date overview of the field. In so doing, we have recognised the need to focus on other areas of research activity such as the biology and biochemistry of Cryptosporidium, host–parasite relationships, and therapy.

2. PHYLOGENETIC RELATIONSHIPS AND TAXONOMY 2.1. Biological and Molecular Characteristics The phylogenetic affinities of Cryptosporidium are intriguing and require re-evaluation in the light of developmental, biochemical and genomic data (see below). Cryptosporidium is traditionally considered as one of the coccidian protists (i.e. a taxonomic sister to the

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intestinal and cyst-forming apicomplexans). However, recent molecular phylogenetic analyses based on a relatively small number of genes have consistently placed the Cryptosporidium genus at the base of the Phylum Apicomplexa (Zhu et al., 2000a), or even as a sister to gregarines (Carreno et al., 1999), suggesting that this group of parasites are evolutionarily divergent not only from the coccidia, but also from almost all other apicomplexans (Tenter et al., 2002). Cryptosporidium was originally classified as a coccidian based on its possession of similar life cycle features (Levine, 1988). However, Cryptosporidium demonstrates several peculiarities that separate it from any other coccidian. These include the location of Cryptosporidium within the host cell where the endogenous developmental stages are confined to the apical surfaces of epithelial cells (intracellular but extracytoplasmic) (Figure 1); the attachment of the parasite to the host cell where a multi-membranous attachment or feeder organelle is formed at the base of the parasitophorous vacuole to facilitate the uptake of nutrients from the host cell; the presence of two morpho-functional types of oocyst, thick- and thin-walled, with the

Figure 1 Diagrammatic representation of the location of Cryptosporidium within the host cell (PV ¼ parasitophorous vacuole; EB ¼ electron-dense band; FO ¼ feeder organelle). Figure drawn by Russ Hobbs.


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Figure 2 Diagrammatic representation of the life cycle of C. parvum in the mucosal epithelium of an infected mammalian host based on information available prior to 2000. After oocyst excystation in the intestinal lumen, sporozoites penetrate the host cell and develop into trophozoites within parasitophorous vacuoles located in the microvillous region of the mucosal epithelium. Trophozoites undergo asexual division (merogony) to form merozoites. After release from type I meronts, merozoites enter adjacent host cells and multiply to form additional type I meronts, or to form type II meronts. Type II meronts do not recycle but enter host cells to commence the sexual phase of the life cycle with the formation of microgamonts and macrogamonts. Most (approximately 80%) of the zygotes formed after fertilisation develop into environmentally resistant, thick-walled oocysts that undergo sporogony to form sporulated oocysts containing four sporozoites. A smaller percentage of zygotes (approximately 20%) form thin-walled oocysts surrounding the four sporozoites that represent the autoinfective life cycle forms that can maintain the parasite within the host without repeated oral exposure to the thick-walled oocysts present in the environment. From Current (1990) and Carey et al. (2004). Figure drawn by Russ Hobbs.

latter responsible for the initiation of the auto-infective cycle in the infected host (Figures 2 and 3 – Figure 3 is Plate 2.3 in the Separate Color Plate section); the small size of the oocyst (7.4 5.6 mm for C. muris and 5.0 4.5 mm for C. parvum), which lacks morphological structures such as sporocyst, micropyle and polar granules; and finally, the insensitivity of Cryptosporidium to almost all anticoccidial

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agents tested so far (Hijjawi et al., 2004; Petry, 2004). In this respect, the unusual biochemical and related organellar characteristics of Cryptosporidium raise further, often controversial issues concerning its phylogenetic relationships (Abrahamsen et al., 2004; Putignani et al., 2004). Beier (2000) considered that seemingly unique characteristics of Cryptosporidium such as its peculiar relationship with the host cell and possession of the so-called ‘feeder organelle’, and two oocyst types containing a small number of sporozoites, are a result of early acquisition in the course of evolution. He did not consider that the molecular differences cast doubt on the coccidian nature of Cryptosporidium. However, The non-coccidian affinities of Cryptosporidium are further supported by recent developmental studies (see Section 3). The ability to observe the life cycle and development of Cryptosporidium in in vitro culture has not only demonstrated the occurrence of previously unrecognised stages in the life cycle but also convincingly demonstrated that Cryptosporidium is not an obligate intracellular parasite (Hijjawi et al., 2004). Recent observations in vitro suggested that pairing which resembles syzygy in gregarines is involved in Cryptosporidium life cycle stages. Pairing of stages of unequal ages and early in development was frequently observed both in cell culture and cell-free culture of Cryptosporidium (Hijjawi et al., 2004). Apart from macro and microgamont stages, pairing was observed between sporozoites, trophozoites as well as merozoites stages of Cryptosporidium. Such a process was recently described in the gregarine species Gregarina tribolorum where pairing behaviour of this species was observed between gamonts of unequal ages, working to erode whatever synchrony might be expected in G. triboliorum development (Watwood et al., 1997). Stages of Cryptosporidium observed in culture show remarkable similarities to those seen in the life cycles of some gregarines. The behaviour of Cryptosporidium sporozoites, once they are released from oocysts and transform into trophozoites and aggregate, leading to two merogony stages with merozoites from meront II initiating the sexual stage in the life cycle is similar to the developmental stages occurring in the life cycle of the gregarine Mattesia dispora (Levine,


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1985; and see below). Cryptosporidium also has an unusual developmental plasticity in its life cycle with the ability to avoid going through all life cycle stages (Hijjawi et al., 2004). Comparing the life cycle of two Mattesia species of gregarine, Mattesia dispora and M. geminate similarities can be seen in their life cycles compared to Cryptosporidium (Figure 4). In M. dispora, the formation of structures after the fusion of sporozoites is similar to what has been observed in the Cryptosporidium life cycle and referred to as the extracellular gamont, like stages in cell culture, because of their ability to grow and develop outside the host cells. Similar stages have been purified from mice infected with C. parvum (Hijjawi et al., 2002). Further, the behaviour of the sporozoites of M. geminate is similar to what has been observed in cell free culture of Cryptosporidium with the formation of circular trophozoites, which aggregate together forming type I meronts. The gliding movements seen in different stages of Cryptosporidium is a behavioural feature also seen in Plasmodium and Toxoplasma. Interestingly, a similar form of gliding motility is exhibited by gregarines (Sibley, 2004). Furthermore, recent molecular and ultrastructural characterisation of the marine aseptate gregarines Selenidium and Lecadina (Leander et al., 2003) provide support for the sister relationship between Cryptosporidium and the gregarines. These unique biological and morphological characteristics of Cryptosporidium have been complemented further by the results of molecular characterisation studies, which consistently group Cryptosporidium as a clade separate from the coccidian taxa (Relman et al., 1996; Barta, 1997; Morrison and Ellis, 1997; Carreno et al., 1998; Lopez et al., 1999). Furthermore, a recent study by Carreno et al. (1999) based on SSU ribosomal RNA sequencing showed that the gregarines and Cryptosporidium formed a clade separate from the other major apicomplexan clades containing the coccidia. On the basis of phylogenetic analysis of molecular data (Morrison and Ellis, 1997; Carreno et al., 1999; Zhu et al., 2000a), Morrison et al. (2004) consider species of Cryptosporidium to be phylogenetically too distant from the coccidia to be considered as the immediate sister group and did not include Cryptosporidium in their recent phylogenetic analysis

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Figure 4 Comparison of the life cycles of (A) Mattesia dispora, note how the sporozoites fuse after their release from oocysts to form a large opaque structure; (B) Mattesia geminate, note how the sporozoites transform into trophozoites after their release from oocysts and fuse together to form a grape-like structure (meront I). Note also the two different types of merozoites formed from type I and type II meronts. (C) Cryptosporidium life cycle in host cellfree culture, note the similarities in both life cycles (A) and (B) and Cryptosporidium. From Levine (1985) and Hijjawi et al. (2004).


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of the coccidia even though they have traditionally been treated as part of the coccidia. Despite the molecular similarities between Cryptosporidium and the gregarines, Carreno et al. (1999) highlighted differences in the developmental cycles between gregarines and Cryptosporidium, namely, the lack of stages of syzygy and other gamont stages from the life cycle of Cryptosporidium. However, as detailed above, the discovery of such stages serves to reinforce the growing view that Cryptosporidium is not a coccidian. Quite clearly, there is a need for more extensive phylogenetic analysis of Cryptosporidium and other apicomplexans, particularly the gregarines. Whether Cryptosporidium warrants inclusion with the gregarines or should be considered as belonging to a new order remains to be determined.

2.2. Current Taxonomy As with many other protozoan parasites with few morphological characters for species discrimination, early workers relied largely on host occurrence in describing species of Cryptosporidium. This resulted in the description of a large number of species and a history of taxonomic confusion and controversy (O’Donoghue, 1995; Thompson, 2002). Today, we recognise that Cryptosporidium is a phenotypically and genotypically heterogeneous assemblage of largely morphologically identical genotypes and species (Morgan et al., 1999; Thompson, 2003a; Monis and Thompson, 2003; Tables 1 and 2). It is not surprising, therefore, that the lack of morphological characters to discriminate variants led to much debate over whether phenotypic differences were ‘real’ and reflected genetic differences or were the result of environmental/host-induced changes (Fall et al., 2003). Clearly, the application of molecular tools has had an enormous impact on our understanding of the nature of variation in Cryptosporidium. In particular, the development of appropriate polymerase-chain reaction (PCR)-based procedures has been of great value with an organism such as Cryptosporidium which, until recently, was largely refractory to laboratory amplification (Hijjawi, 2003;

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Table 1

Currently recognised species of Cryptosporidium

Species

Major hosts

Site

C. C. C. C. C. C. C. C. C. C. C. C. C. C.

Rodents Cattle and other livestock, humans Birds Guinea pigs Cats Reptiles Poultry Lizards

Stomach Small intestine Small intestine Small intestine Small intestine Stomach Bursa Stomach and small intestine Proventriculus Abomasum Small intestine Stomach and small intestine Small intestine Small and large intestine

muris parvum meleagridis wrairi felis serpentis baileyi saurophilum galli andersoni canis molnari hominis suis

Cattle Dogs Fish Humans Pigs

Source: Thompson (2003a, b) and Xiao et al. (2004).

Table 2

Genotypes of C. parvum

Ferret Mouse Skunk Marsupial ( 4) Horse Rabbit Monkey Pig ( 2) Cervid ( 2) Fox Muskrat ( 2)

Deer mice Squirrel Bear Goose ( 2) Duck Bovine Snake Tortoise Lizard Woodcock


Hijjawi et al., 2001, 2002, 2004). The direct characterisation of oocysts recovered from faecal or environmental samples using PCR based procedures has not only had a major impact on resolving the taxonomy of Cryptosporidium at the species level but also on the molecular epidemiology of Cryptosporidium infections. A number of genetic loci have proved particularly useful in studies on Cryptosporidium in terms of reproducibility between different laboratories,


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however, those of most value are the 18S rDNA, HSP70, actin and COWP genes (Monis and Thompson, 2003; Xiao et al., 2004). The majority of species of Cryptosporidium were described in the period following recognition of Cryptosporidium as a human pathogen (Thompson, 2002). However, the taxonomic status of many of these species was questioned in the early 1990s (see O’Donoghue, 1995) and it is only recently with the application of molecular characterisation procedures in different laboratories on a range of isolates, that many of these species have been resurrected and new species described (Table 1). Many of these are morphologically identical but information on host range and other phenotypic characters is slowly being acquired from different parts of the world thus supporting taxonomic validity of the 14 species currently recognised. In addition to these species, a large number of genetically distinct variants, or genotypes, have also been described, which appear to be host adapted, and are thought to represent distinct species (Table 2). However, consideration will have to be given to the epidemiological value of naming multiple species of Cryptosporidium from the same host species. The demonstration of genetic distinctness without significant phenotypic characters to discriminate variants negates the value of naming species. Interestingly, phylogenetic analyses have shown that genetically related hosts often have related forms of Cryptosporidium. For example, marsupials, canids, cervids, primates, lagomorphs and rodents (Xiao et al., 2004). This is considered to support a theory of host–parasite coevolution. However, in some cases, these authors consider that current host occurrence may not reflect the host in which a particular species of Cryptosporidium evolved. For example, C. meleagridis has been proposed to have been a parasite of mammals originally that has been subsequently established in birds (Xiao et al., 2004). It has also been suggested that since Cryptosporidium is not a coccidian, but more closely related to gregarine protozoa, then it is not surprising for Cryptosporidium to have a large number of species, but also that the host range may well include more lower vertebrates than envisaged and possibly species of invertebrate (Hijjawi et al., 2004).

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3. LIFE CYCLE AND DEVELOPMENT 3.1. Establishment Until recently, most studies consistently considered Cryptosporidium to be an obligate intracellular parasite and both light and electron microscopy has confirmed the unique intracellular but extra cytoplasmic location of Cryptosporidium stages within the host cells. In vivo and in vitro studies have shown the penetration and attachment of Cryptosporidium stages to the apical surfaces of host cells. However, the mechanisms by which Cryptosporidium invades host intestinal epithelial cells and establishes this unusual compartment are poorly understood. Initially, Cryptosporidium exhibits actin-dependent gliding and probably uses a parasite-driven process to gain entry into the apical surface of the host intestinal epithelial cells where it rests on a bed of host actin-binding proteins (Sibley, 2004). Endogenous development of Cryptosporidium involves a sequence of events comprising, parasitophorous vacuole formation, development of a unique feeder organelle, and asexual and sexual multiplication of the parasite. Recent studies in which serial electron microscopy was undertaken during a 1 h incubation of C. parvum in cell culture showed that during internalisation, numerous vacuoles covered by the parasite’s plasma membrane are formed and cluster together to establish the parasitophorous vacuole (Huang et al., 2004a). A tunnel directly connecting the parasite to the host cell forms during internalisation and remains when the parasite is totally internalised. The tunnel-like structure would provide efficient communication between the parasite and the infected host cells and may be a transient structure during the invasion process (Chen, X.M., personal communication). The fate of the tunnel-like structure is unknown, but could subsequently be involved in the formation of the feeder organelle. This extracytoplasmic vacuolar niche remains at the apical surface of infected cells in the region of the microvilli (Morrissette and Sibley, 2002), and the apical location and separation from the host cell cytoplasm causes it to bulge into the lumen of the gut (Figure 1).


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The parasite induces host cytoskeletal rearrangements including the formation of branched microvilli clustered around the parasitophorous vacuole (Farney et al., 1999; Lumb et al., 1988). Cryptosporidium also induces the formation of a junctional complex that lies between it and the cytoplasm of the infected epithelial cell, keeping the parasite in the region of the microvilli (Elliott et al., 2001). This electron dense band that separates the parasite from the host cell is composed of host filamentous actin assembled into a plaque-like structure. This parasiteinduced rearrangement of the host cell cytoskeleton involves incorporation of host cell actin and a-actin into a host–parasite junctional complex (Elliott and Clark, 2000).

3.2. Life Cycle The recent discovery of novel extracellular stages in the life cycle of Cryptosporidium coupled with the reported ability of the parasite to complete its entire life cycle without the need of host cells (Hijjawi et al., 2004) demonstrates that Cryptosporidium is not an obligate intracellular parasite, and raises many questions about its developmental biology, nutritional requirements, metabolic pathways, and the unpredictable and plastic reproductive capability of this organism. Cryptosporidium parvum can complete its entire life cycle without the need for host cells, with the presence of all developmental phases including merogony, gametogony, sporogony as well as recently described novel gamont-like stages that in cell culture are predominantly extracellular (Hijjawi et al., 2002, 2004; and see below). The fact that Cryptosporidium is capable of extracellular development and does not exhibit true intracellular development, since it is extracytoplasmic, suggests that from an evolutionary perspective, Cryptosporidium may still be adapting to an intracellular habitat. Since Cryptosporidium does not need to invade a host cell in order to complete its development raises the question why the parasite does so in vivo, and what proportion of the parasite population in vivo is extracellular? The proportion of these populations may vary and be dependent upon immunological or other host factors. Its

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intracellular but extracellular niche may allow the parasite to evade immune surveillance but take advantage of solute transport across the host microvillous membrane or the parasitophorous vacuole (Abrahamsen et al., 2004). The life cycle of Cryptosporidium is complex involving both sexual and asexual developmental stages (Figures 2 and 3). It begins upon the inhalation or the ingestion of sporulated oocysts by a suitable host (Fayer and Unger, 1986). The sporulated oocyst is the only known exogenous stage, consisting of four sporozoites within an environmentally resistant oocyst wall. Once inhaled or ingested, oocysts excyst in the gastrointestinal or respiratory tract releasing infective sporozoites (Reduker et al., 1985; Reduker and Speer, 1985). Once freed, the sporozoites rapidly invade the epithelial cells of the small intestine (mainly the ileum) and respiratory tract where the endogenous phase of the life cycle begins. Within the host cell, the sporozoite transforms into a circular stage known as a trophozoite. The trophozoite undergoes asexual proliferation by merogony (previously called schizogony) to form meronts which contain merozoites. Two types of meronts have been described (Current and Reese, 1986). Type I meronts each form eight merozoites, which are liberated from the parasitophorous vacuoles when mature. Type I merozoites are believed to have the ability to recycle indefinitely under suitable environmental conditions. Some type I merozoites penetrate host cells and form type II meronts containing four merozoites. Once liberated, Type II merozoites enter host cells and start the sexual phase in the life cycle. Once inside host cells, Type II merozoites either enlarge and form macrogametes (macrogametocyte), or undergo cellular fission forming microgametocytes containing 14–16 non-flagellated microgametocytes. Microgametes rupture from microgametocytes and penetrate cells harbouring macrogametocytes, to form a zygote. A resistant oocyst wall is then formed around the zygote and sporozoites are formed by sporogony. Approximately 80% of the oocysts formed after the completion of the life cycle are thick-walled oocysts which are released from the host in the faeces if present in the gastrointestinal tract, or via aerosols or


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nasal secretions if present in the respiratory tract. However, about 20% of oocysts fail to form a thick oocyst wall and are thus known as thin-walled oocysts. Thin-walled oocysts are important in autoinfection through the continuous recycling of sporozoites excysting from ruptured thin-walled oocysts. Thus, Cryptosporidium appears to have two autoinfective cycles mediated by Type I merozoites and thinwalled oocysts.

3.3. Developmental Stages in the Life Cycle Recent advances in the in vitro cultivation of Cryptosporidium (cell culture and cell-free culture) as well as observations from in vivo infections in different hosts have provided more information on its life cycle and necessitate a re-evaluation of the parasite’s phases of development and proliferative potential. The following description of life cycle stages of Cryptosporidium in cell culture and cell-free culture is based on recent studies by Hijjawi et al. (2001, 2002, 2004) highlighting new information and differences to earlier interpretations of the developmental cycle (for detailed reviews see, Upton, 1997; Arrowood, 2002; Hijjawi, 2003). 3.3.1. Trophozoite In cell culture, within HCT-8 cells, trophozoites appear as round or oval uninucleate intracellular forms, 2.7 2.7 mm in diameter (Figures 3, 4C, 5 and 6 – Figures 5 and 6 are Plates 2.5 and 2.6 in the Separate Color Plate section). Trophozoites represent a transitional stage from sporozoites and merozoites to meronts. In cell-free culture, many sporozoites transform into circular to spindle-shaped motile trophozoites measuring 2 1.3 mm in size (Figures 3 and 4C). Trophozoites appear to fuse into aggregates of two or more trophozoites and occasionally large aggregates containing 10–20 stages. After 48–72 h in culture, trophozoites within aggregates develop into meronts (meront I) of variable size depending on the number of initially fused trophozoites (Figures 3 and 4). This

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aggregation was not referred to previously and multiple mitotic division was considered to occur in single trophozoites following penetration of host cells. The aggregation of trophozoites also occurs within HCT-8 cells in culture (Figures 5 and 7 – Figure 7 is Plate 2.7 in the Separate Color Plate section). 3.3.2. Meront I Meront development occurs as a result of multiple mitotic divisions of the fused trophozoites, and there are two different types of meronts (meront I and meront II), which give rise to two morphologically different types of merozoites (Hijjawi et al., 2001, 2002, 2004). Type I meronts appear as grape-like aggregates as early as 48 h post culture inoculation (Figures 3 and 4). Merozoites released from Type I meronts are actively motile, circular to oval in shape and small in size (1.2 1 mm). These merozoites enlarge and clump together to generate type II meronts. The number of merozoites released appears to depend on the initial numbers of stages that fuse together (either trophozoites or merozoites released from meront I) to form these meronts. In vitro studies have shown that the number of merozoites is variable and an unreliable means of differentiating between the two types of meronts. 3.3.3. Meront II Type II meronts (3.1 2.8 mm in diameter), which attain a rosette-like pattern, are first detected after 3 days of culturing (Figure 3). Merozoites released from type II meronts are either broadly spindleshaped with pointed ends measuring 3.5 2 mm in size, or rounded to pleomorphic measuring 1.6 1.5 mm in size. After 7–8 days of culturing large numbers of actively motile merozoites continue to be released from meronts and from 9 days up to 4 months all developmental stages (sporozoites, trophozoites, merozoites, type I and II meronts and sporulated oocysts) have been repeatedly observed in culture. Merozoites released from type II meronts, develop into the


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sexual stages by transforming into macrogamonts and microgamonts (Figure 3). 3.3.4. Merozoites Merozoites are thread-shaped with a central nucleus and pointed ends. Once merozoites are released from the host cell they display vigorous gliding and flexing and penetrate other cells rapidly. Earlier studies on the Cryptosporidium life cycle (Current and Reese, 1986), and others never mentioned the fact that two morphologically different merozoites occur in the life cycle, however, in vitro (cellfree culture) two morphologically different types of merozoites have been observed; merozoites type I which are released from meront I are small in size and very active; however, merozoites type II are bigger in size and sluggish, and give rise to micro and macro gamonts, respectively. 3.3.5. Microgamonts Microgamonts, 5.6 3.4 mm in diameter, containing microgametes (Figure 3) occupy most of the cell around a residuum. Free microgametes as originally described by Current and Reese (1986) are bullet-shaped, and displayed a jerky gliding movement in culture after disruption of the microgamont. The occurrence of budding on the surface of microgamonts might be an attempt by microgametes to escape from the host cell. In cell-free culture, after 6 days of culturing some merozoites released from type II meronts increase in size and develop into microgamonts. Microgamonts are 5.6 5 mm in size, circular in shape and appear very dark at low magnification (Figure 3). Developing microgametes budding from the surface of the microgamont stage are evident 6 days post-culture inoculation (Figure 3). At higher magnifications, microgamonts can be easily differentiated from other stages by the large number of developing microgametes on their surfaces (Figure 3). Microgametes were observed leaving microgamonts via a suture-like opening formed at the surface where clumps of

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motile microgametes, measuring 2.2 1.6 mm in size, could be detected moving freely. 3.3.6. Macrogamonts This stage is distinguished by being large in size (4 4 mm in diameter) and having a large peripheral nucleus (Figure 3). Stages representing macrogamonts with characteristic peripheral nuclei develop after 5 days and are 5 4 mm in size (Figure 3). On several occasions, microgametes were observed adhering to the surface of macrogamonts and in some cases were seen inside a macrogamont. Microgamont/macrogamont pairing has also been observed (Figure 3). Stages resembling zygotes measuring 5 4 mm were also observed after 7–8 days with the appearance of unsporulated oocysts with a large nucleus (Figure 3). The fertilisation process in Cryptosporidium has never directly been observed in previous studies and this has been attributed to two factors. First, because fertilisation is a rapid process and second the interference of the host cells. In cell-free culture, attempts at fertilisation were observed many times (Hijjawi et al., 2004). Microgametes adhering to the surface of macrogamonts were frequently observed and on several occasions a macrogamont with a microgamete inside it was seen. Pairing between gamontlike stages was also observed (Figure 8 is Plate 2.8 in the Separate Color Plate section). Moreover, what appeared to be developing zygotes (Figure 3) resembling unsporulated oocysts were observed confirming that successful fertilisation had occurred in this cell-free culture system. 3.3.7. Oocysts Thick- as well as thin-walled oocysts are usually recovered from culture after 5 days of inoculation. Approximately 80% of the oocysts formed after the completion of the life cycle are thick-walled, whereas about 20% fail to form a thick oocyst wall and are thus known as thin-walled oocysts. Thin-walled oocysts are important in


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autoinfection through the continuous recycling of sporozoites excysting from ruptured thin-walled oocysts. 3.3.8. Sporozoites Sporozoites are 5.2 1.2 mm in size and characterised by having a comma shape with a rounded posterior end and a pointed tapered anterior end. In culture, sporozoites are often seen actively moving inside the oocyst, excysting, and exhibit a gliding movement. The ability to monitor Cryptosporidium in vitro, in cell and cell-free systems has shown that there are at least two ways by which merozoites can be produced during development. The ‘normal’ pathway giving rise to two types of meronts (I and II) as described above, and from sporozoites (Figures 3 and 4). Sporozoites also appear to have the potential to undergo a process similar to multiple syzygy in gregarines in which a large number of sporozoites after excysting from oocysts, fuse together either laterally or end to end to form a large structure up to 35 mm in length (Figures 9 and 10 are Plates 2.9 and 2.10 in the Separate Color Plate section). The size of this stage depends on the number of sporozoites that have been initially fused together. Material secreted by the fused sporozoites causes this structure to become opaque making it difficult to see internal morphology but after a period of time, merozoites are formed and detach from the surface of this novel stage that has not been previously described (Hijjawi et al., 2004).

3.4. Maintenance and Amplification of Cryptosporidium Studies on Cryptosporidium species have been hampered by the limited amount of parasitic stages available for research. One of the major objectives of many laboratories is to amplify the parasite and develop reproducible models of infection to facilitate biological, pathological, immunological and molecular and drug evaluation studies. There are two ways by which Cryptosporidium can be amplified: in vivo, in animal models, and in vitro using different cell lines.

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3.4.1. In vivo maintenance in animal models The majority of in vivo infections have been carried out using the C. parvum cattle genotype. Experimental infections with C. parvum have been successfully established in a variety of domestic and laboratory animals, especially neonates, immunosuppressed or immunodeficient animals (O’Donoghue, 1995). Successful infections of C. parvum have been reported in the neonates of most domestic animals including calves (Tzipori et al., 1983; Fayer et al., 1985), lambs (Tzipori et al., 1981a; Anderson 1982; Snodgrass et al., 1984), goat kids (Tzipori et al., 1982) and piglets (Moon and Bemrick, 1981; Tzipori et al., 1981b, 1994). These models are the only currently available means to produce large number of parasitic stages; however, their maintenance is beyond the capabilities of most laboratories. Small animal models such as neonatal mice have been successfully infected with Cryptosporidium (Sherwood et al., 1982; Ernest et al., 1986: Meloni and Thompson, 1996). A technique for isolating purified oocysts from the gut of mice for further in vitro cultivation, molecular and biochemical studies has been successfully established (Current, 1990; Meloni and Thompson, 1996). Most of the infections in neonate mice have been subclinical and have resolved within 2–4 weeks. In addition, the success of the infection depends on the age of mice; with neonates being susceptible up to 1–2 weeks of age (Sherwood et al., 1982; Ernest et al., 1986; Current, 1990). It has also been found that neonatal mice do not support the infection of all C. parvum genotypes with only the cattle genotype producing infections (Current, 1990; Meloni and Thompson, 1996; Casemore et al., 1997; Peng et al., 1997). Oocysts of C. andersoni are not infective to outbred, inbred, immunocompetent or immunodeficient mice, rats, rabbits or guinea pigs (Koudela et al., 1998; Sre´ter et al., 2000). Differences in the infectivity for laboratory animals also exist between C. hominis and C. parvum cattle genotype with the cattle genotype readily infecting mice and cattle, whereas C. hominis does not (Peng et al., 1997).


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In summary, the amplification of Cryptosporidium in animal models is labour-intensive, expensive beyond the budget of most laboratories, does not support the infection with multiple genotypes of C. parvum and will not support the growth of the parasite for long periods of time. 3.4.2. In vitro cultivation There are many factors that affect the development and proliferation of Cryptosporidium in culture. These include the excystation protocol, age and strain of the parasite, stage and size of the inoculum, host cell type and maturity, and culture conditions such as pH, medium supplements and atmosphere. For more details see review by Hijjawi (2003). The successful cultivation of intracellular protozoan parasites was limited before the 1940s due to the lack of suitable methods of isolating and maintaining host cells in vitro. Thereafter, the necessary conditions to propagate many host cells were defined and led to success in the cultivation of asexual stages of some apicomplexan parasites such as Besnoitia besnoiti, Eimeria tenella and Sarcocystis spp. (Bigalke, 1962; Patton, 1965; Fayer, 1970). Initial attempts to propagate Cryptosporidium were in ovo and used chicken embryos, and resulted in complete development of both C. parvum and C. baileyi (Current and Long, 1983). Both human and calf isolates of C. parvum completed their entire life cycles (from sporozoites to sporulated oocysts) in endoderm cells of the chorioallantoic membrane (CAM) of chicken embryos. The use of in ovo systems to obtain large numbers of Cryptosporidium life cycle developmental stages has been disappointing because of the limited parasite growth and because most oocysts developing in the CAM are not released from the host cells into the allantoic fluid. In addition, separation of developmental stages of the parasite from host tissues is difficult (Current and Garcia, 1991). Sporozoites of C. parvum were reported for the first time to develop into mature meronts after inoculation of human rectal tumour cells (Woodmansee and Pohlenz, 1983; Woodmansee, 1986).

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Oocysts were reported to be produced in vitro using Caco-2 cells, BFTE and MDBK cell lines (Buraud et al., 1991; Yang et al., 1996; Villacorta et al., 1996), however they were few in number, less than the initial inoculum, and failed to maintain the intensity of the infection in vitro and subculturing attempts were unsuccessful (Meloni and Thompson, 1996; Lawton et al., 1997). In most studies, the growth of Cryptosporidium in vitro peaks at 48–72 h post-inoculation and gradually declines. Although, several attempts have been made to improve some of the parameters required to enhance the growth and proliferation in vitro (Upton, 1997), no long-term maintenance or large-scale production of oocysts was achieved and parasite yields were limited. This reduced proliferation of Cryptosporidium in cell culture was attributed to the lack of type I meront recycling and absence of autoinfective thin-walled oocysts (Current and Haynes, 1984). Complete development of C. parvum has been reported in several cell lines with the production of some oocysts, however, the number of oocysts produced was less than that produced in suckling mice or in the CAM of chicken embryos (Current and Haynes, 1984). The lack of appropriate conditions to support the autoinfective cycle as occurs in vivo was considered a serious limitation to the success of continuous culture (Current and Garcia, 1991). Although, major improvements in culturing Cryptosporidium in vitro have occurred since 1984, infections could not be maintained for more than a few days and only the asexual phase of the parasite life cycle could be consistently maintained for short periods (reviewed in Hijjawi, 2003). Routine methods of cell culture and assays for parasite life cycle development have been achieved in different cell lines; however, no continuous culture and efficient complete life cycle development were established until the success of Hijjawi and colleagues (Hijjawi et al., 2001; Hijjawi et al., 2002), who have reported the complete development of all life cycle stages of C. parvum, C. hominis and C. andersoni in cell culture using the HCT-8 cell line. The in vitro cultivation protocols were based on those of Meloni and Thompson (1996), but with important modifications to pH and the monolayer. The pH appeared to play an important role in successfully sustaining growth and development in vitro and successful


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results were dependent on maintaining pH within an optimum range (7.2–7.6). This was achieved by changing the media regularly, every 2–3 days, and adding HEPES buffer at 15 mM. A stable monolayer also appeared to be important and its overgrowth adversely affected parasite development. The problem of monolayer overgrowth can be avoided by subculturing or irradiation of the monolayer. However, the need for host cells has now been shown to be unnecessary for the maintenance of Cryptosporidium in culture. Hijjawi et al. (2004) described the complete in vitro development of C. parvum (cattle genotype) in RPMI-1640 maintenance medium devoid of host cells, the first time Cryptosporidium has been shown to multiply, develop and complete its life cycle without the need for host cells. However, cultivation of Cryptosporidium in diphasic medium consisting of a coagulated new born calf serum base overlaid with maintenance medium greatly increased the total number of Cryptosporidium stages produced in this cell-free system. Both the cell culture and cell-free systems developed by Hijjawi et al. (2001, 2002, 2004) offer the ability to maintain the life cycle in the laboratory for unlimited periods of time but more research is required to optimise conditions in order to increase the numbers of parasite stages produced.

4. HOST–PARASITE RELATIONSHIP 4.1. Pathogenesis The pathogenesis of Cryptosporidium-associated diarrhoea, weight loss and mortality are not well understood but recent research in animal models have provided insight into the pathophysiology of the disease and understanding of the clinical signs. The complicated life cycle, the variety of parasitic forms within the host, the different Cryptosporidium species and the predilection to different tissues in different host species also further complicates the understanding of the host–parasite interactions of Cryptosporidium. Table 3 summarizes the tissues associated with Cryptosporidium infection, which vary among parasite species and animal host.

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Table 3


Tissue specificity of different Cryptosporidium species

Parasite species

Host(s)

Primary tissue

Secondary tissues

C. hominis

Human

Small intestine, large intestine

C. parvum

Small intestine

C. andersoni C. muris C. meleagridis

Human, cattle, sheep, goat, pig, mouse Cattle Mouse Chicken, turkey

Stomach, bile duct and gall bladder, respiratory tract Stomach, bile duct and gall bladder, respiratory tract

C. baileyi

Chicken

C. serpentis C. nasorum C. molnari

Reptiles Fish

Abomasum Stomach Intestine, cloaca, bursa of Fabricius Trachea, bronchi, lung, air sacs Stomach Stomach, intestine

Stomach Intestine, cloaca, bursa of Fabricius

Cryptosporidium parvum causes an acute self-limiting diarrhoea in humans and other mammals. From experimental infection studies, the onset of clinical signs occurs within 2–7 days of challenge and a prepatent period of 7–21 days (Ramirez et al., 2004). Intestinal enterocytes are infected by sporozoites emerging from ingested thickwalled oocysts or from thin-walled oocysts emerging from infected cells where sexual and asexual reproduction has occurred. Other extracellular forms that develop within the intestinal lumen also may infect small intestinal epithelial cells (Hijjawi et al., 2004). Apical complex proteins (e.g. circumsporozoite-like antigen (CSL), Glycoprotein 900 (GP 900), Glycoprotein 40 (GP40, thrombospondinrelated adhesive protein (TRAP C1)), which are numerous have been recognised to facilitate the attachment process of the sporozoite through ligand–host receptor interactions eventually leading to invasion and parasitophorous vacuole formation on the epithelial surface (Ward and Cevallos, 1998; Tzipori and Ward, 2002; Sibley, 2004). Parasite migration across the apical surface of host enterocytes is facilitated by actin polymerisation-dependent motility. The parasite then attaches to host actin filaments and actin-binding proteins where the cytoskeleton is reorganised with the aid of actin polymerisation factors (Sibley, 2004; and see above). After the initial infection, the


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endogenous forms are amplified within the gut leading to dissemination of infection throughout the small and large intestine. Occasionally, the gastric mucosa, bile duct, gall bladder and respiratory epithelium are also infected. Certain species of Cryptosporidium have a predilection for the gastric mucosa of mice (C. muris) or abomasum of ruminants (C. andersoni) and C. baileyi can infect the respiratory tract of chickens, turkeys and ducks. The distribution of infection may determine the intensity of clinical signs as infection of the proximal small intestine frequently leads to severe watery diarrhoea, while infection localised to the distal ileum and large intestine are frequently asymptomatic. Infections of the pyloric region of the stomach or abomasum are usually asymptomatic. The invasion and colonisation of the epithelial surface displaces the microvillus brush border and loss of surface epithelial cells. Villus shortening and fusion as well as crypt hyperplasia also occur as a result of epithelial damage. Colonization of the epithelial surface leads to decreased intestinal surface area, loss of membrane-bound digestive enzymes and impaired nutrient and electrolyte transport (Argenzio et al., 1990; Chen et al., 2002; Buret et al., 2003). A putative enterotoxin has been proposed to lead to chloride secretion resulting in a secretory diarrhoea (Guarino et al., 1995). The proinflammatory mediators interleukin 8, transforming-growth-factor b and chemokines are produced through activation of the N-kB/IkB system through parasite-induced host cell apoptosis (Chen et al., 2002; Buret et al., 2003). Cryptosporidium has recently been shown to disrupt epithelial tight junctions that can lead to increased epithelial permeability (Buret et al., 2003). Disruption of intestinal permeability is a contributing factor in enteric disease caused by Escherichia coli, Clostridium difficile and Giardia duodenalis (Chen et al., 2002; Buret et al., 2003). Cryptosporidiosis is therefore associated with malabsorptive and secretory diarrhoea.

4.2. Immunobiology Cryptosporidiosis is a significant disease in both the immunocompetent and immunocompromised host. Immunocompetent humans and

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animals develop self-limiting infections localised to specific tissues (usually the intestinal tract), while hosts which are immunocompromised by infection (e.g. AIDS patients), chemotherapeutic agents or nutrition develop more severe clinical signs, with parasite dissemination to other tissues and have recurrent and chronic infections. Activation of parasite-specific immune responses provides the only known mechanism for parasite elimination as current chemotherapeutic agents are ineffective or of low efficacy. It is recognised that both the humoral and cell-mediated immune response are involved in parasite elimination and solid immunity is produced as infected hosts with intact immune systems are relatively resistant to reinfection and clinical disease. Immunoprophylaxis is a preferred approach to disease control in livestock species and in certain human populations, but commercial vaccines are not yet available (de Graaf et al., 1999a). Cell-mediated immunity is believed to be the primary method for host elimination of Cryptosporidium spp. infections (Theodos, 1998; Riggs, 2002). This has been established by using animal models with depleted components of their immune system (SCID mice, nude mice, MHC classI/CD8+ T-cell-deficient mice, MHC classI/CD4+ T-celldeficient mice) as well as monitoring the immune response in hosts which have (humans, foals, cats, poultry) deficiencies in cell-mediated immunity due to genetic defects or infection (Riggs, 2002). CD4+ T lymphocytes, and IFN-g are critical in parasite elimination. Humans and animals with depressed CD4+ T cell counts such as patients with AIDS are unable to properly eliminate Cryptosporidium infections, but restoration of CD4+ T cells through provision of antiretroviral therapy or adoptive transfer to the host permits elimination of the parasite (Farthing, 2000; Okhuysen et al., 1998). Peripheral lymphocytes are less important than the intraepithelial lymphocytes (IEL) that are critical for parasite elimination. In the immunocompetent host CD4+ IEL increase early in an infection and CD8+ IEL increase over the recovery phase (Riggs, 2002). It is believed that CD4+ IEL are most important in controlling the parasite early in the infection while CD8+ IEL are important in later phases (Chai et al., 1999). The CD4+ IEL are closely associated with


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infected enterocytes and produce IFN-g: IFN-g acts by inducing host enterocyte resistance to parasite invasion, modification of intracellular Fe2+concentrations and decreased intracellular development (Theodos, 1998; Riggs, 2002). The humoral immune response is considered less important in cryptosporidiosis but there is an elevation in serum and mucosal antibodies associated with parasite elimination and resolution of clinical signs. Antibody responses may be redundant but may also play a role in resistance to infection. Parasite-specific antibody responses have been demonstrated in animals and humans without parasite elimination (Dann et al., 2000) however, in other studies resistance to infection has been associated with local IgA and IgM responses (Okhuysen et al., 1998; Riggs, 2002). A number of immunogenic antigens of Cryptosporidium (Table 4) have been identified and those associated with parasite motility, attachment, invasion and development have been targeted as subunit vaccine

Table 4 Proteins that may be involved in the pathogenesis of cryptosporidiosis and are potential antigens that could be used in vaccines Antigen

Type

Localisation

1300

Glycoprotein

900 47

Glycoprotein Glycoprotein

GP40

40

Glycoprotein

GP15

15

Glycoprotein

Dense granules and micronemes, sporozoite and merozoite stages Micronemes, surface of zoites Apical pedical and subpellicular microtubules Expressed on the surface of zoites Expressed on the surface of zoites Apical pole of sporozoites Oocyst surface Zoite apical and surface antigen complex On zoites surface pellicle and cytoplasmic tubulo-vesicular network

CSL

GP 900 CP47

TRAP-C1 P23 GP 25–200 CPS-500

Molecular weight (kDa)

76 23 25–200 kDa complex

Adhesive protein? Glycoprotein Glycoprotein

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candidates in published and ongoing investigations (Sibley, 2004; Riggs, 2002). Serum and colostrum-derived hyperimmune polyclonal antibodies have been shown to reduce oocyst shedding and clinical signs in experimental models and natural infections. In calves and lambs, ingested colostrum antibodies are absorbed into the circulation through the gut mucosa during the first 24 h of life and later secreted into the intestinal lumen thereby providing passive immunization for 1–2 months. Protection was afforded to neonatal calves provided with polyclonal IgG antibodies to whole oocysts and P23, CP5/60 and CP15 antigens (Riggs, 2002). Similarly, passive immunization using hyperimmune serum has been shown to provide clinical responses in chronically infected humans and animal models (Riggs et al., 2002; Hunt et al., 2002; Arrowood et al., 1989; Doyle et al., 1993). The use of monoclonal antibodies has also been used but with limited success demonstrating that polyvalent neutralising antibodies provide superior protection. In summary, although mechanistic studies are limited it is believed that specific anti-Cryptosporidium antibodies bind one or more of the extracellular life stages and prevent attachment and invasion of the enterocyte. Antibody-coated stages that have invaded enterocytes undergo arrest of intracellular development.

4.3. Infections in Humans In developing countries, cryptosporidiosis is a common cause of diarrhoea in humans, but it is less frequently observed in countries where hygiene, water quality and nutrition are adequate. Severity and duration of clinical signs vary with age and immune status of the host (Ramirez et al., 2004; Chen et al., 2002; Farthing, 2000). It is believed that naive adults and children are equally susceptible to infection, but children are more likely to develop moderate-to-severe clinical signs. The relative immaturity of the immune system, history of previous exposure as well as the likelihood of having a severe challenge due to poor hygiene is believed to account for the difference in clinical signs


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between children and adults. Attention to cryptosporidiosis began with the AIDS epidemic, which began in 1982 as it has been a cause of chronic and fatal diarrhoea in immunosuppressed populations. Patients with CD4+ T cell counts below 150 cells/ml can develop severe life-threatening diarrhoea (Tzipori and Ward, 2002; Chen et al., 2002; Farthing, 2000). There appears to be variation in virulence among different strains of C. parvum. This was clearly demonstrated in experimentally infected volunteers with respect to attack rate and duration of diarrhoea (Tzipori and Ward, 2002). Some strains are more infective to humans than other strains, while different strains produce more severe and prolonged clinical signs suggesting that Cryptosporidium has multiple virulence factors. The time from ingestion of oocysts to the appearance of clinical signs is between 2 and 14 days. The first clinical sign associated with infection is diarrhoea, which may or may not be associated with abdominal cramping. Severity varies from continuous voluminous watery diarrhoea to scant or intermittent diarrhoea. Blood is infrequently observed in the stool. Other clinical signs include general malaise, fever, and fatigue, loss of appetite, nausea and vomiting. Dehydration and weight loss are a direct result from the symptoms. In developing countries, there is an association of cryptosporidiosis with children with stunted growth and who are underweight (Ramirez et al., 2004). Infrequently, symptoms associated with cholecystitis, hepatitis, pancreatitis, reactive arthritis (wrists, hands, knees and ankles) and respiratory problems are observed. In the immunocompetent host clinical signs last for 10–14 days, but in the immunocompromised individual they can persist for months to years.

4.4. Infections in Cattle The most common enteropathogen in calves during the first week of life is considered to be C. parvum although it may be associated with other viral, bacterial and parasitic pathogens (O’Handley et al., 1999; Fayer et al., 1998; de Graaf et al., 1999b). Two species of

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Cryptosporidium have been identified in cattle, C. parvum and C. andersoni. An additional genotype of C. parvum has also been recently reported in adult cattle (Santin et al., 2004). C. parvum primarily infects the distal small intestine of young calves, while C. andersoni colonises the abomasum of weaned calves and adult cattle. Calves can begin shedding C. parvum oocysts as early as 2 days of age but the peak shedding occurs at approximately 14 days of age (Fayer et al., 1998; O’Handley et al., 1999; Becher et al., 2004). Typically, shedding is observed between 1 and 4 weeks of age with infection lasting for about 2 weeks. The disease is manifested by diarrhoea (varies from pale yellow with mucus to profuse watery diarrhoea), depression, anorexia and abdominal pain. Clinical signs can persist for 4–14 days and the severity and duration is highly variable. The pathogenesis of disease is frequently complicated by concurrent viral (rotavirus, cornavirus), bacterial (E. coli, Salmonella) and parasitic (Giardia) infections (Fayer et al., 1998; O’Handley et al., 1999; de Graaf et al., 1999b). Calves can die from dehydration and cardiovascular collapse but cryptosporidiosis mortalities are highly variable. In endemic herds, morbidity rates are usually 100%, but mortalities are infrequently observed. Calves from naive herds are susceptible to high mortalities (up to 30%) and certain beef breeds (Belgian blue, Limousin and Charolais) appear more vulnerable to mortalities (de Graaf et al., 1999b). Colostrum antibodies and milk antibodies protect calves from developing severe clinical signs by blocking parasite invasion and immobilisation of gut luminal parasitic forms. Micronutrients may also play a significant role in the clinical outcome of cryptosporidiosis as severe clinical signs were observed in calves with selenium deficiency (Olson et al., 2004a; McAllister et al., 2005). This has been confirmed in experimental animal models (Huang and Yang, 2002). Calves that recover from C. parvum do not have recurrent cryptosporidiosis-associated diarrhoea and do not appear to shed oocysts for the rest of their life. Cryptosporidiosis in young calves is a costly disease to farmers because of its high prevalence and losses associated with mortalities, body weight loss, impaired body weight gain and costs of treatment.


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An economic analysis of cryptosporidiosis in calves is necessary to clearly demonstrate the high costs of this parasitic infection in beef and dairy cattle production. Adult cattle shedding C. parvum-like oocysts appear to have a unique genotype of C. parvum that is not associated with clinical signs (Santin et al., 2004). The biology and clinical significance of this novel C. parvum in adult cattle is currently being investigated. C. andersoni has been reported worldwide in post-weaned beef and dairy cattle (Olson et al., 2004a; Enemark et al., 2002). The infection persists for months to years. C. andersoni invades the peptic and pyloric glands of the ruminant abomasum causing glandular dilation and hypertrophy of the gastric mucosa and thinning of the epithelial lining (Anderson, 1987; Anderson, 1998; Kvac and Vitovec, 2003). Diarrhoea is not observed in cattle with C. andersoni infections, but infection is associated with maldigestion. C. andersoni can cause moderate-to-severe impairment of weight gain, decreased feed efficiency and reduced milk production (3.2 kg/day) (Anderson, 1987; 1998; Esteban and Anderson, 1995; Ralston et al., 2003). The mechanism of impaired performance is through inhibition of protein digestion by decreased gastric proteolytic function and increased gastric pH. Feedlot cattle and milking dairy cattle are provided high protein rations to optimize meat and milk production and presumably C. andersoni infections inhibit the digestion of these high protein rations. A report did not show any difference between rumen microflora, volatile fatty acid production in normal and infected animals (Holko et al., 2004), which suggests that the pathogenesis of production losses may be restricted to impairment of protein digestion. C. andersoni may have a significant economic impact in beef and dairy operations; however, analysis of the cost of this disease has not been undertaken.

4.5. Infections in Sheep and Goats Cryptosporidium parvum and a novel C. parvum genotype are responsible for infections in sheep and goats (Ramirez et al., 2004;

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de Graaf et al., 1999b; Olson et al., 2004b; Chalmers et al., 2002). C. andersoni does not appear to infect sheep and goats and is unique to the abomasum of cattle. The prepatent period is approximately 4 days in sheep and goats and clinical signs are more severe in young lambs and kids. Typically, animals between 1 and 5 weeks of age are infected with C. parvum and animals demonstrate clinical signs for 5 days to 2 weeks. The prominent symptom is mild-to-severe diarrhoea, but other clinical signs include depression, anorexia and abdominal pain. The diarrhoea is typically yellow, soft to liquid and has a strong odour. Large numbers of oocysts (108–1010 oocysts/g) are present in the diarrhoeatic stool. As in cattle, clinical signs and clinical outcome may be influenced with concurrent viral (rotavirus), bacterial (E. coli, Clostridium perfringes, Salmonella) and parasitic (Giardia, Eimeria) gastrointestinal infections. Mortality is more frequently observed in animals with concurrent infections and in outbreaks in naı¨ ve flocks. There appear to be differences in clinical signs and mortalities among isolates of ruminant C. parvum, but virulence factors have not been identified.

4.6. Infections in Pigs Pigs are naturally infected with C. suis and C. parvum, but can be experimentally infected with C. hominis (Ortega-Mora and Wright, 1994; Pereira et al., 2002; Ryan et al., 2004). Animals experimentally infected with C. hominis are typically asymptomatic and the parasites are displaced when subsequently challenged with C. parvum (Akiyoshi et al., 2003). Prevalence and transmission studies suggest that C. hominis and possibly C. parvum are poorly host adapted to pigs. Nursing piglets are infrequently infected with Cryptosporidium spp. and infection usually appears shortly after weaning (Tacal et al., 1987; Giselle et al., 2003). This suggests that maternal milk antibodies may protect animals from developing infections. Pigs do not shed high numbers of oocysts, but infections can be prolonged, lasting for 3–5 weeks. Infections are typically asymptomatic, however, performance data on the effects of cryptosporidiosis is limited to small studies


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where animals have other enteric pathogens (Tacal et al., 1987; Giselle et al., 2003). Larger studies on SPF pigs are necessary to determine the impact of this parasite on performance.

4.7. Infections in Poultry Poultry can be infected with C. meleagridis and C. baileyi. C. meleagridis colonises the intestinal tract, bursa of Fabricius and the cloaca of chickens and turkeys (de Graaf et al., 1999b; Sreter and Varga, 2000; Ramirez et al., 2004). C. meleagridis infections are asymptomatic in chickens, but in turkeys leads to diarrhoea with occasional mortalities. The pathophysiology of disease is similar to that in mammalian species. This species has been reported to cause clinical disease in humans, which may suggest that it has novel virulence factors and the ability to readily cross species barriers. This has been recently confirmed in transmission studies where a human C. meleagridis isolate successfully infected chickens, mice, piglets and calves (Akiyoshi et al., 2003). Cryptosporidium baileyi primarily infects the respiratory tract of chickens turkeys and ducks, but it can also colonise the intestine (bursa of Fabricius and cloaca) and kidney. Chickens become infected through inhalation or ingestion of oocysts within the environment (Current et al., 1986; Lindsay et al., 1988; de Graaf et al., 1999b; Ramirez et al., 2004). As in mammalian species there is an age-related susceptibility to clinical disease with young chicks (7–14 day old) showing more severe symptoms. Chickens greater than 11 weeks of age appear to be resistant to infection. Infected poultry cough, sneeze and are dyspneic associated with the accumulation of mucoid exudates and inflammation within the respiratory tract. Deciliation of respiratory epithelium, epithelial hypertrophy and hyperplasia are pathological features of birds with respiratory cryptosporidiosis. Mortalities are associated with respiratory cryptosporidiosis but are usually low. Birds with respiratory cryptosporidiosis become depressed, anorexic and emaciated and producer losses are associated with impaired growth rate and carcass condemnation.

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4.8. Infection in Horses Horses with cryptosporidiosis are typically asymptomatic and are observed in foals 5–8 weeks of age. Animals, immunocompromised by stress or genetic disorders may develop severe diarrhoea which can be fatal (Xiao and Herd, 1994). Severe diarrhoea in foals has been associated with C. parvum (Grinberg et al., 2003) and a host-specific strain has not yet been characterised.

4.9. Infection in Dogs and Cats Dogs infected with C. canis are generally asymptomatic (Irwin, 2002). Animals less than 6 months of age are more frequently infected, but adult dogs are frequently infected. Infected dogs can shed oocysts for weeks to months. C. felis infections in cats is more frequently observed in kittens and is frequently associated with diarrhoea (Hill et al., 2000; Irwin, 2002). Cryptosporidiosis in cats is characterised by oocyst shedding that can persist for months with recurrent clinical signs (Olson et al., 2004b). There does not appear to be an association between feline leukaemia virus and C. felis infections, but concurrent gastrointestinal infections may play a role in symptoms in cats (Olson et al., 2004b). Kittens which are stressed by weaning, nutritional deficiency and by introduction to a new environment are more frequently infected with C. felis.

4.10. Infections in Reptiles A number of reptilian species can be infected with C. serpentis including snakes, lizards and tortoises. Reptiles frequently develop chronic infections where shedding is observed for several years (Brownstein et al., 1977; Upton et al., 1989). Reptiles with cryptosporidiosis typically develop gastric hypertrophy resulting in regurgitation 2–3 days after a meal. Reptiles with chronic cryptosporidiosis have moderate-to-severe weight loss. Infections are frequently


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observed in animals, which have been recently captured or imported suggesting that stress has an important role in infection susceptibility.

4.11. Infections in Fish Cryptosporidium nasorum and C. molnari has been reported in the stomach, intestinal tract and faeces of wild caught, captive and ornamental fish (Hoover et al., 1981; Landsberg and Paperna, 1986; Muench and White, 1997; Alvarez-Pellitero and Sitja-Bobadilla, 2002). It is probable that fish are infected with more than one species of Cryptosporidium spp. and cross-species transmission may or may not occur. Clinical signs associated with infections have not been reported, but performance effects might be expected. The pathophysiology and clinical impact of cryptosporidiosis in fish needs further investigation as the parasite infects wild and farmed species.

5. THE REGULATION OF BIOCHEMICAL PROCESSES Although detailed ultrastructural descriptions of the different asexual and sexual developmental stages have been reported (Fayer et al., 1997), protocols for efficient purification of the various asexual, sexual and intracellular developmental stages of the parasite are lacking. As a consequence, experimental approaches for characterisation of C. parvum biochemical pathways and direct knowledge of C. parvum biology has been limited. Recently, the complete genome sequence of C. parvum has been elucidated (Abrahamsen et al., 2004), confirming many of the early speculations on this apicomplexan, but also revealing many unexpected and unique biochemical activities of this important pathogen. In this section, we will highlight the major pathways involved in energy metabolism and discuss some of the novel aspects of C. parvum biology revealed by the genome project that provide a better understanding of the nature of extreme parasitism and have important implications for future efforts towards development of effective therapies.

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5.1. Cryptosporidium parvum Genome To provide for a better understanding of the complicated biology and metabolic activities of this pathogen, the complete genome sequence of C. parvum has been obtained and analysed (Abrahamsen et al., 2004). The 9.1 Mb (megabase) genome is distributed on eight chromosomes and contains an estimated 3807 genes. In silico reconstitution of C. parvum biochemical properties reveals that basic metabolic pathways are extremely streamlined and indicate adaptation to an anaerobic and high nutrient environment of the intestinal epithelium (Figure 11). The parasite lacks a tricarboxylic acid cycle (TCA) cycle and much of the oxidative phosphorylation pathway, but retains evidence of a remnant mitochondrion. A complete glycolytic pathway is present (Figure 12); however, atypical enzymes are used to maximise ATP conservation. Adaptation to extreme parasitism is reflected in its inability to synthesise amino acids, and the presence of an expanded array of amino acid and sugar transporters presumably for mediating the uptake of these key metabolites from the host cell. Structurally, the genome is quite compact relative to the 23-Mb, 14-chromosome genome of the related apicomplexan Plasmodium falciparum (Gardner et al., 2002), and in contrast to other apicomplexans including Plasmodium, Toxoplasma and Eimeria, C. parvum lacks both apicoplast and mitochondrial genomes and most of the nuclear genes that normally function in these compartments (Zhu et al., 2000b; Abrahamsen et al., 2004). The compactness of the C. parvum genome is evidenced by significantly fewer (3807 vs. 5268) and shorter (1795 vs. 2283 bp, excluding introns) genes than P. falciparum. In addition, the distance between putative C. parvum coding sequences (566 vs. 1694 bp) and the number of C. parvum genes estimated to contain introns (190 vs. 2840) is significantly smaller than P. falciparum. Further, in contrast to P. falciparum, the C. parvum genome is essentially devoid of repetitive DNA. The majority of the 1400 ‘‘missing genes’’ of C. parvum relative to P. falciparum can be accounted for by the lack of mitochondrial targeted/encoded (246) and apicoplast targeted/encoded (581) genes, and the absence of the large families of extracellular proteins (236)


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Figure 11 Overview of metabolic and transport activities highlighting the extreme parasitic life style of C. parvum. The parasite lacks an apicoplast organelle, but retains a remnant mitochondrion in which Krebs cycle and electron transport components are lacking. Amino acid and nucleotide metabolisms include salvage and interconversion pathways, but no capacity for de novo synthesis. Uptake of sugars, amino acids and nucleotides are facilitated by gene amplifications of specific transporters, but the uptake mechanism of fatty acids is yet undetermined. Also indicated are a capacity for polysaccharide synthesis, amylopectin and trehalose.

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Figure 12 Cryptosporidium parvum carbohydrate metabolism and its major connections to other pathways. End products are boxed. Arrows with dashed lines indicated connections to other major pathways.


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analogous to vars, rifins and stevor that are found in P. falciparum (Gardner et al., 2002). In addition, only 167 of the annotated C. parvum genes were able to be assigned Enzyme Commission (EC) numbers, with many biochemical pathways being completely absent, indicating that specific genes were not missed due to sequence gaps or divergence from other organisms. Furthermore, C. parvum contains a significantly smaller number (925 vs. 3208) and lower percentage (24.3 vs. 60.9%) of genes as compared with P. falciparum that annotate only as hypothetical proteins, likely a reflection of a simpler life cycle, lacking an insect vector stage and multiple tissue stages, and streamlined biosynthetic capabilities. Rather unexpectedly, accompanying the massive gene loss and the absence of two organellar genomes is the gain of a significant number of genes by duplication events and the apparent acquisition of genes from bacteria (Deng et al., 2002; Striepen et al., 2002; Huang et al., 2004b; Madern et al., 2004; Templeton et al., 2004). These events have significantly altered the genetic repertoire of C. parvum, resulting in specific enzymatic activities and metabolic processes that are distinct from those found in mammals, providing unique opportunities for chemotherapy.

5.2. Core Energy Metabolism The completion of the C. parvum genome has revealed a mosaic collection of animal, plant and bacterial-like genes that coexist, even within a single biochemical pathway, as a result of the evolutionary forces that have selected for an obligate intracellular pathogen with minimal biosynthetic capabilities. In general, C. parvum has lost the ability to synthesise most basic metabolites, relying instead on an extensive collection of transporters and salvage pathways to meet its basic metabolic requirements. It appears that this adaptation to extreme parasitism has been facilitated by the acquisition of novel bacterial-like genes that have replaced traditional eukaryotic versions of basic metabolic enzymes.

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Analysis of the genome sequence confirms that C. parvum relies solely on cytosolic glycolysis to fulfill its energy needs. Such a notion had been previously hypothesised based on biochemical assays using oocysts and in vitro inhibitory analyses (Denton et al., 1996; Entrala and Mascaro, 1997). C. parvum differs from other apicomplexans by lacking a Krebs cycle and respiratory chain, indicating that its carbon sources are not fully oxidated for generating energy. In contrast, all major components for the glycolysis of glucose to pyruvate are present in the genome (Figure 12). C. parvum can utilise amylopectin to produce glucose-1P via glycogen phosphorylase. Glucose and fructose may be scavenged from host cells (or the intestinal lumen), or produced from maltose or sucrose. Although mannose is also a potential source for generating energy, its primary use is likely for synthesising N-glycans, an important component of glycolipids in C. parvum surface antigens (Priest et al., 2001). This parasite is capable of synthesising amylopectin and trehalose. The former is common in apicomplexans as energy storage, whereas the latter is unique to C. parvum, and may play a similar role as mannitol in Eimeria oocysts (Schmatz, 1997). C. parvum lacks enzymes for the oxidation of fatty acids, suggesting that this parasite cannot metabolise fatty acids to produce energy, and glycolysis is likely the only source of energy for this parasite. There are two pyrophosphate-dependent phosphofructokinase (PPi–PFK) homologues whose activity was previously reported in C. parvum (Denton et al., 1996). PPi–PFK differs from the ATPdependent PFK in humans and animals and is an economically beneficial enzyme to an organism relying on glycolysis to produce limited numbers of ATP molecules. The conversion from phosphoenolpyruvate (PEP) into pyruvate can be catalysed by pyruvate kinase (PK) to yield an ATP or mediated by PEP carboxylase (PEPCL), malate dehydrogenase (MDH) and malic enzyme (ME) in an apparent bypass pathway to recycle NADH to NAD+. Pyruvate can be converted by an unusual bifunctional pyruvate-NADP+ oxidoreductase (CpPNO) into acetyl-CoA (Rotte et al., 2001), which can be subsequently converted by cytosolic acetyl CoA carboxylase (ACCase) into malonylCoA, an essential building block in fatty acid and polyketide syntheses.


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There are three potential end products to keep the major carbon flow through the glycolytic pathway. Acetate can be produced from acetyl-CoA by an AMP-dependent acetyl-CoA synthase that is coupled with ATP production. Lactate is produced by lactate dehydrogenase (CpLDH1) that also recycles NADH to NAD+. The third end product is ethanol, which can be produced from pyruvate by pyruvate decarboxylase (PDC) and a monofunctional alcohol dehydrogenase (adh2), or from acetyl-CoA by a bifunctional alcohol dehydrogenase (adhE) that is found in several other anaerobic protists (e.g. Entamoeba and Giardia) (Bruchhaus and Tannich, 1994; Dan and Wang, 2000) but not in other apicomplexans. In contrast to a previous report of weak glycerol kinase (GK) activity (Entrala and Mascaro, 1997), no GK gene is found in the C. parvum genome, suggesting that this parasite does not produce glycerol. Probably a reflection of the evolutionary selection pressure for adaptation to this extremely parasitic life style, several of the enzymes within the C. parvum glycolytic pathway are distinct from those found in other apicomplexans or mammals. The bifunctional CpPNO is unique and only reported from another distant protist Euglena gracilis (Rotte et al., 2001). Phylogenetic analysis has indicated that CpLDH1 and CpMDH1 genes likely evolved from the same ancestor by a recent event of gene duplication after Cryptosporidium diverged from other apicomplexans (Madern et al., 2004). Furthermore, CpLDH1 and CpMDH1, together with other apicomplexan LDH and MDH enzymes are phylogenetically divergent from other eukaryotic LDH/MDH homologues, but related to MDHs from a-proteobacteria, suggesting that they might be acquired from an a-proteobacterial ancestor (Zhu and Keithly, 2002). Despite the absence of Krebs cycle and cytochrome respiratory pathway, C. parvum possesses the two headpieces (i.e. a- and b-subunits) of a bacterial-type ATP synthase. However, no putative homologues for the stalk components and other subunits appear to be present, and the genome provides no other clues for speculating the function of these headpieces. In addition, an alternative oxidase (AOX) is present in the C. parvum genome (Roberts et al., 2004; Suzuki et al., 2004). AOX is a cyanide-resistant mitochondrial protein

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that has been found in a number of organisms including fungi, plants and trypanosomes. Like several other mitochondrial proteins including CpHSP70 and CpHSP60 (Riordan et al., 2003; Slapeta and Keithly, 2004), CpAOX contains a putative mitochondrial targeting sequence, although its exact intracellular location is yet undetermined. AOX belongs to the di-iron carboxylate protein family and catalyses the reduction of oxygen by ubiquinol to produce water (Nihei et al., 2002; Rosenfeld and Beauvoit, 2003; Kuntz, 2004). The process may be coupled with an inner-membrane NADH dehydrogenase to reduce ubiquinone back to ubiquinol. In plants, yeast and trypanosomes that possess a complete respiratory chain, AOX functions as an alternative to the cytochrome chain, in which the electron flux is not coupled with the ATP production. The C. parvum AOX could also act as a detoxifying enzyme as an adaptation to the micro-anaerobic environment, perhaps to reduce O2 that may harm mitochondrial (Fe–S) cluster assembly, or as a redox sink as suggested by the studies performed on other organisms.

5.3. Fatty Acid and Polyketide Synthesis The fatty acid biosynthesis in C. parvum differs from that of other apicomplexans (Zhu, 2004). First, as expected due to the absence of an apicoplast (Zhu et al., 2000b), C. parvum lacks discrete type II fatty acid synthases (FAS) that are involved in the de novo fatty acid synthesis in the apicoplast of P. falciparum and T. gondii. Secondly, fatty acid synthesis is mediated by a large, modular type I FAS (CpFAS1) encoded by a 25 kb ORF (Zhu et al., 2000c). Recently, the CpPFAS1 loading unit, three internal fatty acid elongation modules, and the terminating reductase, have been expressed in bacteria and the activities of enzymatic domains were characterised (Zhu et al., 2004). The most preferred substrate for the CpFAS1 acyl ligase domain within the loading unit was determined to be a C16:0 palmitic acid, suggesting CpFAS1 may function as long-chain fatty acid ‘elongase’. Together with the lack of a type II FAS, these data indicate that C. parvum is probably unable to synthesize fatty acids de


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novo. More recently, type I modular FAS genes have been identified from T. gondii and E. tenella genomes (Crawford, Zhu, & Roos 2003). However, these megasynthases differ from CpFAS1 by possessing different numbers of elongation modules. In contrast to P. falciparum and C. parvum that possess only type II and type I FAS, respectively (Keeling, 2004), it is likely that T. gondii and E. tenella utilise type I FAS for the elongation of fatty acids synthesised de novo by type II FAS. In addition to CpFAS1, the C. parvum genome also contains a 40-kb intronless ORF that encodes a 1500 kDa, putative polyketide synthase (CpPKS1) that consists of 29 separate enzymatic domains (Zhu et al., 2002). Similar to CpFAS1, the CpPKS1 domains are organised into an N-terminal loading unit, 7 polyketide chain elongation modules, and a carboxy terminator unit. The biosynthesis of polyketides resembles that of fatty acids except that incomplete reduction and/or dehydration frequently occur during the acyl chain elongations. It is noticeable that C. parvum has devoted 0.7% of its small genome (9.1 Mb) to CpFAS1 (25 kb) and CpPKS1 (40 kb), suggesting these two genes play an essential role in this parasite. Both CpFAS1 and CpPKS1 possess an evolutionarily related C-terminal reductase to release their final products, which differs from that of human FAS that uses a thioesterase (TE) domain (Zhu et al., 2000c; Zhu et al., 2002). Data mining of the complete C. parvum genome also failed to detect any TE homologues, suggesting this parasite also lacks a discrete TE enzyme. The released fatty acyl or polyketide products by a reductase could be either an aldehyde (one-step reduction) or alcohol (two-step reduction) (Gaitatzis et al., 2001). If aldehydes are produced, they are usually unstable and toxic to cells, and C. parvum must immediately convert these aldehydes produced by CpPKS1 or CpFAS1 into acids or alcohols. The biological functions of CpFAS1 and CpPKS1 remain to be fully determined. While very long-chain fatty acid(s) or fatty alcohol(s) produced by CpFAS1 may be utilised in the remodelling and/or regeneration of biomembranes, the function of a polyketide by C. parvum is yet unknown. Polyketides are a diverse group of secondary metabolites, and their functions vary from defensive

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molecules in microbes to playing a role in cell-to-cell and organismto-organism signalling pathways (Rawlings, 1997; Staunton and Weissman, 2001). Although it is as yet unclear why C. parvum possesses a polyketide synthetic pathway, it is interesting to speculate that these products may significantly improve the ability of C. parvum to colonise the small intestine and/or to successfully compete with other microbes in this highly desirable niche. Another interesting possibility is that the CpPKS1-produced polyketide is a toxin-like molecule that plays a critical role in the C. parvum-unique severe watery diarrhoea, for which the causative mechanism is not fully understood (Leav et al., 2003; Robinson et al., 2s03). Although an early study reported an enterotoxic-like activity associated with C. parvum infection (Guarino et al., 1995), this finding is still controversial and the identity of the enterotoxin is still elusive. However, polyketide toxins are commonly produced in dinoflagellates, a group of protists closely related to apicomplexans (Rein and Borrone, 1999). Since the watery diarrhoea is unique to C. parvum, and a PKS is only found in this parasite, rather than any other well-studied apicomplexans, it will be interesting to test whether a polyketide toxin is responsible for the severe diarrhoea associated with C. parvum infection in humans and animals.

5.4. Nucleic Acid Metabolism The genes employed for DNA and RNA replication in C. parvum are typical of most eukaryotes (Abrahamsen et al., 2004; Zhu and Abrahamsen, 2004). However, similar to other essential biochemical processes, C. parvum differs from other apicomplexan in regards to how nucleic acid precursors are obtained and modified. As expected, the complete genome sequence confirms that C. parvum has no ability for the de novo synthesis of purines or pyrimidines, and is dependent on salvaging the required nucleic acid precursors from the host cell. All of the enzymes necessary to produce ATP, GTP, dATP and dGTP from adenosine are present in the genome: a 50 nucleotidase, an adenosine transporter, adenosine kinase, AMP deaminase, inosine


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monophosphate dehydrogenase (IMPDH) and GMP synthase. However, in contrast to other apicomplexans, C. parvum is lacking a phosphoribosyl transferase, indicating that other purine nucleobases cannot be salvaged from the host cell and that there is no redundancy in this pathway. Also lacking are the enzymes that catabolise purine nucleobases. Therefore, C. parvum is absolutely dependent on the adenosine salvage pathway as a source of purines. Within this pathway, previous phylogenetic studies have demonstrated that CpIMPDH is not of eukaryotic nuclear descent or even mitochondrial origin (since it is not specifically related to the a-proteobaterial homologues), but instead was probably obtained by lateral gene transfer from a e-proteobacterium (Striepen et al., 2002). This is not the case for P. falciparum or T. gondii IMPDH that are consistently grouped with eukaryotic versions of the enzyme. In contrast to all other apicomplexans examined to date, C. parvum lacks the entire set of genes encoding enzymes necessary for de novo pyrimidine synthesis. However, all of the enzymes necessary to salvage pyrimidines, and to produce UTP, CTP, TTP and dCTP from uracil, are present in the C. parvum genome, including thymidine kinase to facilitate recycling of thymidine directly. Careful phylogenetic analysis indicates that several of these pyrimidine salvage enzymes have been acquired via gene transfer events (Striepen et al., 2004). Consistent with the lack of de novo synthesis capabilities for pyrimidines, C. parvum lack the entire pentose phosphate pathway.

5.5. Amino Acid Metabolism Similar to purine and pyrimidine metabolism, C. parvum has lost the ability for de novo amino acid synthesis, further increasing its dependence on the host cell as a source of key metabolites. C. parvum does maintain the ability for interconversion between a limited number of amino acids, but the majority of these activities appear to be necessary for generating co-factors for other reactions. For example, conversion of glutamine into glutamate is coupled to a GMP synthetase reaction, and serine to glycine conversion results in the

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generation of methylene-tetrahydrofolate (MTHF) for TMP synthetase (see below). One exception is the ability to convert glutamine into proline. All three enzymes necessary for this conversion are present (glutamate kinase, g-glutamyl phosphate reductase, pyrroline-5-carboxylate reductase) in C. parvum. In contrast, this activity and enzymes are lacking in P. falciparum, suggesting that these enzymes, which are highly conserved across species, may be another example of a biochemical process that has been acquired by a gene transfer event. As expected, C. parvum lacks the necessary biochemical pathways for the de novo synthesis of folate. However, even in organisms that lack the ability to synthesise folate, this key co-factor is required for linking nucleotide metabolism and amino acid metabolism. In C. parvum, folate utilisation appears to solely support the synthesis of TMP from UMP. C. parvum does retain the ability for the interconversion of serine into glycine, and thus regenerate MTHF. However, C. parvum lack methionine synthetase and MTHF reductase that are required for the recycling of methionine, suggesting that methionine is obtained from the host cell. To apparently compensate for this lack of ability for de novo synthesis and catabolism of amino acids, C. parvum has an extensive set of putative amino acid transporters. Simple blast searching of the genome identifies 11 different amino acid transporters randomly distributed throughout the genome (Figure 11), which is in stark contrast to P. falciparum, which appears to only have a single amino acid transporter. In addition, C. parvum lacks the extensive set of dedicated proteases used by P. falciparum to generate amino acids from haemoglobin, further evidence of its dependence on the host cell and the importance of this expanded set of amino acid transporters.

5.6. Conclusions The highly compact C. parvum genome encodes an extremely streamlined metabolism, in which a great number of conventional biosynthetic pathways are completely lost or simplified (Figure 11). The


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overriding theme that has emerged is that C. parvum has evolved to maximise the number of biosynthetic molecules that are salvaged from the host, and has retained the capacity for biosynthesis of only those metabolites that are unavailable, or require too much energy to transport. In addition, C. parvum appears to have obtained novel biochemical abilities or modified existing metabolic processes through several distinct gene transfer events. Many of these acquisitions have been apparently accompanied by a subsequent loss of genetic information, resulting in C. parvum being very metabolically different from the other characterised apicomplexans. As a result, many of the conventional drug targets being pursued for apicomplexans are not present in C. parvum (e.g. cytochrome respiration, type II FAS, HXGPRT, shikimate synthesis). However, these novel metabolic pathways and distinct genes (e.g. IMPDH, adhE, LDH, PNO, CpFAS1, CpPKS1 and AOX) may serve as novel targets for drug development against cryptosporidiosis. All these notions are also supported by the more recently released genome sequence of C. hominis (previously referred to as Type I C. parvum), which is 95–97% identical to that of C. parvum and comprised of the same sets of major metabolic pathways (Xu et al., 2004).

6. EPIDEMIOLOGY AND TRANSMISSION 6.1. Cycles of Transmission and Zoonotic Potential Transmission of Cryptosporidium may be direct, from host to host; through contact with contaminated materials, for example, soiled nappies in day-care centres; through the ingestion of contaminated food or water; or, like many other parasites that produce resistant stages that are passed in the faeces into the environment, vectors such as arthropods or even birds may play a role as mechanical agents of transmission. The demonstration of numerous species and genotypes of Cryptosporidium, that appear to be largely host-specific suggests a multitude of transmission cycles involving different species of vertebrate host.

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However, most interest has focussed on C. parvum, which is zoonotic, and appears to have the broadest host range of the currently recognised species of Cryptosporidium. The first human case of infection with Cryptosporidium was described in 1976, and considerable circumstantial evidence accumulated of zoonotic exposure associated with farms and farm animals, riding stables, animal manure and contaminated water (Fayer et al., 2000). Early reports drew attention to the association of human infection with exposure to infected livestock, particularly young cattle or sheep. The occurrence of secondary spread within households or play-groups following such zoonotic exposure has also been reported (Casemore et al., 1997). Although farm workers and visitors to farms are considered to have contracted cryptosporidiosis by direct contact, indirect zoonotic transmission of Cryptosporidium of livestock origin via water has been considered to be the most important zoonotic source of human infection. However, such conclusions were often only circumstantial, with presumptions being made that run-off from pasture used for cattle, was the pre-disposing factor. In 1991, restriction fragment length polymorphism (RFLP) analysis revealed differences between Cryptosporidium of cattle and human origin (Ortega et al., 1991). A series of studies between 1995 and 1997 confirmed this result and more importantly revealed that humans were susceptible to infection with two genotypes of Cryptosporidium; one that also infected livestock, principally cattle, and the other that only infected humans (Awad-el-Kariem et al., 1995, Morgan et al., 1995, 1997). Molecular epidemiological tools have now provided evidence to support the existence of at least two distinct life cycles of Cryptosporidium involving humans (Figure 13). This information was first put into an epidemiological context in 1997 in determining the source of contamination of the notorious Milwaukee outbreak (Peng et al., 1997), and subsequently in a series of outbreaks some of which were shown to be of zoonotic origin (Table 5). Interestingly, although cattle have been repeatedly implicated as sources of water-borne outbreaks, the application of genotyping procedures to the contaminating isolate(s) has often incriminated human effluent as the source. For example, cattle have not been


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Figure 13 Cycles of transmission of C. parvum and C. hominis. Figure drawn by Russ Hobbs. Table 5

Cryptosporidium

Outbreak

Transmission

Species

1993 1993 1996 1997 1997 1998 2000 2002 2003

Waterborne Foodborne Waterborne Animal contact Waterborne Foodborne Waterborne 2 Foodborne Waterborne

C. C. C. C. C. C. C. C. C.

Milwaukee Maine British Columbia Pennsylvania UK Washington Northern Ireland Queensland France

hominis parvum parvum (Cranbrook); C. hominis (Kelowna) parvum hominis hominis parvum/C. hominis parvum hominis


conclusively identified as the source of any water-borne outbreak within the United States, and in Canada, an outbreak in Cranbrook, BC, is the only water-borne outbreak in North America in which oocysts of C. parvum have been identified (Fayer et al., 2000). However, there have been outbreaks caused by C. parvum in North America linked to direct contact with animals or contaminated food such as the Maine Apple cider outbreak in 1995, the Pennsylvania rural family outbreak in 1997 and the Minnesota Zoo outbreak in 1997 (Sulaiman et al., 1999). Although the species was not identified,

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the fact that the source of infection was linked to drinking unpasteurised cow milk suggests C. parvum was responsible for a recent outbreak of cryptosporidiosis in Australia (Harper et al., 2002). Recently, in Slovenia, 49 of 338 human cases of cryptosporidiosis were found to be associated with animal contact and a molecular epidemiological investigation revealed C. parvum in both humans and livestock (Stantic-Pavlinic et al., 2003). In addition to cattle, other species of livestock such as horses and deer are also susceptible to infection with C. parvum. A recent molecular epidemiological study in the Czech republic confirmed that cattle as well as horses and deer were potential zoonotic sources of infection with Cryptosporidium (Hajdusek et al., 2004). Non-zoonotic outbreaks of cryptosporidiosis have also been reported between different animal species. A recent outbreak of severe neonatal diarrhoea in foals in New Zealand was shown to be caused by C. parvum and the source of infection thought to be co-existing cattle (Grinberg et al., 2003). Companion animals have long been considered potential sources of human infection. However, despite the frequency with which pets are present in households of infected patients, rarely have they been implicated as a source of infection. Until recently, surveys of dogs and cats in most developed countries revealed Cryptosporidium to be prevalent, but no information was provided on the genotypes present. Similarly, a recent survey of equine cryptosporidiosis in Poland demonstrated that 9.4% of 43 horses were infected, and although raising the possibility of zoonotic transmission, the genotype(s) affecting the horses was not determined (Majewska et al., 1999). Recent studies in which oocysts recovered from dogs and cats have been genotyped have shown that they are most commonly infected with what appear to be predominantly host-adapted species; C. canis and C. felis (Abe et al., 2002; Thompson, 2003a, b). The study by Abe et al. (2002) in Osaka, Japan, is an excellent illustration of how molecular epidemiological techniques can provide far more meaningful data to what otherwise would have been a much less valuable survey. These authors examined samples from 140 stray adult dogs captured in the city of Osaka and of the 13 positive dogs all were shown by PCR to harbour C. canis. Thus, dogs and cats and possibly other companion


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animals may not be important zoonotic reservoirs of Cryptosporidium infection. However, molecular characterisation of oocysts recovered from infected animals in many more endemic areas is required before this assumption can be verified. It should also be emphasised that companion animals, particularly dogs and cats, may act as mechanical vectors for Cryptosporidium, with oocysts they have ingested passing through the gut intact and acting as a source of infection either through environmental contamination or directly. This has been demonstrated with other parasites such as Ascaris lumbricoides (Traub et al., 2002). The frequency that this may occur with Cryptosporidium is not clear, but the potential was demonstrated recently with the recovery of both C. baileyi and C. muris oocysts from cat faeces (McGlade et al., 2003). Seasonal shifts in prevalence and oocyst shedding have been identified in human and wildlife populations associated with warmer weather (Roy et al., 2004; Atwill et al., 2004). However, such seasonal fluctuations have not been reported in cattle (Becher et al., 2004).

6.2. Epidemiology of Infections in Humans There has been a steady accumulation of epidemiological data during the last 5 years in which isolates of Cryptosporidium from human cases have been genotyped (Table 6). This has revealed some interesting differences between the situation in Australia and North America, where most cases appear to be of human origin, and in Europe where zoonotic sources of infection appear to be more common. These can only be general observations at present and more focused molecular epidemiological studies in defined endemic foci are required to gain a better understanding of transmission. For example, the study by Read et al. (2001) in day-care centres found that all infected children harboured the human genotype of C. parvum (i.e. C. hominis), a result to be expected in an environment favouring direct, person-to-person transmission. In contrast, although only a few cases were examined, the study by Fretz et al. (2003) in Switzerland (Table 6), where there is a reliance on surface waters and where there are large numbers of

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Table 6


C. parvum and C. hominis in human beings Location

C. parvum (%)

C. hominis (%)

Morgan et al. (1998), n ¼ 36 Sulaiman et al. (1998), n ¼ 50 Ong et al. (2002), n ¼ 150 Xiao et al. (2001), n ¼ 132 Read et al. (2002), n ¼ 39

Western Australia United States Canada Peru Western Australia

17 18 19 12.6 0

83 83 72 71.7 100

Pedraza-Diaz et al. (2001), n ¼ 2057 Lowery et al. (2001), n ¼ 39 Fretz et al. (2003), n ¼ 12

UK Northern Ireland Switzerland

60 87.2 100

38 12.8 0

Yagita et al. (2001), n ¼ 22 Gatei et al. (2002), n ¼ 33 Goh et al. (2004), n ¼ 67

Japan Kenya UK

16 26 84

84 74 16

cattle in close association with water sources and people, waterborne zoonotic transmission would not be unexpected. A recent case-control study in the North West of England and Wales identified different risk factors for C. hominis and C. parvum. For the former, overseas travel and changing diapers of children less than 5 years of age were strongly significant risk factors, whereas for C. parvum, touching farm animals was associated with infection (Hunter et al., 2004). International travel and contact with cattle were also identified as risk factors for sporadic cryptosporidiosis in the United States, but no information was obtained on the causative species (Roy et al., 2004). In a study undertaken in Scotland between 1996 and 2000, of patients with cryptosporidiosis, C. parvum was shown to be the causative agent in 84% of 67 cases, supporting livestock faecal pollution of water sources as the leading cause of human sporadic cryptosporidiosis in the study population (Goh et al., 2004). It is interesting therefore, that in the United Kingdom, where zoonotic transmission has been considered the major route of cryptosporidial infection in humans, regulations imposed during the recent outbreak of foot and mouth disease are thought to be the reason for the recent decline in cases of cryptosporidiosis in humans. These regulations removed access to the countryside thus preventing humans from coming into contact with farms, wild animals and their excrement (Hunter et al., 2003).


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In addition to C. parvum and C. hominis, humans have been shown to be susceptible to other species of Cryptosporidium (Table 7). However, until recently, it was assumed that because such infections were confined to certain groups, principally the immunocompromised and children living in disadvantaged environments, susceptibility was enhanced by deficiencies in host immunity. However, this may not be the case for C. meleagridis which has recently been recovered from immunocompetent individuals. There is thus a need to further evaluate the public health significance of C. meleagridis, and in particular prevalence and sources of infection. Table 8 also summarises the range of species and genotypes of Cryptosporidium that have been isolated from water. In addition to clearly identifiable species of public health significance, numerous novel genotypes have been isolated for which we have no information on host range or prevalence. It has always been assumed that if C. parvum was introduced into an appropriate endemic focus, such as a day-care centre, then it could be maintained by subsequent human to human transfer (Figure 13). However, several studies have provided data supporting the existence Table 7 beings

Prevalence of other species of Cryptosporidium found in human HIV patients

C. meleagridis C. felis C. canis

Children (%)

Normal (%)

8 1 2

2–14 o1 o1

0–21% 3–23% 0–8%


Table 8 C. C. C. C. C. C. C.

Species and genotypes of Cryptosporidium in natural water

parvum hominis andersoni felis canis muris baileyi Source: Thompson (2003a, b) and Xiao et al. (2004).

Cervine genotype Mouse genotype 412 novel genotypes

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of anthroponotic variants of C. parvum. Using loci such as the GP60 gene (Strong et al., 2000), several studies have demonstrated the occurrence of sub-genotypes within C. parvum (Alves et al., 2003; Peng et al., 2003; Xiao et al., 2004). Some of these have so far only been isolated from humans raising questions about their zoonotic potential. Similarly, using a heteroduplex mobility assay and nucleic acid sequencing based on a small double-stranded RNA element, Leoni et al. (2003) detected subgenotypes of C. parvum from humans not detected in livestock. If some subgenotypes of C. parvum are restricted to humans, then the occurrence of C. parvum in water is not necessarily indicative of a non-human source of contamination. Indeed, some subgenotypes of C. parvum may only be capable of infecting humans and possibly others only cattle or other species of mammal. This obviously raises questions concerning their taxonomic status and emphasises the need for caution in describing species before sufficient information on host range has been obtained.

7. CONTROL 7.1. Detection A number of recent articles have provided comprehensive reviews of diagnostic methods for detecting Cryptosporidium in clinical samples and the environment (Caccio, 2003, 2004; Carey et al., 2004). In the clinical setting, whether concerned with humans, domestic animals or wildlife, there is a need for rapid, sensitive and specific diagnostic tools that can guide appropriate therapy, and possibly provide information of epidemiological value. With the former, current laboratory methods generally rely on microscopic examination of faecal samples for detecting Cryptosporidium oocysts. The oocysts of C. parvum, C. hominis and many other species and genotypes of Cryptosporidium are morphologically indistinguishable in terms of size and it is only the larger oocysts of C. andersoni and C. muris that can be reliably distinguished from these. In addition, microscopy has suffered from the problem of distinguishing Cryptosporidium from


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other faecal components of similar size and shape such as yeasts and algae. A number of staining techniques have been developed but many suffer from problems of sensitivity and specificity and variable results between laboratories is common (Elliot et al., 1999). The most recent staining method to have been described for detecting Cryptosporidium in stools employs a negative staining technique using malachite green with superior results to other methods (Elliot et al., 1999). However, microscopy, even when combined with immunofluorescence, is relatively insensitive and prone to ‘operator variability’. PCR-based procedures have proved to have greater sensitivity and specificity than ‘conventional’ diagnostics that are reliant on microscopy (Morgan et al., 1998). Molecular techniques can also provide information on the genotype or species present by combining PCR with restriction fragment length polymorphism (RFLP) analysis, without having to resort to costly and time-consuming sequencing. In addition to their excellent sensitivity and specificity, such procedures are quick and amenable to large sample throughput. One of the major advantages of such PCR-based procedures is the ease of interpretation and it is only a matter of time before molecular diagnostic procedures become common in medical diagnostic laboratories, whereas microscopy will remain the mainstay for the routine diagnosis of Cryptosporidium infections in animals. Real-time PCR offers the prospect for quantification, but also greater reproducibility and speed, and with the use of specific probes, multiplex PCR and melting-curve analysis has also been applied to real-time PCR (Tanriverdi et al., 2002; Amar et al., 2003; Widmer et al., 2004). Such procedures combine speed, reproducibility and sensitivity with the ability to discriminate at the species and genotypic levels for Cryptosporidium, as well as the opportunity to develop assays for the simultaneous detection of other enteric pathogens routinely screened for in clinical diagnostic laboratories such as Giardia and Blastocystis. There have been a number of recent reviews on environmental, particularly water, detection of Cryptosporidium. The aim of recent research is directed at developing rapid methods for the detection of viable and infective oocysts (Carey et al., 2004). Again, molecularbased procedures, such as RT-PCR offer the most promise with the

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discovery of target sequences potentially indicative of viability, as well as the ability now to combine PCR procedures (see above) with in vitro viability assays. However, there is still considerable research required to automate these procedures so that they can form part of the routine surveillance required by the water industry. There is also a need to develop or modify existing methods that can be applied to effluents, particularly those from abattoirs, that present different problems to those encountered with water. The future value of immunological detection procedures will depend on improving the affinity and specificity of the antibody preparation. High-affinity antibodies which give rise to minimal crossreactivity with algae, autofluorescing microorganisms and mineral contaminants, are essential for the specific detection of small amounts of Cryptosporidium in environmental samples (Carey et al., 2004). There is therefore an urgent need for more robust and specific antibodies, not only for the detection of oocysts and sporozoites, but also for other recently described stages in the life cycle of Cryptosporidium (see Section 3.3).

7.2. Chemotherapy In spite of extensive screening of a large number of chemotherapeutic agents there is no reliable curative treatment for cryptosporidiosis (Armson et al., 2003). The poor response to many chemotherapeutic agents has been explained by the parasite’s unique intracellular location (Sterling, 2000), which may serve as an ‘escape mechanism’ to protect the parasite from anticryptosporidial drugs. Research on the metabolism and biochemistry of the different forms of Cryptosporidium has been limited by the inability to in vitro culture the parasite in cell-free media. The recent description of cell-free culturing of Cryptosporidium (Hijjawi et al., 2004), and access to the genome sequence database (Striepen and Kissinger, 2004; see Section 5) should aid in understanding the metabolic requirements of different parasitic forms and aid in the development of novel chemotherapeutic agents. Most of the chemotherapeutic agents that have been


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shown to be effective in controlling coccidiosis in cattle, pigs and poultry have limited or no efficacy against cryptosporidiosis in spite of the similarities in the life cycle of the parasite. It has been proposed that Cryptosporidium spp are more closely related to gregarines that infect invertebrates than coccidian parasites (Tenter et al., 2002; Hijjawi et al., 2004). This may explain the lack of efficacy of anticoccidian chemotherapeutic agents as gregarines have different life cycle stages, biochemistry and metabolic requirements than eimeriid coccidia (Tenter et al., 2002; Keeling and Fast, 2002; see Section 5). Chemotherapeutic agents can now be screened using cell-free cultures or using insect gregarine models. Treatment has currently been focussed around rehydration and electrolyte replenishment until the patient has developed sufficient specific immunity to clear the infection. Non-specific antidiarrhoeal agents such as kaolin and pectin and loperamide are also useful in reducing the severity of diarrhoea. In immunocompromised patients, increasing the function of the immune system is an important part of cryptosporidiosis therapy. In AIDS patients, antiretroviral therapy results in a reduced viral load rise in CD4 lymphocyte counts, reduction of oocyst excretion and improvement in gastrointestinal clinical signs (Foundraine et al., 1998). Certain antiretroviral protease inhibitors such as Indinavir also act directly on C. parvum by interfering with the parasite life cycle (Mele et al., 2003). A limited number of chemotherapeutic agents have demonstrated efficacy in animal models and under clinical trials. Most of these agents (Table 9) do not eliminate the parasite from the host but reduce oocyst shedding with resolution or improvement of clinical signs. Paromomycin (an aminoglycoside antibiotic) and azithromycin (a macrolide antibiotic) have been used in humans with intestinal, biliary and pancreatic cryptosporidiosis (Fahey, 2003). Paromomycin is poorly absorbed within the gastrointestinal tract but small quantities cross the apical membrane surrounding the parasite. It may act against extracellular and intracellular parasitic forms. Clinical trials in humans have shown variable results. Some studies have reported beneficial clinical effects and parasite clearance, while other studies show no difference between controls. Paromomycin has been shown

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Table 9 Cryptosporidium therapies that demonstrate clinical efficacy in humans and animals Drug name

Trade name Human dose

Paromomycin

Humatin

Azithromycin

Zithromax

Roxithromycin

Numerous

Halofuginone lactate

Halocur

Nitazoanide

Alinia

Letrazuril Decocquinate

b-Cyclodextrin Hyperimmune egg yokes Bovine antiCryptosporidium

Immunoglobulin

Deccox

Animal dose

References

500–750 mg PO Calf: 100 mg/kg/day Fahey, 2003; Fayer tid/qid (adult) PO and Ellis, 1993; Viu 25–35 mg/kg/day Lamb: 100–200 mg/ et al., 2002; PO divided tid kg/day PO Mancassola et al., (Paediatric) 1993 Goat kid: 100 mg/kg/ day PO 500 mg PO qd Fahey, 2003 (adult) 300 mg bid PO Uip et al., 1998 (adult) Calf, lamb, goat Joachim, 2003; kid: 100 mg/kg sid Villacorta et al., for 7 days 1991 100–200 mg PO Rossignol et al., 2001 (adult) 50–100 mg/day Alak et al., 1999 (adult) Calf: 2.5–10 mg/kg Villacorta et al., Goat: 2.5 mg/kg 1991; Mancassola et al., 1997 Sheep: 500 mg/kg Rossignol et al., 2001 sid for 3 days Morrison, 1998 60 ml/day 5 daily for 3 weeks 20 ml/h for Calf: 200 ml bid Tzipori et al., 1987; 10 days Nord et al., 1990; Harp et al., 1989; Fayer et al., 1989 20–80 g/day Lamb: 50 ml bid

to be effective in reducing clinical signs and oocyst shedding in calves, lambs and kids (Fayer and Ellis, 1993; Viu et al., 2002; Mancassola et al., 1993). Azithromycin and roxithromycin are macrolide antibiotics, which act in bacteria by blocking dissociation of ribosomal t-RNA leading to inhibition of protein synthesis. Azithromycin has been used alone or in combination with paromomycin in humans with clinical improvement


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(Fahey, 2003). Clinical improvement and parasite elimination has been observed in AIDS patients receiving roxithromycin (Uip et al., 1998). The nitrothiazolylsalicylamide derivative, nitazoxanide, has shown broad-spectrum parasiticidal activity against protozoa, nematodes, trematodes and cestodes and has shown in vitro activity against sexual and asexual stages of C. parvum by inhibiting parasite development (Gargala et al., 2000). Nitazoxanide was efficacious in animal models of cryptosporidiosis (Theodos et al., 1998). In human clinical trials, nitazoxanide has shown clinical efficacy in reducing severity of diarrhoea and reducing or eliminating oocyst shedding (Harris et al., 1994; Doumbo et al., 1997; Rossignol et al., 2001). Diclazuril and Letrazuril are benzene acetonitrile derivatives with documented efficacy against animal Eimeria protozoa and have activity against Cryptosporidium spp. As Letrazuril is more bioavailable, clinical trails have been conducted with this drug. Letrazuril has been shown to improve clinical signs and eliminate oocysts in AIDS patients with cryptosporidiosis (Amadi et al., 2002). b-Cyclodextrin has been shown to reduce clinical signs and oocyst shedding in experimentally infected mice and naturally infected lambs (Castro-ermida et al., 2001). b-Cyclodextrin is an excipient used in the pharmaceutical industry to improve stability and solubility of certain drugs. It has been suggested that b-cyclodextrin acts by washing out oocysts and other extracellular forms through an osmotic diarrhoea. Halfuginone lactate (Halocur, Intervet) has been used as an anticoccidial agent in poultry and has recently been registered in Europe as a chemotherapeutic agent for cryptosporidiosis in domestic cattle. It has also been used in sheep and goat kids. Halfuginone has a cryptosporidiostatic effect on sporozoite and merozoite stages of the parasite. It has been shown to reduce incidence and severity of diarrhoea, but does not prevent oocyst shedding (Villacorta et al., 1991; Joachim, 2003). The safety margin of Halfuginone is low and it is necessary to use only the recommended dosage. Non-clinical infection in calves may be beneficial as it allows animals to develop longterm immunological protection.

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Decoquinate is an anticoccidial premix that has been used for the prevention of coccidiosis caused by Eimeria spp in goats, sheep and cattle. It has been reported to decrease oocyst shedding and improve the frequency and severity of diarrhoea in goat kids (Mancassola et al., 1997). Recently, researchers have been unable to document efficacy in challenged neonatal calves or in rodent models (Moore et al., 2003).

7.3. Immunotherapy and Immunoprophylaxis Hyperimmune bovine colostrum is produced by vaccinating cows during their pregnancy and collecting the postnatal colostrum, which is rich in immunoglobulins (primarily IgG). Provision of hyperimmune colostrum per os to patients with cryptosporidiosis has resulted in some patients with no improvement in clinical signs or parasite burden, while others had resolution of diarrhoea and cessation of oocyst shedding (Saxon and Weinstein, 1987; Tzipori et al., 1987; Nord et al., 1990; Greenberg and Cello, 1996). Nausea, vomiting and cramps are frequently observed in humans receiving bovine colostrum as this product can be distasteful. Administration of normal colostrum did not protect calves or lambs from natural Cryptosporidium infections (Harp et al., 1989) but improvement in clinical signs and reduced oocyst shedding were observed in lambs and calves provided with hyperimmune colostrum (Fayer et al., 1989; Naciri et al., 1994). Hyperimmune bovine colostrum was therapeutically beneficial to geckos and snakes infected with Cryptosporidium (Graczyk et al., 1998; Graczyk et al., 1999). The difference in clinical response of hyperimmune bovine colostrum between humans and animals is probably due to the specificity of the antibody in the host and differences in gut physiology. Non-specific agents in milk such as lactoferrin, lactoperoxidase, lysozyme and growth factors (e.g. EGF) have been shown to protect enterocytes from pathogen-induced intestinal alterations (Buret et al., 2003). Hyperimmune egg yolks are produced by vaccination of hens with Cryptosporidium antigen and collection of eggs. Egg yolks contain high concentrations of immunoglobulin Y (IgY), which can provide


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immunological protection similar to cow’s colostrum. Patients receiving hyperimmune egg yolks had reduced clinical signs, but faecal oocysts were not reduced (Morrison, 1998). Hyperimmune egg yolks were not well tolerated by patients due to the high fat and cholesterol levels. Probiotics have been employed as a prophylactic and therapeutic treatment for cryptosporidiosis. Lactobacillus reuteri, L. acidophilus, Bifidobacterium breve and B. longum have been shown to aid in the alleviation of clinical signs and reduce or eliminate faecal oocysts in humans and animal models (Alak et al., 1999; Waters et al., 1999; Rotkiewicz et al., 2001; Pickerd and Tuthill, 2004). Bacterial supernatants also have been shown to inactivate C. parvum oocysts (Foster et al., 2003). It is believed that bacterial proteases and numerous antimicrobial substances are toxic to C. parvum. These probiotic bacteria also induce enterocyte proliferation and enhanced turnover rates which assist in expelling the parasite. Vaccination has been proposed as a method to control cryptosporidiosis in animal populations. Whole oocyst preparations, subunit vaccines and DNA vaccines have been prepared and vaccination trials have been conducted in mice and calves (Harp and Goff, 1998; de Graaf et al., 1999a; Perryman et al., 1999; Sagodira et al., 1999; Jenkins, 2001). Vaccines have been shown to reduce clinical signs, but in most cases have not eliminated or reduced oocyst shedding. Although IgG, IgA and IgM responses are developed following vaccination the significance of antibodies in parasite clearance and prevention of clinical signs is unclear. Indeed, cell-mediated immunity may in fact be more important for prevention of clinical signs and elimination of the parasite. As calves, lambs and goats are infected with Cryptosporidium spp during the first or second week of life, passive immune protection by vaccination of dams, is the approach for these species (Harp and Goff, 1998). Colostrum containing a high concentration of IgG antibodies to Cryptosporidium (hyperimmune colostrum) has been reported to reduce diarrhoea, and oocyst shedding in calves and lambs (Harp et al., 1989; Fayer et al., 1989; Naciri et al., 1994). Vaccination of dams will enable dams to produce protective hyperimmune colostrum.

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8. PERSPECTIVES FOR THE FUTURE Much of the last 10 years has been devoted to the development and application of molecular tools to solve questions about the epidemiology and zoonotic potential of Cryptosporidium. This research has been very successful. As well as helping to elucidate cycles of transmission, it has also served to resolve many taxonomic issues. However, significant progress has also been made on other fronts, particularly in developmental biology and genomics. The results of this research have revealed fundamental questions that must now be addressed about host–parasite relationships, biochemical strategies and evolutionary biology. The overriding picture to emerge is of an organism that is unique and whose phylogenetic affinities require reevaluation. A shift in research focus towards the biology of Cryptosporidium seems warranted and is likely to dominate what will be an exciting era of investigation. Advances in in vitro cultivation and the data now available from the genome sequencing project will support this research. The ability to maintain, observe and manipulate the parasite in the laboratory under defined conditions allowing all aspects of the life cycle to be studied in detail will be of enormous benefit, as will the availability of genome sequence data and the capacity to relate this to phenotypic characters. Advances in proteomics will enhance this process, particularly in terms of a better understanding of metabolic pathways and their relationship with Cryptosporidium’s novel parasitic existence, and in the search for new drug targets and vaccine candidates.

ACKNOWLEDGEMENTS Studies referred to in this review were supported in part by grants from the Australian Research Council, Sydney Water and National Institute of Allergy and Infectious Diseases (NIAID) at the National Institutes of Health (NIH), U.S. Department of Human Health Services (DHHS) (R01 AI44594, R21 AI055278 to G.Z. and U01


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AI046397, R21 AI057216 to M.S.A.). We should like to thank Russ Hobbs for his contribution to the artwork.

REFERENCES Abe, N., Sawano, Y., Yamada, K., Kimata, I. and Iseki, M. (2002). Cryptosporidium infection in dogs in Osaka, Japan. Veterinary Parasitology 108, 185–193. Abrahamsen, M.S., Templeton, T.J., Enomoto, S., Abrahante, J.E., Zhu, G., Lancto, C.A., Deng, M., Liu, C., Widmer, G., Tzipori, S., Buck, G.A., Xu, P., Bankier, A.T., Dear, P.H., Konfortov, B.A., Spriggs, H.F., Iyer, L., Anantharaman, V., Aravind, L. and Kapur, V. (2004). Complete genome sequence of the apicomplexan, Cryptosporidium parvum. Science 304, 441–445. Akiyoshi, D.E., Mor, S. and Tzipori, S. (2003). Rapid displacement of Cryptosporidium parvum type 1 by type 2 in mixed infections in piglets. Infection and Immunity 71, 5765–5771. Alak, J.I., Wolf, B.W., Mdurvwa, E.G., Pimentel-Smith, G.E., Kolavala, S., Abdelrahman, H. and Suppiramaniam, V. (1999). Supplementation with Lactobacillus reuteri or L. acidophilus reduced intestinal shedding of Cryptosporidium parvum oocysts in immunodeficient C57BL/6 mice. Cellular and Molecular Biology 45, 855–863. Alvarez-Pellitero, P. and Sitja-Bobadilla, A. (2002). Cryptosporidium molnari n. sp. (Apicomplexa: Cryptosporidiidae) infecting two marine fish species, Sparus aurata L. and Dicentrarchus labrax L. International Journal for Parasitology 32, 1007–1021. Alves, M., Xiao, L., Sulaiman, I., Lal, A.A., Matos, O. and Antunes, F. (2003). Subgenotype analysis of Cryptosporidium isolates from humans, cattle, and zoo ruminants in Portugal. Journal of Clinical Microbiology 41, 2744–2747. Amadi, B., Mwiya, M. and Musuku, J. (2002). Effect of nitazoxanide on morbidity and mortality in Zambian children with cryptosporidiosis: a randomised controlled trial. Lancet 360, 1375–1380. Amar, C.F., Dear, P.H. and McLauchlin, J. (2003). Detection and genotyping by real-time PCR/RFLP analyses of Giardia duodenalis from human faeces. Journal of Medical Microbiology 52, 681–683. Anderson, B.C. (1982). Cryptosporidiosis in Idaho Lambs: natural and experimental infections. Journal of the American Veterinary Medical Association 181, 151–153. Anderson, B.C. (1987). Abomasal cryptosporidiosis in cattle. Veterinary Pathology 24, 235–238.

140


Anderson, B.C. (1998). Cryptosporidiosis in bovine and human health. Journal of Dairy Science 81, 3036–3041. Argenzio, R.A., Liacos, J.A., Levy, M.L., Meuten, D.J., Lecce, J.G. and Powell, D.W. (1990). Villus atrophy, crypt hyperplasia, cellular infiltration, and impaired glucose-Na absorption in enteric cryptosporidiosis of pigs. Gastroenterology 98, 1129–1140. Armson, A., Reynoldson, J.A. and Thompson, R.C.A. (2003). A review of chemotherapeutic approaches to the treatment of Cryptosporidium. In: Cryptosporidium from Molecules to Disease (R.C.A. Thompson, A. Armson and U.M. Morgan-Ryan, eds), pp. 395–403. Amsterdam: Elsevier. Arrowood, M.J. (2002). In vitro cultivation of Cryptosporidium species. Clinical Microbiology Review 15, 390–400. Arrowood, M.J., Mead, J.R., Mahrt, J.L. and Sterling, C.R. (1989). Effects of immune colostrum and orally administered antisporozoite monoclonal antibodies on the outcome of Cryptosporidium parvum infections in neonatal mice. Infection and Immunity 57, 2283–2288. Atwill, E.R., Phillips, R., Das Gracas, M., Pereira, C., Li, X. and McCowan, B. (2004). Seasonal shedding of multiple Cryptosporidium genotypes in California ground squirrels (Spermophilus beecheyi). Applied and Environmental Microbiology 70, 6748–6752. Awad-el-Kariem, F.M., Robinson, H.A., Dyson, D.A., Evans, D., Wright, S., Fox, M.T. and McDonald, V. (1995). Differentiation between human and animal strains of Cryptosporidium parvum using isoenzyme typing. Parasitology 110, 129–132. Barta, J.R. (1997). Investigating phylogenetic relationships within the Apicomplexa using sequence data: the search for homology. Methods 13, 81–88. Becher, K.A., Robertson, I.D., Fraser, D.M., Palmer, D.G. and Thompson, R.C.A. (2004). Molecular epidemiology of Giardia and Cryptosporidium infections in dairy calves originating from three sources in Western Australia. Veterinary Parasitology 123, 1–9. Beier, T.V. (2000). Further comment on the coccidian nature of cryptosporidia (Sporozoa: Apicomplexa). Parazitologiia 34, 183–195. Bigalke, R.D. (1962). Preliminary communication on the cultivation of Besnoitia besnoiti (Marotel, 1912) in tissue culture and embryonated eggs. South African Medical Journal 23, 523. Brownstein, D.G., Strandberg, J.D., Montali, R.J., Bush, M. and Fortner, J. (1977). Cryptosporidium in snakes with hypertrophic gastritis. Veterinary Pathology 14, 606–617. Bruchhaus, I. and Tannich, E. (1994). Purification and molecular characterization of the NAD(+)-dependent acetaldehyde/alcohol dehydrogenase from Entamoeba histolytica. Biochemical Journal 303, 743–748.


141

Buraud, M., Forget, E., Favennec, L., Bizet, J., Gobert, J. and Deluol, A. (1991). Sexual stages of development of Cryptosporidia in the Caco-2 cell line. Infectious Immunology 59, 4610. Buret, A.G., Chin, A.C. and Scott, K.G.E. (2003). Infection of human and bovine epithelial cells with Cryptosporidium andersoni induces apoptosis and disrupts tight junction ZO-1: effects of epidermal growth factor. International Journal for Parasitology 33, 1363–1371. Caccio, S.M. (2003). Molecular techniques to detect and identify protozoan parasites in the environment. Acta Microbiologica Polonica 52, 23–34. Caccio, S.M. (2004). New methods for the diagnosis of Cryptosporidium and Giardia. Parasitologia 46, 151–155. Carey, C.M., Lee, H. and Trevors, J.T. (2004). Biology, persistence and detection of Cryptosporidium parvum and Cryptosporidium hominis oocyst. Water Research 38, 818–862. Carreno, R.A., Martin, D.S. and Barta, J.R. (1999). Cryptosporidium is more closely related to gregarines than to coccidia as shown by phylogenetic analysis of apicomplexan parasites inferred using small-subunit ribosomal RNA gene sequences. Parasitology Research 85, 899–904. Carreno, R.A., Schnitzler, B.E., Jeffries, A.C., Tenter, A.M., Johnson, A.M. and Barta, J.R. (1998). Phylogenetic analysis of coccidia based on the 18S rDNA sequence comparison indicates that Isospora is more closely related to Toxoplasma and Neospora. Journal of Eukaryotic Microbiology 45, 184–188. Casemore, D.P., Wright, J.T. and Coop, R.L. (1997). Cryptosporidiosishuman and animal epidemiology. In: Cryptosporidium and Cryptosporidiosis (R. Fayer, ed.), pp. 65–92. FL: CRC Press. Castro-ermida, J.A., Quilez-Cinca, J., Lopez-Bernad, F., Sanchez-Acedo, C., Freire-Santos, F. and Ares-Mazas, E. (2001). Treatment with b-cyclodextrin of natural Cryptosporidium parvum infections in lambs under field conditions. International Journal for Parasitology 31, 1134–1137. Chai, J.Y., Guk, S.M., Han, H.K. and Yun, C.K. (1999). Role of intraepithelial lymphocytes in mucosal immune responses of mice experimentally infected with Cryptosporidium parvum. Journal of Parasitology 85, 234–239. Chalmers, R.M., Elwin, K., Reilly, W.J., Irvine, H., Thomas, A.L. and Hunter, P.R. (2002). Cryptosporidium in farmed animals: the detection of a novel isolate in sheep. International Journal for Parasitology 32, 21–26. Chen, A.X., Keithly, J.S., Paya, C.V. and LaRusso, N.F. (2002). Cryptosporidiosis. The New England Journal of Medicine 346, 1723–1731. Crawford, M.J., Zhu, G. and Roos, D.S. (2003). Both Type I and Type II fatty acid synthases in Toxoplasma gondii. Molecular Parasitology Meeting XIV (2003), Abstract No. 14C.

142


Current, W.L. (1990). Techniques and laboratory maintenance of Cryptosporidium. In: Cryptosporidiosis of man and animals (J.P. Dubey, C.A. Speer and R. Fayer, eds), pp. 31–49. Boca Raton, FL: CRC Press. Current, W.L. and Garcia, L.S. (1991). Cryptosporidiosis. Clinical Microbiological Reviews 4, 325–358. Current, W.L. and Haynes, T.B. (1984). Complete development of Cryptosporidium in cell culture. Science 224, 603–605. Current, W.L. and Long, P.L. (1983). Development of human and calf Cryptosporidium in chicken embryos. Journal of Infectious Diseases 148, 1108. Current, W.L. and Reese, N.C. (1986). A comparison of endogenous development of three isolates of Cryptosporidium in suckling mice. Journal of Protozoology 33, 98–108. Current, W.L., Upton, S.J. and Haynes, T.B. (1986). The life cycle of Cryptosporidium baileyi n. sp. (Apicomplexa, Cryptosporidiidae) infecting chickens. The Journal of Protozoology 33, 289–296. Dan, M. and Wang, C.C. (2000). Role of alcohol dehydrogenase E (ADHE) in the energy metabolism of Giardia lamblia. Molecular and Biochemical Parasitology 109, 25–36. Dann, S.M., Okhuysen, P.C., Salameh, B.M., DuPont, D.L. and Chappell, C.L. (2000). Fecal antibodies to Cryptosporidium parvum in healthy volunteers. Infection and Immunity 68, 5068–5074. de Graaf, D.C., Spano, F., Petry, F., Sagodira, S. and Bonnin, A. (1999a). Speculation on whether a vaccine against cryptosporidiosis is a reality or fantasy. International Journal for Parasitology 29, 1289–1306. de Graaf, D.C., Vanopdenbosch, E., Ortega-Mora, L.M., Abbassi, H. and Peeters, J.E. (1999b). A review of the importance of cryptosporidiosis in farm animals. International Journal for Parasitology 29, 1269–1287. Deng, M.Q., Templeton, T.J., London, N.R., Bauer, C., Schroeder, A.A. and Abrahamsen, M.S. (2002). Cryptosporidium parvum genes containing thrombospondin type 1 domains. Infection and Immunity 70, 6987–6995. Denton, H., Brown, S.M., Roberts, C.W., Alexander, J., McDonald, V., Thong, K.W. and Coombs, G.H. (1996). Comparison of the phosphofructokinase and pyruvate kinase activities of Cryptosporidium parvum Eimeria tenella and Toxoplasma gondii. Molecular and Biochemical Parasitology 76, 23–29. Doumbo, O., Rossignol, J.F., Pichard, E., Traore, H.A., Dembele, M., Diakite, M., Traore, F. and Diallo, D.A. (1997). Nitazoxanide in the treatment of cryptosporidial diarrhoea and other intestinal parasitic infections associated with acquired immunodeficiency syndrome in tropical Africa. American Journal of Tropical Medicine and Hygiene 56, 637–639.


143

Doyle, P.S., Crabb, J. and Petersen, C. (1993). Anti-Cryptosporidium parvum antibodies inhibit infectivity in vitro and in vivo. Infection and Immunity 61, 4079–4084. Elliot, A., Morgan, U.M. and Thompson, R.C.A. (1999). Improved staining method for detecting Cryptosporidium oocysts in stools using Malachite Green. Journal of General and Applied Microbiology 45, 139–142. Elliott, D.A. and Clark, D.P. (2000). Cryptosporidium parvum induces host cell actin accumulation at the host–parasite interface. Infection and Immunity 68, 2315–2322. Elliott, D.A., Coleman, D.J., Lane, M.A., May, R.C., Machesky, L.M. and Clark, D.P. (2001). Cryptosporidium parvum infection requires host cell actin polymerisation. Infection and Immunity 69, 5940–5942. Enemark, H.L., Ahrens, L.P., Lowery, C.J., Thamsborg, S.M., Enemark, J.M.D., Bille-Hansen, V. and Lind, P. (2002). Cryptosporidium andersoni from a Danish cattle herd: identification and preliminary characterization. Veterinary Parasitology 107, 37–49. Entrala, E. and Mascaro, C. (1997). Glycolytic enzyme activities in Cryptosporidium parvum oocysts. FEMS Microbiology Letters 151, 51–57. Ernest, J.A., Blagburn, B.L., Lindsay, D.S. and Current, W.L. (1986). Infection dynamics of Cryptosporidium parvum (Apicomplexa: Cryptosporidiidae) in neonatal mice (Mus musculus). Journal of Parasitology 72, 796–798. Esteban, E. and Anderson, B.C. (1995). Cryptosporidium muris: prevalence, persistency and detrimental effects on milk production in a drylot dairy. Journal of Dairy Science 78, 1068–1072. Fahey, T. (2003). Cryptosporidiosis Prim. Care Update Ob/Gyns 2, 75–80. Fall, A., Thompson, R.C.A., Hobbs, R.P. and Morgan-Ryan, U. (2003). Morphology is not a reliable tool for delineating species within Cryptosporidium. Journal of Parasitology 89, 399–402. Farney, J.R., DeWald, D.B., Yang, S., Speer, C.A. and Healey, M.C. (1999). A role for host phosphoinositide 3-kinase and cytoskeletal remodelling during Cryptosporidium parvum infection. Infection and Immunology 67, 844–852. Farthing, M.J. (2000). Clinical aspects of human cryptosporidiosis. Contributions to Microbiology 6, 50–74. Fayer, R. (1970). Sarcocystis: development in cultured avian and mammalian cells. Science 168, 1104. Fayer, R., Andrews, C., Ungar, B.L.P. and Blagburn, B. (1989). Efficacy of hyperimmune bovine colostrum for prophylaxis of cryptosporidiosis in neonatal calves. Journal of Parasitology 75, 393–397. Fayer, R. and Ellis, W. (1993). Paromomycin is effective as prophylaxis for cryptosporidiosis in dairy calves. Journal of Parasitology 79, 771–774.

144


Fayer, R., Ernest, J.V., Miller, R.G. and Leek, R.G. (1985). Factors contributing to clinical illness in calves experimentally infected with a bovine isolate of Cryptosporidium. Proceedings of the Helminthological Society of Washington 52, 64–70. Fayer, R., Gasbarre, L., Pasquali, P., Canals, A., Almeria, S. and Zarlenga, D. (1998). Cryptosporidium parvum infection in bovine neonates: dynamic, clinical, parasitic and immunologic patterns. International Journal for Parasitology 28, 49–56. Fayer, R., Morgan, U. and Upton, S.J. (2000). Epidemiology of Cryptosporidium: transmission, detection and identification. International Journal for Parasitology 30, 1305–1322. Fayer, R., Speer, C.A. and Dubey, J.P. (1997). The general biology of Cryptosporidium. In: Cryptosporidium and Cryptosporidiosis (R. Fayer, ed.), pp. 1–42. Boca Raton, FL: CRC Press. Fayer, R. and Unger, B.L. (1986). Cryptosporidium spp. and Cryptosporidiosis. Microbiological Reviews 50, 458–483. Foster, J.C., Glass, M.D., Courtney, P.D. and Ward, L.A. (2003). Effect of Lactobacillus and Bifidobacterium on Cryptosporidium parvum oocyst viability. Food Microbiology 20, 351–357. Foundraine, N.A., Weverling, G.J., van Gool, T., Roos, M.T., de Wolf, F., Koopmans, P.P., van den Broek, P.J., Meenhorst, P.L., van Leeuwen, R., Lang, J.M. and Reiss, P. (1998). Improvement of chronic diarrhoea in patients with advanced HIV-1 infection during potent antiretroviral therapy. AIDS 12, 35–41. Fretz, R., Svoboda, P., Ryan, U.M., Thompson, R.C.A., Tanner, M. and Baumgartner, A. (2003). Genotyping of Cryptosporidium spp. isolated from human stool samples in Switzerland. Epidemiology and Infection 131, 663–667. Gaitatzis, N., Kunze, B. and Muller, R. (2001). In vitro reconstitution of the myxochelin biosynthetic machinery of Stigmatella aurantiaca Sg a15: Biochemical characterization of a reductive release mechanism from nonribosomal peptide synthetases. Proceedings of the National Academy of Sciences of the United States of America 98, 11136–11141. Gardner, M.J., Hall, N., Fung, E., White, O., Berriman, M., Hyman, R.W., Carlton, J.M., Pain, A., Nelson, K.E., Bowman, S., Paulsen, I.T., James, K., Eisen, J.A., Rutherford, K., Salzberg, S.L., Craig, A., Kyes, S., Chan, M.S., Nene, V., Shallom, S.J., Suh, B., Peterson, J., Angiuoli, S., Pertea, M., Allen, J., Selengut, J., Haft, D., Mather, M.W., Vaidya, A.B., Martin, D.M., Fairlamb, A.H., Fraunholz, M.J., Roos, D.S., Ralph, S.A., McFadden, G.I., Cummings, L.M., Subramanian, G.M., Mungall, C., Venter, J.C., Carucci, D.J., Hoffman, S.L., Newbold, C., Davis, R.W., Fraser, C.M. and Barrell, B. (2002). Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419, 498–511.


145

Gargala, G., Delaunay, A., Li, X., Brasseur, P., Favennec, L. and Ballet, J.J. (2000). Efficacy of nitazoxanide, tizoxanide and tizoxanide glucuronide against Cryptosporidium parvum development in sporozoite-infected HCT-8 enterocyte cells. Journal of Antimicrobial Chemotherapy 46, 57–60. Gatei, W., Ashford, R.W., Beeching, J.J., Kamwati, S.K., Greensill, J. and Hart, C.A. (2002). Cryptosporidium muris infection in an HIV-infected adult, Kenya. Emerging Infectious Diseases 8, 204–206. Giselle, N.J., Appelbee, A.J. and Olson, M.E. (2003). Biology of Cryptosporidium parvum in pigs from weaning to market. Veterinary Parasitology 113, 7–18. Goh, S., Reacher, M., Casemore, D.P., Verlander, N.Q., Chalmers, R., Knowles, M., Williams, J., Osborn, K. and Richards, S. (2004). Sporadic Cryptosporidiosis, North Cumbria, England, 1996–2000. Emerging Infectious Diseases 10, 1007–1015. Graczyk, T.K., Cranfield, M.R. and Bostwick, E.F. (1999). Hyperimmune bovine colostrum treatment of moribund Leopard geckos infected with Cryptosporidium sp. Veterinary Research 30, 377–382. Graczyk, T.K., Cranfield, M.R., Helmer, P., Fayer, R. and Bostwick, E.F. (1998). Therapeutic efficacy of hyperimmune bovine colostrum treatment against clinical and subclinical Cryptosporidium serpentis infection in captive snakes. Veterinary Parasitology 71, 123–132. Greenberg, P.D. and Cello, J.P. (1996). Treatment of severe diarrhoea caused by Cryptosporidium parvum with oral bovine immunoglobulin concentrate in patients with AIDS. AIDS Research and Human Retroviruses 13, 348–354. Grinberg, A., Oliver, L., Learmonth, J.J., Leyland, M., Roe, W. and Pomroy, W.E. (2003). Identification of Cryptosporidium parvum ‘cattle’ genotype from a severe outbreak of neonatal foal diarrhoea. Veterinary Record 153, 628–631. Guarino, A., Canani, R.B., Casola, A., Pozio, E., Russo, R., Bruzzese, E., Fontana, M. and Rubino, A. (1995). Human intestinal cryptosporidiosis: secretory diarrhoea and enterotoxic activity in Caco-2 cells. Journal of Infectious Diseases 171, 976–983. Hajdusek, O., Ditrich, O. and Slapeta, J. (2004). Molecular identification of Cryptosporidium spp. in animal and human hosts from the Czech Republic. Veterinary Parasitology 122, 183–192. Harp, J.A. and Goff, J.G. (1998). Strategies for the control of Cryptosporidium parvum infection in calves. Journal of Dairy Science 81, 289–294. Harp, J.A., Woodmansee, D.B. and Moon, H.W. (1989). Effect of colostral antibody on susceptibility of calves to Cryptosporidium parvum infection. American Journal Research 50, 2117–2119.

146


Harper, C.M., Cowell, N.A., Adams, B.C., Langley, A.J. and Wohlsen, T.D. (2002). Outbreak of Cryptosporidium linked to drinking unpasteurised milk. Communicable Disease Intelligence 26, 449–450. Harris, M., Deutsch, G., MacLean, J.D. and Tsoukas, C.M. (1994). A phase I study of letrazuril in AIDS-related cryptosporidiosis. AIDS 8, 1109–1113. Hijjawi, N.S. (2003). In vitro cultivation and development of Cryptosporidium in cell culture. In: Cryptosporidium from Molecules to Disease (R.C.A. Thompson, A. Armson and U.M. Morgan-Ryan, eds), pp. 233–253. Amsterdam: Elsevier. Hijjawi, N.S., Meloni, B.P., Morgan, U.M., Olson, M.E. and Thompson, R.C.A. (2002). Successful in vitro cultivation of Cryptosporidium andersoni with evidence for the existence of novel extracellular stages in the Cryptosporidium life cycle. International Journal for Parasitology 32, 1719–1726. Hijjawi, N.S., Meloni, B.P., Morgan, U.M. and Thompson, R.C.A. (2001). Complete development and long-term maintenance of Cryptosporidium parvum human and cattle genotypes in cell culture. International Journal for Parasitology 31, 1048–1055. Hijjawi, N.S., Meloni, B.P., Ng’anzo, M., Ryan, U.M., Olson, M.E., Cox, P.T., Monis, P.T. and Thompson, R.C.A. (2004). Complete development of Cryptosporidium parvum in host cell-free culture. International Journal for Parasitology 34, 769–777. Hill, S.L., Cheney, J.M., Taton-Allen, G.F., Reiff, J.S., Bruns, C. and Lappin, M.R. (2000). Prevalence of enteric zoonotic organisms in cats. Journal of the American Veterinary Medical Association 216, 687–692. Holko, I., Pavlasek, I., Barton, L. and Kmet, V. (2004). Comparison of digestive microflora between cattle with and without infection by Cryptosporidium andersoni 49, 165–168. Hoover, D.M., Hoerr, F.J., Carlton, W.W., Hisman, E.J. and Ferguson, H.W. (1981). Enteric cryptosporidiosis in a naso tang, Naso lituratus Bloch and Snider. Journal of Fish Diseases 4, 425–428. Huang, B.Q., Chen, X.M. and LaRusso, N.F. (2004a). Cryptosporidium parvum attachment to and internalisation by human biliary epithelia in vitro: a morphologic study. Journal of Parasitology 90, 212–221. Huang, J., Mullapudi, N., Lancto, C.A., Scott, M., Abrahamsen, M.S. and Kissinger, J.C. (2004b). Phylogenomic evidence supports past endosymbiosis, intracellular and horizontal gene transfer in Cryptosporidium parvum. Genome Biology 5(R88), 1–13. Huang, K. and Yang, S. (2002). Inhibitory effect of selenium on Cryptosporidium parvum infection in vitro and in vivo. Biological Trace Element Research 90, 261–272.


147

Hunt, E., Fu, Q., Armstrong, M.U., Rennix, D.K., Webster, D.W., Galanko, J.A., Chen, W., Weaver, E.M., Argenzio, R.A. and Rhoads, J.M. (2002). Oral bovine serum concentrate improves cryptosporidial enteritis in calves. Pediatric Research 51, 370–376. Hunter, P.R., Chalmers, R.M., Syed, Q., Hughes, L.S., Woodhouse, S. and Swift, L. (2003). Foot and mouth disease and cryptosporidiosis: possible interaction between two emerging infectious diseases. Emerging Infectious Diseases 9, 109–112. Hunter, P.R., Hughes, S., Woodhouse, S., Syed, Q., Verlander, N.Q., Chalmers, R.M., Morgan, K., Nichols, G., Beeching, N. and Osborn, K. (2004). Sporadic cryptosporidiosis case–control study with genotyping. Emerging Infectious Diseases 10, 1241–1249. Irwin, P.J. (2002). Companion animal parasitology: a clinical perspective. International Journal for Parasitology 32, 581–593. Jenkins, M.C. (2001). Advances and prospects for subunit vaccines against protozoa of veterinary importance. Veterinary Parasitology 101, 291–310. Joachim, A. (2003). Prevalence and control of bovine cryptosporidiosis in German dairy herds. Veterinary Parasitology 112, 277–288. Keeling, P.J. (2004). Reduction and compaction in the genome of the apicomplexan parasite Cryptosporidium parvum. Developmental Cell 6, 614–616. Keeling, P.J. and Fast, N.M. (2002). Microsporidia: biology and evolution of highly reduced intracellular parasites. Annual Review of Microbiology 56, 93–116. Koudela, B., Modry, D. and Vitovec, J. (1998). Infectivity of Cryptosporidium muris isolated from cattle. Veterinary Parasitology 76, 181–188. Kuntz, M. (2004). Plastid terminal oxidase and its biological significance. Planta 218, 896–899. Kvac, M. and Vitovec, J. (2003). Prevalence and pathogenicity of Cryptosporidium andersoni in one herd of beef. Journal of Veterinary Medicine. B, Infectious Diseases and Veterinary Public Health 50, 451–457. Landsberg, J.H. and Paperna, I. (1986). Ultrastructural-study of the coccidian Cryptosporidium sp. from stomachs of juvenile cichlid fish. Diseases of Aquatic Organisms 2, 13–20. Lawton, P., Naciri, M., Mancassola, R. and Petavy, A. (1997). In vitro cultivation of Cryptosporidium parvum in the non-adherent human monocytic THP-1 cell line. Journal of Eukaryotic Microbiology 6, 44. Leander, B.S., Harper, J.T. and Keeling, P.J. (2003). Molecular phylogeny and surface morphology of marine aseptate gregarines (Apicomplexa): Selenidium spp. and Lecudina spp. Journal of Parasitology 89, 1191–1205. Leav, B.A., Mackay, M. and Ward, H.D. (2003). Cryptosporidium species: new insights and old challenges. Clinical Infectious Diseases 36, 903–908.

148


Leoni, F., Gallimore, C.I., Green, J. and McLauchlin, J. (2003). Molecular epidemiological analysis of Cryptosporidium isolates from humans and animals by using a heteroduplex mobility assay and nucleic acid sequencing based on a small double-stranded RNA element. Journal of Clinical Microbiology 41, 981–992. Levine, N.D. (1985). Phylum II. Apicomplexa Levine 1970. In: Illustrated Guide to the Protozoa (J.J. Lee, S.H. Hunter and E.C. Bovee, eds), pp. 322–374. Lawrence: Society of Protozoologists. Levine, N.D., ed. (1988). The Protozoan Phylum Apicomplexa. FL: CRC Press. Lindsay, D.S., Blagburn, B.L., Sundermann, C.A. and Giambrone, J.J. (1988). Effect of broiler chicken age on susceptibility to experimentally induced Cryptosporidium baileyi infection. American Journal of Veterinary Research 49, 1412–1414. Lopez, F.A., Manglicmot, J., Schmidt, T.M., Yeh, C., Smith, H.V. and Relman, D.A. (1999). Molecular characterization of Cyclospora like organisms from baboons. Journal of Infectious Diseases 179, 670–676. Lowery, C.J., Millar, B.C., Moore, J.E., Xu, J., Xiao, L., Rooner, P.J., Crothers, L. and Dooley, J.S. (2001). Molecular genotyping of human cryptosporidiosis in Northern Ireland: epidemiological aspects and review. Irish Journal of Medical Science 170, 246–250. Lumb, R., Smith, K., O’Donoghue, P.J. and Lanser. (1988). Ultrastructure of the attachment of Cryptosporidium sporozoites to tissue culture cells. Parasitology Research 74, 531–536. Madern, D., Cai, X., Abrahamsen, M.S. and Zhu, G. (2004). Evolution of Cryptosporidium parvum lactate dehydrogenase from malate dehydrogenase by a very recent event of gene duplication. Molecular Biology and Evolution 21, 489–497. Majewska, A.C., Werner, A., Sulima, P. and Luty, T. (1999). Survey on equine cryptosporidiosis in Poland and the possibility of zoonotic transmission. Annals of Agriculture and Environmental Medicine 6, 161–165. Mancassola, R., Reperant, J.M., Naciri, M. and Chartier, C. (1993). Chemoprophylaxis of Cryptosporidium parvum infection with paromomycin in kids and immunological study. Antimicrobiology Agents and Chemotherapy 39, 75–78. Mancassola, L., Richard, A. and Naciri, M. (1997). Evaluation of decoquinate to treat experimental cryptosporidiosis in kids. Veterinary Parasitology 69, 31–37. McAllister, T.A., Olson, M.E., Fletch, A., Wetzstein, M. and Entz, T. (2005). Prevalence of Giardia and Cryptosporidium in beef cows in southern Ontario and in beef calves in southern British Columbia. Canadian Veterinary Journal 46, 47–55.


149

McGlade, T.R., Robertson, I.D., Elliot, A.D. and Thompson, R.C.A. (2003). High prevalence of Giardia detected in cats by PCR. Veterinary Parasitology 110, 197–205. Mele, R., Gomez, M.A., Tosini, F. and Pozio, E. (2003). Indinavir reduces Cryptosporidium parvum infection in both in vitro and in vivo models. International Journal for Parasitology 33, 757–764. Meloni, B.P. and Thompson, R.C.A. (1996). Simplified method for obtaining purified oocysts from mice and growing Cryptosporidium parvum in vitro. Journal of Parasitology 82, 757–762. Monis, P.T. and Thompson, R.C. (2003). Cryptosporidium and Giardia zoonoses: fact or fiction? Infection Genetics and Evolution 3, 233–244. Moon, H.W. and Bemrick, W.J. (1981). Fecal transmission of calf cryptosporidia between calves and pigs. Veterinary Pathology 18, 248–255. Moore, D.A., Atwill, E.R., Kirk, J.H., Brahmbhatt, D., Herrera Alonso, L., Hou, L., Singer, M.D. and Miller, T.D. (2003). Prophylactic use of decoquinate for infections with Cryptosporidium parvum in experimentally challenged neonatal calves. Journal of the American Veterinary Medical Association 223, 839–845. Morgan, U.M., Constantine, C.C., O’Donoghue, P., O’Brien, P.A. and Thompson, R.C.A. (1995). RAPD (random amplified polymorphic DNA) analysis of Cryptosporidium isolates. American Journal of Tropical Medicine and Hygiene 52, 559–564. Morgan, U.M., Constantine, C.C., Forbes, D.A. and Thompson, R.C.A. (1997). Differentiation between human and animal isolates of Cryptosporidium parvum using rDNA sequencing and direct PCR analysis. Journal of Parasitology 83, 825–830. Morgan, U.M., Pallant, L., Dwyer, B., Forbes, D.A. and Thompson, R.C.A. (1998). Comparison of PCR versus microscopy for the detection of Cryptosporidium—a clinical trial. Journal of Clinical Microbiology 36, 995–998. Morgan, U.M., Xiao, L., Fayer, R., Lal, A.A. and Thompson, R.C.A. (1999). Variation in Cryptosporidium: towards a taxonomic revision of the genus. International Journal for Parasitology 29, 1733–1751. Morrison, D.A. and Ellis, J.T. (1997). Effects of nucleotide sequence alignment on phylogeny estimation: a case study of 18 rDNA of Apicomplexa. Molecular Biology and Evolution 14, 428–441. Morrison, D.A., Bornstein, S., Thebo, P., Wernery, U., Kinne, J. and Mattsson, J.G. (2004). The current status of the small subunit rRNA phylogeny of the coccidia (Sporozoa). International Journal for Parasitology 34, 501–514. Morrison, L. (1998). Cryptosporidiosis in a Critical Review of the Treatment and Prophylaxis of AIDS-related Opportunistic Infections. New York: TAG. http://www.aidsinfonyc.org/tag/comp/ois98/14.html

150


Morrissette, N.S. and Sibley, L.D. (2002). Cytoskeleton of apicomplexan parasites. Microbiology and Molecular Biology Reviews 66, 21–38. Muench, T.R. and White, M.R. (1997). Cryptosporidiosis in a tropical freshwater catfish (Plecostomus spp.). Journal of Veterinary Diagnostic Investigation 9, 87–90. Naciri, M., Mancassola, R., Reperant, J.M., Canivez, O., Quinque, B. and Yvore, P. (1994). Treatment of experimental ovine cryptosporidiosis with ovine or bovine hyperimmune colostrum. Veterinary Parasitology 53, 173–190. Nihei, C., Fukai, Y. and Kita, K. (2002). Trypanosome alternative oxidase as a target of chemotherapy. Biochimica et Biophysica Acta 1587, 234–239. Nord, J., Ma, P., Di John, D., Tzipori, S., Sahner, D. and Shieb, G. (1990). Treatment with bovine hyperimmune colostrum of cryptosporidial diarrhoea in AIDS patients. AIDS 4, 581–584. O’Donoghue, P.J. (1995). Cryptosporidium and Cryptosporidiosis in man and animals. International Journal for Parasitology 85, 525–530. O’ Handley, R.M., Cockwill, C., Mcallister, T.A., Jelinski, M., Morck, D.W. and Olson, M.E. (1999). Duration of naturally acquired giardiasis and cryptosporidiosis in dairy calves and their association with diarrhoea. Journal of the American Veterinary Medical Association 214, 391–396. Okhuysen, P.C., Chappell, C.L., Crabb, J., Valdez, L.M., Douglass, E.T. and DuPont, H.L. (1998). Prophylactic effect of bovine antiCryptosporidium hyperimmune colostrum immunoglobulin in healthy volunteers challenged with Cryptosporidium parvum. Clinical Infectious Diseases 26, 1324–1329. Olson, M.E., O’Handley, R.M., Ralston, B.J., McAllister, T.A. and Thompson, R.C.A. (2004a). Update on Cryptosporidiosis in cattle. Trends in Parasitology 20, 185–191. Olson, M.E., Ralston, B.J., O’Handley, R., Guselle, N.J. and Appelbee, A.J. (2004b). What is the clinical and zoonotic significance of cryptosporidiosis in domestic animals and wildlife. In: Cryptosporidium: from Molecules to Disease (R.C.A. Thompson, ed.), pp. 51–68. Amsterdam: Elsevier. Ong, C.S., Eisler, D.L., Alikhani, A., Fung, V.W., Tomblin, J., Bowie, W.R. and Isaac-Renton, J.L. (2002). Novel Cryptosporidium genotypes in sporadic cryptosporidiosis cases: first report of human infections with a cervine genotype. Emerging Infectious Diseases 8, 263–268. Ortega, Y.R., Sheehy, R.R., Cama, V.A., Oishi, K.K. and Sterling, C.R. (1991). Restriction fragment length polymorphism analysis of Cryptosporidium parvum isolates of bovine and human origin. Journal of Protozoology 38, 40–41.


151

Ortega-Mora, L.M. and Wright, S.E. (1994). Age-related resistance in ovine cryptosporidiosis: patterns of infection and humoral immune response. Infection and Immunity 62, 5003–5009. Patton, W.H. (1965). Eimeria tenella: cultivation of the asexual stages in cultured animal cells. Science 150, 767. Pedraza-Diaz, S., Amar, C., Iversen, A.M., Stanley, P.J. and McLauchlin, J. (2001). Unusual Cryptosporidium species recovered from human faeces: first description of Cryptosporidium felis and Cryptosporidium ‘dog type’ from patients in England. Journal of Medical Microbiology 50, 293–296. Peng, M.M., Wilson, M.L., Holland, R.E., Meshnich, S.R., Lal, A.A. and Xiao, L. (2003). Genetic diversity of Cryptosporidium spp. in cattle in Michigan: implications for understanding the transmission dynamics. Parasitological Research 90, 175–180. Peng, M.M., Xiao, L. and Freeman, A.R. (1997). Genetic polymorphism among Cryptosporidium parvum isolates: evidence of two distinct human transmission cycles. Emerging Infectious Diseases 3, 567–573. Pereira, S.J., Ramirez, N.E., Xiao, L. and Ward, L.A. (2002). Pathogenesis of human and bovine Cryptosporidium parvum in gnotobiotic pigs. Journal of Infectious Diseases 186, 715–718. Perryman, L.E., Kapil, S.J., Jones, M.L. and Hunt, E.L. (1999). Protection of calves against cryptosporidiosis with immune bovine colostrum induced by a Cryptosporidium parvum recombinant protein. Vaccine 17, 2142–2149. Petry, F. (2004). Structural analysis of Cryptosporidium parvum. Microscopy and Microanalysis 10, 586–601. Pickerd, N. and Tuthill, D. (2004). Resolution of cryptosporidiosis with probiotic treatment. Postgraduate Medical Journal 80, 112–113. Priest, J.W., Xie, L.T., Arrowood, M.J. and Lammie, P.J. (2001). The immunodominant 17-kDa antigen from Cryptosporidium parvum is glycosylphosphatidylinositol-anchored. Molecular and Biochemical Parasitology 113, 117–126. Putignani, L., Tait, A., Smith, H.V., Horner, D., Tovar, J., Tetley, L. and Wastling, J.M. (2004). Characterization of a mitochondrion-like organelle in Cryptosporidium parvum. Parasitology 129, 1–18. Ralston, B.J., Cockwill, C.L., Guselle, N.J., Van Herk, F.H., McAllister, T.A. and Olson, M.E. (2003). Prevalence of Giardia and Cryptosporidium andersoni and their effects on performance in feedlot beef calves. Canadian Journal of Animal Science 83, 153–159. Ramirez, N.E., Ward, L.A. and Sreevatan, S. (2004). A review of the biology and epidemiology of cryptosporidiosis in humans and animals. Microbes and Infection 6, 773–785. Rawlings, B.J. (1997). Biosynthsis of polyketides. Natural Product Reports 14, 523–556.

152


Read, C., Walters, J., Robertson, I.D. and Thompson, R.C.A. (2002). Correlation between genotypes of Giardia duodenalis and diarrhoea. International Journal for Parasitology 32, 229–231. Reduker, D.W. and Speer, C.A. (1985). Factors influencing excystation in Cryptosporidium oocysts from cattle. Journal of Parasitology 71, 112–115. Reduker, D.W., Speer, C.A. and Blixt, J.A. (1985). Ultra structural changes in the oocyst wall during excystation of Cryptosporidium parvum (Apicomplexa; Eucoccidiorida). Canadian Journal of Zoology 63, 1892–1896. Rein, K.S. and Borrone, J. (1999). Polyketides from dinoflagellates: origins, pharmacology and biosynthesis. Comparative Biochemistry and Physiology B—Biochemistry and Molecular Biology 124, 117–131. Relman, D.A., Schmidt, T.M., Gajadhar, A., Sogin, M., Cross, J., Yoder, K., Sethabutr, O. and Echerevrria, P. (1996). Molecular phylogenetic analysis of Cyclospora, the human intestinal pathogen, suggests that it is closely related to Eimeria species. Journal of Infectious Diseases 173, 440–445. Riggs, M.W. (2002). Recent advances in cryptosporidiosis: the immune response. Microbes and Infection 4, 1067–1080. Riggs, M.W., Schaefer, D.A., Kapil, S.J., Barley-Maloney, L. and Perryman, L.E. (2002). Efficacy of monoclonal antibodies against defined antigens for passive immunotherapy of chronic gastrointestinal cryptosporidiosis. Antimicrobial Agents and Chemotherapy 46, 275–282. Riordan, C.E., Ault, J.G., Langreth, S.G. and Keithly, J.S. (2003). Cryptosporidium parvum Cpn60 targets a relict organelle. Current Genetics 44, 138–147. Roberts, C.W., Roberts, F., Henriquez, F.L., Akiyoshi, D., Samuel, B.U., Richards, T.A., Milhous, W., Kyle, D., McIntosh, L., Hill, G.C., Chaudhuri, M., Tzipori, S. and McLeod, R. (2004). Evidence for mitochondrial-derived alternative oxidase in the apicomplexan parasite Cryptosporidium parvum: a potential anti-microbial agent target. International Journal for Parasitology 34, 297–308. Robinson, P., Okhuysen, P.C., Chappell, C.L., Weinstock, J.V., Lewis, D.E., Actor, J.K. and White, A.C., Jr. (2003). Substance P expression correlates with severity of diarrhoea in cryptosporidiosis. Journal of Infectious Diseases 188, 290–296. Rosenfeld, E. and Beauvoit, B. (2003). Role of the non-respiratory pathways in the utilization of molecular oxygen by Saccharomyces cerevisiae. Yeast 20, 1115–1144. Rossignol, J.F., Ayoub, A. and Ayers, M.S. (2001). Treatment of diarrhoea caused by Cryptosporidium parvum: a prospective randomized, doubleblind, placebo-controlled study of nitazoxanide. Journal of Infectious Diseases 184, 103–106.


153

Rotkiewicz, T., Rotkiewicz, Z., Depta, A. and Kander, M. (2001). Effect of Lactobacillus acidophilus and Bifidobacterium sp. on the course of Cryptosporidium parvum invasion in new-born piglets. Bulletin Veterinary Institute Pulawy 45, 187–195. Rotte, C., Stejskal, F., Zhu, G., Keithly, J.S. and Martin, W. (2001). Pyruvate:NADP+ oxidoreductase from the mitochondrion of Euglena gracilis and from the apicomplexan Cryptosporidium parvum: a biochemical relic linking pyruvate metabolism in mitochondriate and amitochondriate protists. Molecular Biology and Evolution 18, 710–720. Roy, S.L., DeLong, S.M., Stenzel, S.A., Shiferaw, B., Roberts, J.M., Khalakdina, A., Marcus, R., Segler, S.D., Shah, D.D., Thomas, S., Vugia, D.J., Zansky, S.M., Dietz, V., Beach, M.J. and the Emerging Infections Program FoodNet Working Group. (2004). Risk factors for sporadic Cryptosporidiosis among immunocompetent persons in the United States from 1999–2001. Journal of Clinical Microbiology 42, 2944–2951. Ryan, U.M., Monis, P., Enemark, H.L., Sulaoman, I., Samarasinghe, B., Read, C., Buddle, R., Robertson, I., Lal, A.A., Thompson, R.C.A. and Xiao, L. (2004). Cryptosporidium suis sp. (Apicomplexa; Cryptosporidiidae) in pigs (Sus scrofa). Journal of Parasitology 90, 769–773. Sagodira, S., Buzoni-Gatel, D., Lockmann, S., Naciri, M. and Bout, D. (1999). Protection of kids against Cryptosporidium parvum infection after immunization of dams with CP15-DNA. Vaccine 17, 2346–2355. Santin, M., Trout, J.M., Xiao, L., Zhou, L., Greiner, E. and Fayer, R. (2004). Prevalence and age-related variation of Cryptosporidium species and genotypes in dairy calves. Veterinary Parasitology 122, 103–117. Saxon, A. and Weinstein, W. (1987). Oral administration of bovine colostrum anti-cryptosporidia antibody fails to alter the course of human cryptosporidiosis. Journal of Parasitology 73, 415–417. Schmatz, D.M., (1997). The mannitol cycle in Eimeria. Parasitology 114 Supplement, S81–S89. Sherwood, D., Angus, K.W., Snodgrass, D.R. and Tzipori, S. (1982). Experimental cryptosporidiosis in laboratory mice. Infection and Immunity 38, 471–475. Sibley, L.D. (2004). Intracellular parasite invasion strategies. Science 304, 248–253. Slapeta, J. and Keithly, J.S. (2004). Cryptosporidium parvum mitochondrialtype HSP70 targets homologous and heterologous mitochondria. Eukaryotic Cell 3, 483–494. Snodgrass, D.R., Angus, K.W. and Gray, E.W. (1984). Experimental cryptosporidiosis in germ-free lambs. Journal of Comparative Pathology 94, 141–152. Sreter, T. and Varga, I. (2000). Cryptosporidiosis in birds—a review. Veterinary Parasitology 87, 261–279.

154


Sreter, T., Egyed, Z., Szell, Z., Kovacs, G., Nikolausz, M., Marialigeti, K. and Varga, I. (2000). Morphologic, host specificity, and genetic characterization of a European Cryptosporidium andersoni isolate. Journal of Parasitology 86, 1244–1249. Stantic-Pavlinic, M., Xiao, L., Glaberman, S., Lal, A.A., Orazen, T., RatajVerglez, A., Logar, J. and Berce, I. (2003). Cryptosporidiosis associated with animal contacts. Wien Klin Wochenschrift 115, 125–127. Staunton, J. and Weissman, K.J. (2001). Polyketide biosynthesis: a millennium review. Natural Product Reports 18, 380–416. Sterling, C.S. (2000). Cryptosporidiosis: the treatment dilemma. Journal of Medical Microbiology 49, 2007–2008. Striepen, B. and Kissinger, J.C. (2004). Genomics meets transgenics in search of the elusive Cryptosporidium drug target. Trends in Parasitology 20, 355–358. Striepen, B., Pruijssers, A.J., Huang, J., Li, C., Gubbels, M.J., Umejiego, N.N., Hedstrom, L. and Kissinger, J.C. (2004). Gene transfer in the evolution of parasite nucleotide biosynthesis. Proceedings of the National Academy of Sciences of the United States of America 101, 3154–3159. Striepen, B., White, M.W., Li, C., Guerini, M.N., Malik, S.B., Logsdon, J.M., Jr., Liu, C. and Abrahamsen, M.S. (2002). Genetic complementation in apicomplexan parasites. Proceedings of the National Academy of Sciences of the United States of America 99, 6304–6309. Strong, W.B., Gut, J. and Nelson, R.G. (2000). Cloning and sequence analysis of a highly polymorphic Cryptosporidium parvum gene encoding a 60-kilodalton glycoprotein and characterization of its 15- and 45kilodalton zoite surface antigen products. Infection and Immunity 68, 4117–4134. Sulaiman, I.M., Xiao, L. and Lal, A.A. (1999). Evaluation of Cryptosporidium parvum genotyping techniques. Applied Environmental Microbiology 65, 4431–4435. Sulaiman, I.M., Xiao, L., Yang, C., Escalante, L., Moore, A., Beard, C.B., Arrowood, M.J. and Lal, A.A. (1998). Differentiating human from animal isolates of Cryptosporidium parvum. Emerging Infectious Diseases 4, 681–685. Suzuki, T., Hashimoto, T., Yabu, Y., Kido, Y., Sakamoto, K., Nihei, C., Hato, M., Suzuki, S., Amano, Y., Nagai, K., Hosokawa, T., Minagawa, N., Ohta, N. and Kita, K. (2004). Direct evidence for cyanide-insensitive quinol oxidase (alternative oxidase) in apicomplexan parasite Cryptosporidium parvum: phylogenetic and therapeutic implications. Biochemical and Biophysical Research Communications 313, 1044–1052. Tacal, J.V., Jr., Sobieh, M. and el-Ahraf, A. (1987). Cryptosporidium in market pigs in southern California, USA. Veterinary Record 120, 615–616.


155

Tanriverdi, S., Tanyeli, A., Baslamish, F., Koksal, F., Kilinc, Y., Feng, X., Batzer, G., Tzipori, S. and Widmer, G. (2002). Detection and genotyping of oocysts of Cryptosporidium parvum by real-time PCR and melting curve analysis. Journal of Clinical Microbiology 40, 3237–3244. Templeton, T.J., Iyer, L.M., Vivek Anantharaman, V., Enomoto, S., Abrahante, J.E., Subramanian, G.M., Hoffman, S.L., Abrahamsen, M.S. and Aravind, L. (2004). Comparative analysis of apicomplexa and genomic diversity in eukaryotes. Genome Research 14, 1686–1695. Tenter, A.M., Barta, J.R., Beveridge, I., Duszynski, D.W., Mehlhorn, H., Morrison, D.A., Thompson, R.C.A. and Conrad, P.A. (2002). The conceptual basis for a new classification of the coccidia. International Journal for Parasitology 32, 595–616. Theodos, C.M. (1998). Innate and cell-mediated immune responses to Cryptosporidium parvum. Advances in Parasitology 40, 121–149. Theodos, C.M., Griffiths, J.K., D’Onfro, J., Fairfield, A. and Tzipori, S. (1998). Efficacy of nitazoxanide against Cryptosporidium parvum in cell culture and in animal models. Antimicrobial Agents and Chemotherapy 42, 1959–1965. Thompson, R.C.A. (2002). Presidential address: rediscovering parasites using molecular tools- towards revising the taxonomy of Echinococcus, Giardia and Cryptosporidium. International Journal for Parasitology 32, 493–496. Thompson, R.C.A. (2003a). Molecular epidemiology of Giardia and Cryptosporidium infections. Journal of Parasitology 89, S134–S140. Thompson, R.C.A. (2003b). The zoonotic potential of Cryptosporidium. In: Cryptosporidium from Molecules to Disease (R.C.A. Thompson, A. Armson and U.M. Morgan-Ryan, eds), pp. 113–119. Amsterdam: Elsevier. Traub, R.J., Robertson, I.D., Irwin, P., Mencke, N. and Thompson, R.C.A. (2002). The role of dogs in transmission of gastrointestinal parasites in a remote tea-growing community in northeast India. American Journal of Tropical Medicine and Hygiene 67, 539–545. Tzipori, S. and Ward, H. (2002). Cryptosporidiosis: biology, pathogenesis and disease. Microbes and Infection 4, 1047–1058. Tzipori, S., Angus, K.W., Gray, E.W., Campbell, I. and Allan, F. (1981a). Diarrhoea in lambs experimentally infected with Cryptosporidium isolated from calves. American Journal of Veterinary Research 42, 1400–1404. Tzipori, S., Larsen, J., Smith, M. and Leuff, R. (1982). Diarrhoea in goat kids attributed to Cryptosporidium infection. Veterinary Record 111, 35–36. Tzipori, S., McCartney, E., Lawson, G.H.K. and Rowland, A.C. (1981b). Experimental infection of piglets with Cryptosporidium. Research in Veterinary Science 31, 358–368.

156


Tzipori, S., Rand, W., Griffiths, J., Widmer, G. and Crabb, J. (1994). Evaluation of an animal model system for Cryptosporidiosis: therapeutic efficacy of paromomycin and hyperimmune bovine colostrumimmunoglobulin. Clinical and Diagnostic Laboratory Immunology 1, 450–463. Tzipori, S., Robertson, D. and Cooper, D. (1987). Chronic cryptosporidial diarrhoea and hyperimmune cow colostrum. Lancet II 2(8554), 344–345. Tzipori, S., Smith, M., Halpin, C., Angus, K.W., Sherwood, D. and Campbell, I. (1983). Experimental cryptosporidiosis in calves: clinical manifestation and pathological findings. Veterinary Record 112, 116–120. Uip, D.E., Lima, A.L., Amato, V.S., Boulos, M., Neto, V.A. and Bem, D.D. (1998). Roxithromycin treatment for diarrhoea caused by Cryptosporidium spp. in patients with AIDS. The Journal of Antimicrobial Chemotherapy 41, Supplement B, 93–97. Upton, S.J. (1997). In vitro cultivation. In: Cryptosporidiosis of Humans and Animals (R. Fayer, ed.), pp. 181–207. FL: CRC Press. Upton, S.J., McAllister, C.J., Freed, P.S. and Barnard, S.M. (1989). Cryptosporidium spp. in wild and captive reptiles. Journal of Wildlife Diseases 25, 20–30. Villacorta, I., Graaf, D., Charlier, G. and Peeters, J.E. (1996). Complete development of Cryptosporidium parvum in MDBK cells. FEMS Microbiological Letters 142, 129–132. Villacorta, I., Peeters, J.E., Vanopdenbosch, E., Ares-Mazas, E. and Theys, H. (1991). Efficacy of halofuginone lactate against Cryptosporidium parvum in calves. Antimicrobial Agents and Chemotherapy 35, 283–287. Viu, M., Quilez, J., Sanchez-Acedo, C., Del Cacho, E. and Lopez-Bernad, F. (2002). Field trial on the therapeutic efficacy of paromomycin on natural Cryptosporidium parvum infections in lambs. Veterinary Parasitology 90, 163–170. Ward, H. and Cevallos, A.M. (1998). Cryptosporidium: molecular basis of host-parasite interaction. Advances in Parasitology 40, 151–185. Waters, W.R., Harp, J.A., Wannemuehler, M.J., Carbajal, N.Y. and Casas, I.A. (1999). Effects of Lactobacillus reuteri on Cryptosporidium parvum infection of gnotobiotic TCR-alpha-deficient mice. Journal Eukaryotic Microbiology 46, 60S–61S. Watwood, S., Janovy, Jr., Peterson, E. and Addison, M.A. (1997). Gregarina tribolium (Eugregarindae) n.sp. from Tribolium confusum and resolution of the confused taxonomic history of Gregarina minuta Ishii, 1914. Journal of Parasitology 83, 502–507. Widmer, G., Feng, X. and Tanriverdi, S. (2004). Genotyping of Cryptosporidium parvum with microsatellite markers. Methods Molecular Biology 268, 177–187.


157

Woodmansee, D.B. (1986). Isolation, in vitro excystation, and in vitro development of Cryptosporidium sp. from calves. Ph.D. Dissertation. Iowa State University. Woodmansee, D.B. and Pohlenz, J.F. (1983). Development of Cryptosporidium sp. in a human rectal tumour cell line. Proceedings of the 4th International Symposium on Neonatal Diarrhoea. University of Saskatchewan, Canada. Xiao, L. and Herd, R.P. (1994). Review of equine Cryptosporidium infection. Equine Veterinary Journal 26, 9–13. Xiao, L., Bern, C., Limor, J., Sulaiman, I., Roberts, J., Checkley, W., Cabrera, L., Gilman, R.H. and Lal, A.A. (2001). Identification of 5 types of Cryptosporidium parasites in children in Lima, peru. Journal of Infectious Diseases 183, 492–497. Xiao, L., Fayer, R., Ryan, U. and Upton, S.J. (2004). Cryptosporidium Taxonomy: recent advances and implications for public health. Clinical Microbiology Reviews 17, 72–97. Xu, P., Widmer, G., Wang, Y., Ozaki, L.S., Alves, J.M., Serrano, M.G., Puiu, D., Manque, P., Akiyoshi, D., Mackey, A.J., Pearson, W.R., Dear, P.H., Bankier, A.T., Peterson, D.L., Abrahamsen, M.S., Kapur, V., Tzipori, S. and Buck, G.A. (2004). The genome of Cryptosporidium hominis. Nature 431, 1107–1112. Yagita, K., Izumiyama, S., Tachibana, H., Masuda, G., Iseki, M., Furuya, K., Kameoka, Y., Kuroki, T., Itagaki, T. and Endo, T. (2001). Molecular characterization of Cryptosporidium isolates obtained from human and bovine infections in Japan. Parasitology Research 87, 950–955. Yang, S., Healey, M.C., Du, C. and Zhang, J. (1996). Complete development of Cryptosporidium parvum in bovine fallopian tube epithelial cells. Infectious Immunology 64, 349. Zhu, G. (2004). Current progress in the fatty acid metabolism in Cryptosporidium parvum. Journal of Eukaryotic Microbiology 51, 381–388. Zhu, G. and Abrahamsen, M.S. (2004). Cryptosporidium parvum genomics: impact on research and control. In: World Class Parasites: The Pathogenic Enteric Protozoa: Giardia, Entamoeba, Cryptosporidium and Cyclospora (C.R. Sterling and R.D. Adam, eds), Vol. 8, pp. 153–163. Kluwer: Norwell, MA. Zhu, G. and Keithly, J.S. (2002). Alpha-proteobacterial relationship of apicomplexan lactate and malate dehydrogenases. Journal of Eukaryotic Microbiology 49, 255–261. Zhu, G., Keithly, J.S. and Philippe, H. (2000a). What is the phylogenetic position of Cryptosporidium? International Journal of Systematic and Evolutionary Microbiology 50, 1673–1681.

158


Zhu, G., LaGier, M.J., Stejskal, F., Millership, J.J., Cai, X. and Keithly, J.S. (2002). Cryptosporidium parvum: the first protist known to encode a putative polyketide synthase. Gene 298, 79–89. Zhu, G., Li, Y., Cai, X., Millership, J.J., Marchewka, M.J. and Keithly, J.S. (2004). Expression and functional characterization of a giant Type I fatty acid synthase (CpFAS1) gene from Cryptosporidium parvum. Molecular and Biochemical Parasitology 134, 127–135. Zhu, G., Marchewka, M.J. and Keithly, J.S. (2000b). Cryptosporidium parvum appears to lack a plastid genome. Microbiology 146, 315–321. Zhu, G., Marchewka, M.J., Woods, K.M., Upton, S.J. and Keithly, J.S. (2000c). Molecular analysis of a Type I fatty acid synthase in Cryptosporidium parvum. Molecular and Biochemical Parasitology 105, 253–260.

Plate 2.3 Cryptosporidium parvum life cycle in host cell-free culture (after Hijjawi et al., 2004).

Plate 2.5 Light photomicrograph of HCT-8 cell monolayer after 24 h of infection with C. parvum (cattle genotype). Circular structures are the trophozoite stage.

Plate 2.6 Nomarski interference – contrast photomicrographs of HCT-8 cell monolayer 72 h post-infection with C. parvum.

Plate 2.7 Location of Cryptosporidium stages within host cells are always superficial as shown by light microscopy in HCT-8 cells infected with C. parvum and at the ultrastructural level.

Plate 2.8 Pairing of Cryptosporidium stages at different stages of maturity and between different developmental stages.

Plate 2.9 Multiple pairing (syzygy) in sporozoites of C. parvum 24 h after excystation from oocysts in cell-free culture.

Plate 2.10 Novel, multinucleated structures formed upon the fusion of sporozoites of C. parvum.

Plate 4.3 Histological section of developing Paragordius varius males and females within the definitive host Gryllus firmus, 25 days post-exposure.

Ichthyophthirius multifiliis Fouquet and Ichthyophthiriosis in Freshwater Teleosts R.A. Matthews School of Biological Sciences, University of Plymouth, Drake Circus, Plymouth PL4 8AA, UK

Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Importance and Global Distribution . . . . . . . . . . 1.2. Life Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Historical Background and Taxonomy . . . . . . . . 2. Free-Living Stages . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Tomont . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Theront . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Host Finding and Contact . . . . . . . . . . . . . . . . . 3. Infection of the Fish Host . . . . . . . . . . . . . . . . . . . . . 3.1. Invasion of the Fish Epidermis . . . . . . . . . . . . . 3.2. Site of Infection . . . . . . . . . . . . . . . . . . . . . . . . 4. Trophont . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Structure and Function . . . . . . . . . . . . . . . . . . . 4.2. Growth and Development . . . . . . . . . . . . . . . . . 4.3. Feeding and Digestion . . . . . . . . . . . . . . . . . . . 4.4. Reproduction within the Fish Host . . . . . . . . . . . 4.5. Exit from the Fish Host. . . . . . . . . . . . . . . . . . . 4.6. Culture in vitro and Cryopreservation. . . . . . . . . 5. Immunity to Ichthyophthirius multifiliis . . . . . . . . . . . . 5.1. i-Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Protective Immunity . . . . . . . . . . . . . . . . . . . . . 5.3. Parasite Evasion of the Host Immune Response

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ABSTRACT The ciliate Ichthyophthirius multifiliis is an important pathogen of freshwater teleosts occurring in both temperate and tropical regions throughout the world. The disease, ichthyophthiriosis, accounts for significant economic losses to the aquaculture industry, including the ornamental fish trade, and epizootics in wild fish populations can result in mass kills. This review attempts to provide a comprehensive overview of the biology of the parasite, covering the free-living and parasitic stages in the life cycle, host–parasite interactions, and the immune response of host and immune evasion strategies by the parasite. Emphasis on the immunological aspects of infection within the fish host, including molecular studies of i-antigens, reflects the current interest in this subject area and the quest to develop a recombinant vaccine against the disease. The current status of methods for the control of ichthyophthiriosis is discussed, together with new approaches in combating this important disease.

1. INTRODUCTION 1.1. Importance and Global Distribution The ciliate Ichthyophthirius multifiliis Fouquet, 1876, commonly called ‘ich’ (pronounced ‘ik’), is probably the most widespread parasite of freshwater teleosts with a geographical range extending from the tropics to temperate regions, northwards in Europe to the Arctic Circle. The first records of the characteristic ‘white spots’ in fish, which mark the location of the parasite in the skin (Figure 1), were from China before AD 1126

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Figure 1 Caudal fin of juvenile carp with 6-day-old trophonts of Ichthyophthirius multifiliis. Note two trophonts within a single chamber (arrowed) and infiltration of host cells into surrounding tissue.

(Dashu and Lien-Siang, 1960). These observations support the view that I. multifiliis was originally endemic in the Far East, being introduced to Europe in the Middle Ages with the development of carp culture (Hoffman, 1970b) and to many other countries, including the United States, through the importation of goldfish, Carassius auratus (see Hoffman, 1970a, 1978). The parasite was probably introduced to South Africa in the 18th century with importation of carp (Huchzermeyer, 1994;

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Mouton et al., 2001), this fish now being established in temperate waters of the continent (De Moor and Bruton, 1988). Paperna (1972, 1991) has reviewed the occurrence of I. multifiliis in warmer regions of the world. Key factors in the current worldwide distribution of I. multifiliis in freshwater teleosts, including cold water and tropical species, are wide temperature tolerance together with low degree of host specificity and direct life cycle. In the Far East, I. multifiliis is well adapted to survive the seasonal changes in temperature (4 to 281C) experienced by its common host, Cyprinus carpio. Nigrelli et al. (1976) considered the possibility of multiple physiological races and even different species of Ichthyophthirius, adapted to different temperatures. Two new species of ‘ich’-like parasites have been described, namely, Ichthyophthirioides browni Roque and de Puytorac, 1968 and Neoichthyophthirius scholtfeldti Bauer and Yunchis, 2001 from tropical fish; however, strains or races of I. multifiliis based upon physiochemical characters have yet to be defined. Dickerson et al. (1993) have demonstrated serotypic variation among isolates of the parasite, which they suggested could find application as biochemical markers in strain identification and in epidemiological studies of the parasite. Recent studies by Aihua and Buchmann (2001) on the development of a Nordic isolate of I. multifiliis provided a valuable approach to defining strains, based on physiological features of the free-living stages in the life cycle. The disease ichthyophthiriosis, or ‘white spot’, probably accounts for more damage to freshwater fish populations worldwide than any other eukaryote pathogen (Hines and Spira, 1973a; Rogers and Gaines, 1975). Aquarists have been aware of the condition in ornamental species since the turn of the century, associating mortalities with the appearance of the white spots within the skin and gills. Today, ichthyophthiriosis is a significant factor not only in the freshwater ornamental industry but also in the intensive farming of salmonids (Valtonen and Kera¨nen, 1981; Wahli and Meier, 1987), carp (Hines and Spira, 1973a; Ko¨rting, 1984), channel catfish (Klesius and Rogers, 1995), eels (Egusa et al., 1970), and Tilapia species (Subasinghe and Sommerville, 1986). Although epizootics have been less frequently observed in wild fish populations, they are usually associated with mass kills (Elser, 1955; Allison and Kelly, 1963; Kozel,


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1976; Wurtsbaugh and Tapia, 1988) following recent introduction (Hoffman, 1970b). Nevertheless, that the disease can have a significant impact on commercial fisheries is indicated by a more recent outbreak in pre-spawning and spawning sockeye salmon, when an estimated 153.6 million fewer fry were produced as a result of ichthyophthiriosis (Traxler et al., 1998). I. multifiliis has been used as a model in fundamental studies of fish behaviour and fish immunology. Milinski and Bakker (1990) used the parasite to investigate the function of secondary sexual ornamentation in teleosts. They found that infections influenced female choice of partner in stickleback populations, the white spots associated with the parasite in the epidermis reducing the intensity of male red breeding coloration. Dickerson et al. (1997) and Dickerson and Clark (1998) have discussed the development of I. multifiliis as an experimental system for the investigation of cutaneous immunity in fish.

1.2. Life Cycle MacLennan (1935a) and Butcher (1943) provided interesting accounts of the early studies on the life cycle of I. multifiliis, later work on the subject being reviewed by Matthews, R.A. (1994) and Dickerson and Dawe (1995). I. multifiliis is an endoparasite in which the trophont, or feeding stage, occurs within the epidermis of the fish host. The life cycle is direct (Figure 2). The trophont, following a period of growth and development, transforms to the tomont, which actively leaves the host tissues, encysting within the aquatic environment. The tomont undergoes a rapid phase of division, normally within the cyst, with the production of daughter cells called tomites. Following a set number of divisions each tomite differentiates into a theront, the stage infective to the fish host.

1.3. Historical Background and Taxonomy According to Fouquet (1876), Hilgendorf and Paulicki were probably the first to publish (in 1869) details concerning both the morphology

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tion? n juga ductio con repro

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Figure 2

Main events in the life cycle of Ichthyophthirius multifiliis.

and life history of I. multifiliis, noting somatic cilia, macronucleus, contractile vacuoles and granules, and that the trophont stage leaves the fish host to encyst and undergo reproduction as a free-living stage. Although they had suggested that the ciliate might be related to Opantotricha Ehrenberg, Fouquet (1876) proposed the name


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I. multifiliis for this parasite, the specific name relating to the large number of daughter cells (tomites) produced at encystment. Fouquet (1876) described the parasite from juvenile trout, noting further details of morphology including mucocysts and cytopharynx. The oral ciliature, as described by Roque et al. (1967), forms the basis for the inclusion of I. multifiliis within the class Oligohymenophorea. The relationship of I. multifiliis to other histophagous parasites of fish within the Hymenostomatida is shown in Figure 3. Canella (1964) established the suborder Ophryoglenina to include all species with an

Figure 3 Phylogenetic relationship of Ichthyophthirius multifiliis among the major lineages of the Ciliophora derived from 18S rRNA gene sequences. (Reproduced and modified with permission from Wright and Corlorni, 2002.)

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organelle of Lieberku¨hn, this structure being recognized as an important taxonomic feature. Lynn et al. (1991) confirmed the suborder as a monophyletic taxon following ultrastructural investigations of the organelle within the tomont of Ophryoglena sp. and theront of I. multifiliis. Separation of Ophryoglenina from Tetrahymenina is clearly supported by studies of ciliate phylogeny based upon the characterization of the histone H3/H4 gene region (Van den Bussche et al., 2000) and similarities in small subunit rRNA gene sequences (Wright and Lynn, 1995, 1997; Hammerschmidt et al., 1996). The family Ichthyophthiriidae Kent, 1881 was erected solely for I. multifiliis; the status of Ichthyophthirioides browni has still to be confirmed, as there has been no further record of the species since its description by Roque and de Puytorac (1968). Cryptocaryon irritans, the cause of white spot in marine fish, is no longer included within Hymenostomatidae (Diggles and Adlard, 1995; Wright and Colorni, 2002). Similarities with Ichthyophthirius multifiliis concerning life cycle and course of infection are attributed to parallel evolution. Recently, Wright and Colorni (2002) sequenced the complete 18S rRNA gene of C. irritans and on the basis of phylogenetic analysis have re-assigned this parasite to the class Protostomatea, erecting a new family, Cryptocaryonidae, within the order Prorodontida.

2. FREE-LIVING STAGES 2.1. Tomont 2.1.1. Behaviour and encystment The tomont culminates the growth phase within the fish host (Figure 2), attaining a size and stage of development sufficient to complete the aquatic phase, including location of substrate, encystment and production of viable theronts (Ewing and Kocan, 1992). This free-swimming stage encysts within 15 min to 6 h of leaving the host fish epidermis (MacLennan, 1937). Nickell and Ewing (1989) demonstrated a marked photoresponse to substrate colour, significantly larger


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numbers encysting on light surfaces than on dark. They suggested that such behaviour might be of survival value as the greyishcoloured cyst would be less obvious to predators against a lighter background. Attraction to light-reflecting objects, however, might also be advantageous in providing distinct targets for encystment, reducing unnecessary searching and entry into dark areas of deeper water. The cyst wall, secreted by mucocysts, is composed of two layers, an inner homogeneous proteinaceous layer and a less dense outer layer (MacLennan, 1937; Ewing et al., 1983). It is capable of adhering to a wide range of substrates, including plants and snail shells (Wagner, 1960) and filter paper (Ewing et al., 1983); the surface may become overlaid with debris and bacteria. Although tomont division normally occurs following the secretion of the cystogenous material (MacLennan, 1937), the successful production of viable theronts is not subsequently dependent upon the integrity of the cyst wall (Ewing et al., 1983; Aihua and Buchmann, 2001). In the wild, however, it is likely that free tomites would be dispersed in water currents, falling prey to micropredators before transformation to the theront. The theront is unable to swim against even slow water currents (MacLennan, 1935a). The successful exploitation of fish hosts in riparian systems must therefore be attributed to the adhesive properties of the cyst and to the retention of developmental stages up to theront production within the immediate vicinity of likely fish hosts, probably in shallow waters.

2.1.2. Reproduction Reproduction of I. multifiliis within the aquatic environment is by palitomy (Lynn and Corliss, 1991). Following encystment the tomont undergoes a rapid sequence of binary fission, with no intervening growth, culminating in the production of large numbers of tomites. The plane of cleavage at cell division is transverse, perkinetal. According to Uspenskaja and Ovchinnikova (1966), DNA synthesis stops at the 8–16 tomite stage and from then on there is equal distribution of the genomes among the daughters. MacLennan (1935b, 1936) recorded

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a degree of dedifferentiation of some organelles, including the neuromotor system; however, at no stage are somatic cilia lost. Ewing et al. (1986) have undertaken a comprehensive study of tomont development, relating this to period of residency within host, size, and temperature; tomonts were recovered from channel catfish held at 21 and 241C. A period of 3 days within the host was considered critical for maximum survival (97–100%) of the tomonts leaving the fish and producing theronts. By this stage tomonts would have attained diameters of 100–150 mm; a small number of tomonts originating from 2-day infections and tomonts as small as 55 mm were also capable of limited theront production. The reproductive potential of tomonts was also influenced by the duration of trophont residency and temperature during the infection (Ewing et al., 1986). In general, longer periods within the host at higher temperatures resulted in larger tomonts, which produced greater numbers of theronts. Aihua and Buchmann (2001) investigated the effect of environmental factors, including temperature and salinity, on tomont development following their collection from a Danish isolate of I. multifiliis experimentally maintained at 11.61C in rainbow trout. In this study, tomonts were incubated at a range of temperatures from 5 to 301C, and time from encystment to theront release was recorded together with the number of theronts produced and the theront size. The number of theronts recovered from one cyst varied significantly with temperature, from 329 at 51C to more than 642 at 171C and then declined to 458 at 251C. The number of theronts/tomonts recorded by previous workers ranged from 64 (MacLennan, 1937) to 1000 (Hoffman, 1970a), with a possible upper limit of 3000 from over-wintering trophonts (Wagner, 1960). Such variations are not surprising in view of the multiple factors influencing tomont reproduction (Ewing et al., 1986; Aihua and Buchmann, 2001).

2.1.3. Survival conditions The free-living stages of I. multifiliis (Figure 2) are essentially adapted for survival in freshwater, development being inhibited at


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concentrations 45 p.p.m. NaCl (Aihua and Buchmann, 2001). Water conditions described by Dickerson et al. (1984) for the long-term maintenance of this parasite in channel catfish held in aquaria included total hardness of 28 p.p.m. as CaCO3, alkalinity of 30 p.p.m. as CaCO3, and pH 8. Ewing et al. (1991) demonstrated that tomonts reproduced successfully when deprived of extracellular calcium and successfully produced theronts. The viability of the latter, however, was significantly reduced at calcium concentrations below 0.81 mM Ca. Doubling the magnesium concentration to increase water hardness did not improve viability in the absence of calcium. According to Wagner (1960), I. multifiliis can survive within a pH range of 5.5–10.1 and requires oxygen levels of at least 0.6–0.8 mg/L at 151C to complete development within the aquatic environment. The tomont survives and reproduces within a wide temperature range (Suzuki, 1935; Wagner, 1960; Bauer et al., 1973; Aihua and Buchmann, 2001). Aihua and Buchmann (2001) usefully brought together previous studies and provided a detailed profile of development for the Nordic isolate; development time decreased inversely with temperature ranging from 9 days at 51C to 18 h at 25–301C. Although some reproduction can occur at temperatures as low as 31C (Wagner, 1960; Bauer et al., 1973), this does not proceed to the production of viable theronts, tomites dying within 8 days (Nigrelli et al., 1976). The tomonts would not normally have left the host under such conditions, as transmission is arrested in locations where water temperatures fall below 51C.

2.2. Theront Theronts are short-lived and must locate a fish host without delay. McCallum (1982) recorded a significant decline in viability after 12 h at 201C with a life expectancy of 22.5 h, the latter agreeing with earlier studies by Suzuki (1935). Isolates investigated by MacLennan (1935a) and Wagner (1960) survived for up to 96 h at similar temperatures. According to Bauer (1958), survival at higher temperatures up to 281C is no more than 10 h and Van Duijn (1967) considered 301C

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to be lethal to the theront; strain variability could account for differences in both viability and longevity of the free-living stages. Reduced life span under conditions of continuous light recorded by Bauer (1958) probably resulted from increased activity and more rapid depletion of energy reserves. The precise mechanism involved in theront emergence from the cyst is unknown. It seems probable, however, that the highly active theronts are capable of swimming through the cyst wall (MacLennan, 1937), the first few to penetrate it leaving escape routes for the remainder (Dickerson and Dawe, 1995). Ewing et al. (1983) noted a significant reduction in cyst wall thickness by the time of theront emergence. The free-swimming theront is cylindrical in shape (Figure 4) with slightly dilated anterior and posterior regions, the former terminating in the perforatorium (Roque et al., 1967; Canella and Rocchi-Canella, 1976). The length of the theront is generally within a range of 30–50 mm, but this varies depending on the size of the tomont (MacLennan, 1942; Canella and Rocchi-Canella, 1976) and temperature (Aihua and Buchmann, 2001). Aihua and Buchmann (2001) recorded lengths of 28.64–57.42 mm in theronts, following inactivation with a drop of 4% phosphate-buffered formaldehyde. These workers also noted that theront size was inversely related to the temperature at which the tomont developed, with mean lengths of 28.64 mm at 301C and 57.42 mm at 51C. Structural features of the theront are summarized in Figure 4. Distinctive features include somatic cilia arranged in longitudinal diagonal rows originating from the troughs of strongly developed pellicular ridges (Canella and Rocchi-Canella, 1976; Crilley and Buckelew, 1977); trailing cilium, 20 mm in length, arising obtusely at the posterior end (Canella and Rocchi-Canella, 1976; McCartney et al., 1985; Geisslinger, 1987); and high density of mucocysts in the anterior region. Lynn et al. (1991) provided a detailed description of the oral cavity, including the organelle of Lieberku¨hn and somatic and the oral kineties. They confirmed the organelle of Lieberku¨hn as a synapomorph for the Ophryoglenina. Its function, however, remains unclear although it is probably connected with the phototropic response of the theront in the aquatic environment. Further ultrastructural details are given in works relating to the


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p

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Figure 4 Theront stage of Ichthyophthirius multifiliis: (a) Diagrammatic representation and (b) horizontal section; note densely packed mucocysts in anterior region of body. Abbreviations: c, cilia; cc, caudal cilium; cp, cytoproct; cs, cytostome; cv, contractile vacuole; ma, macronucleus; mc, mucocyst; mcs, densely packed mucocysts within anterior region of body; mi, micronucleus; mt, mitochondrion; oc, oral cavity; ol, organelle of Lieberku¨hn; p, perforatorium; r, refractile granules; s, spongiome of excretory system; sv, small vacuole. (Diagram (a) reproduced and modified from Matthews, R.A., 1994, with permission from Samara Publishing Ltd.)

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infection of fish hosts (Ewing et al., 1985; Ewing and Kocan, 1992; Matthews, R.A., 1994).

2.3. Host Finding and Contact Entry to the host is initially in response to environmental factors, in particular light as theronts show positive phototaxis (Lom and Cˇerkasovova´, 1974; Wahli et al., 1991). Wahli et al. (1991) demonstrated that the attraction was not initially towards the host; when given a choice between an illuminated and darkened region of an aquarium, a significant number of theronts was located within the former within 45–70 min, whereas the distribution was unchanged after introduction of a fish to the darkened area. The theront is unable to detect hosts by long-range chemotaxis (Lom and Cˇerkasovova´, 1974; Haas et al., 1998, 1999). Indeed, little would be gained by I. multifiliis by such a strategy in view of the relative swimming speeds of host and parasite in addition to the existence of water currents, even when fish are at rest. Such a positive response to light, however, would ensure the parasite’s upward movement to the surface, increasing opportunities for host contact. It is possible that fish are at greatest risk of infection on rising to the surface to take food, as Hines and Spira (1973a) recorded significantly larger numbers of trophonts within the dorsal skin of carp. Matthews, R.A. (1994) noted that skin penetration was usually preceded by a bout of fast swimming activity within the immediate vicinity of the host surface. At such close range the parasite responds to chemical signals unique to teleosts (Lom and Cˇerkasovova´, 1974; Buchmann and Nielsen, 1999; Haas et al., 1999). Haas et al. (1999) described 12 types of behaviour in free-swimming theronts, eight of these in response to fractions of a ‘carp skin surface mucus homogenate’. Buchmann and Nielsen (1999) confirmed the positive response of I. multifiliis to fish serum (Lom and Cˇerkasovova´, 1974) and mucus using a novel bioassay for the presentation of the potential chemoattractants. Partial purification of sera from Salmo trutta by gel filtration showed immunoglobulin and some undetermined


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proteins to be attractants. Low molecular weight molecules, however, were not attractive to theronts, although amino acids can affect swimming behaviour (Haas et al., 1999). The possible involvement of immunoglobulin is of interest as it would provide a unique signal for aquatic vertebrates and it is released at the host surface by cutaneous secretion (Xu and Klesius, 2003). Such an attractant molecule would explain the low degree of host specificity of I. multifiliis within teleosts (Buchmann and Nielsen, 1999) and the fact that infections have been established in the tadpoles of the striped marshfrog (Gleeson, 1999). Interestingly, Haas et al. (1999) noted weak but positive behavioural responses of theronts to mucus from the frog Rana esculenta, although not to mucus from invertebrates in the aquatic environment. Buchmann and Nielsen (1999) proposed mucus cells as candidate targets for theront invasion of the fish host on the bases that these contain serum proteins and intensities of I. multifiliis are greatest in regions of fish epidermis with the highest densities of goblet cells (Buchmann et al., 1999).

3. INFECTION OF THE FISH HOST 3.1. Invasion of the Fish Epidermis Although there have been many observations concerning the entry of I. multifiliis into the epidermis of host fish, there is little agreement concerning the mechanism involved (Matthews, R.A. 1994). MacLennan (1935b), in the first major work on the parasite, considered that the theront bored into the epidermis using ‘the non-ciliated anterior region’ as a wedge to force the tissues apart, noting that ‘the closely set cilia are more powerful and active than those of the average ciliate’. Roque et al. (1967) supported this view in describing the perforatorium as an organelle serving to prise apart host cells at their intercellular junctions. The perforatorium must play an important role in host entry, being the first point of contact with the fish epidermis (Matthews, R.A., 1994). Nigrelli et al. (1976) stated that entry was achieved by means of modified anterior cilia and a cup-like

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mouth, although there is no evidence in support of this view. A further suggestion proposed by Canella and Rocchi-Canella (1976) implicated longitudinal pellicular ridges as serving to rasp away surrounding tissues. The ridges, however, are angled so as to present a smooth slope towards the direction of rotation and, being partly occluded by the relatively dense somatic ciliature, are unlikely to be abrasive. Hines and Spira (1974a) suggested that invasion might be aided by the release of lytic enzymes, hyaluronidase activity being detected in theronts and theront water by Uspenskaja (1963). Unfortunately, the precise location of hyaluronidase within I. multifiliis was not determined by Uspenskaja (1963) and remains unknown. Nevertheless, that such enzymes may be involved in penetration of the fish epidermis by I. multifiliis gains some support from ultrastructural studies in which host cells adjacent to the invading theront showed evidence of lytic necrosis (Matthews, R.A. and Matthews, B.F., 1984; Matthews, R.A., 1994), although the damage could be attributed to other factors including osmotic imbalance following destruction of the surface coat by the perforatorium and ingress of water (Matthews, R.A., 1994). Buchmann et al. (1999) suggested that theronts enter the fish epidermis via the pores of goblet cells, having detected these by chemotaxy (Buchmann and Nielsen, 1999). Although host–parasite docking might occur at such locations, with insertion of the perforatorium into the pore, penetration of the epidermis is associated with considerable disruption and erosion of surface cells over a relatively wide area of the epidermis (Ewing et al., 1985; Matthews, R.A., 1994). Nevertheless, the targeting of mucous cells, and release of chemoattractants, is consistent with the aggregated patterns of theront invasion recorded in carp by Matthews, R.A. (1994). Xu et al. (2001) have demonstrated the presence of monosaccharides and amino acid derivatives on the surface of the theront with the aid of lectins. The binding of lentil agglutinin, gorse agglutinin and wheat germ agglutinin, but not soybean agglutinin, significantly reduced the ability of theronts to invade the host and reduced the development of trophonts. The discharge of the mucocysts during initial invasion of the epidermis has led to further speculative interpretations of the infection


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process (Matthews, R.A. and Matthews, B.F., 1984; Ewing et al., 1985; Ewing and Kocan, 1992). Matthews, R.A. and Matthews, B.F. (1984) speculated that these organelles could be the source of the hyaluronidase detected in theront water by Uspenskaja (1963). Ewing and Kocan (1992) suggested that mucocyst material might aid theront adhesion to the outer surface of the fish host. That these organelles secrete a sticky cyst wall in the free-living trophont would not be inconsistent with such a view. Such material might also aid in the disruption of host cells by swelling on contact with water. Such a mechanism has been proposed for the blood fluke, Schistosoma mansoni, in which hydroscopic materials released from cercarial preacetabular glands may assist skin penetration of mammalian hosts by separating the squales (McClaren, 1980). Although the function of mucocysts remains unclear, their importance in the infection process is not in doubt. Ewing et al. (1991) suggested that the reduced infectivity recorded in theronts deprived of calcium may have been associated with impaired exocytosis and that significantly fewer mucocysts were produced in these individuals compared to controls. Whether mucocysts are homologous in both theront and trophont is open to question, as they are involved in two distinct activities in the life cycle, namely entry to the fish host and encystment within the aquatic environment. Two types of morphologically identical mucocysts have been identified in the ciliate Balantidium coli, a parasite of pigs and humans, on the basis of the cytoenzymatic reaction of the membrane to b-glucuronidase (Skotarczak, 1999). The organelle of Lieberku¨hn is unlikely to be involved in the early invasion process as it was still intact in a significant number of trophonts up to 7.5 h after infection (Matthews, R.A., et al., 1996). The major role of this structure probably lies in the phototropic response of the theront in the aquatic environment and its disappearance shortly following invasion might therefore be expected. Nevertheless, the presence of strongly developed ridges facing the oral cavity led Lynn et al. (1991) to suggest an additional function in the acquisition and ingestion of host tissues, possibly in the young trophont.

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3.2. Site of Infection Trophonts occur within the epidermis (Chapman, 1984), adjacent to the basal lamina, throughout the body of the fish host, and also in the epithelia of gills and buccal and pharyngeal cavities; rarely in the nasal pits and circumorbital clefts (Ventura and Paperna, 1985). Invasions of subepidermal tissues (Bauer, 1958; Reichenbach-Klinke and Landolt, 1965; Ventura and Paperna, 1985) have previously been attributed to advanced stages of the disease when underlying tissues are exposed by erosion and peeling of the epithelium (Hines and Spira, 1994a). Nevertheless, the occurrence of trophonts within the peritoneal cavity of fish may be a normal event in the life cycle, having been recorded in wild sticklebacks by Hoffman (1967) and recently in juvenile channel catfish, following natural exposure to theronts via a standardized protocol, by Maki et al. (2001). Dickerson et al. (1985) have shown that the parasite can develop within the peritoneal cavity of fish following intraperitoneal injection of theronts. The parasites survived at least 20 days at 201C and produced viable theronts when surgically removed to water. How trophonts reach such a location is unknown; however, their presence there would be significant in stimulating protective immunity within the host. Hines and Spira (1973a) noted that skin trophonts were 3 times more prevalent in the dorsal region than elsewhere on the body of experimentally infected carp. This non-random distribution was attributed to this being the site at which theronts contacted the fish host rather than to regional differences in the nature of the epidermal layer. That theronts can invade the skin in any region of the body has been shown following experimental exposure of carp in the dark (Matthews, R.A., personal observation). The occurrence of trophonts in aggregated groups within the epidermis, numerous individuals sharing a common chamber or gallery, is well recognized, having been first recorded by Stiles (1893). That such aggregations can be attributed to theronts invading the epidermis at single sites was noted by Buschkiel (1910) and Canella and Rocchi-Canella (1976) and further confirmed by Matthews, R.A. (1994), in post-larval carp


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following their first exposure to the parasite. It might be significant that Buchmann et al. (1999) have demonstrated a positive correlation between trophont numbers and mucous cell density in primary infections of rainbow trout. Young trophonts are capable of significant migrations within the host epidermis (Matthews, R.A., personal observation). Ewing and Kocan (1986) noted a shift of trophonts within the gill epithelium during the 5-day infection period to positions close to the afferent blood vessel. In heavy infections, trophonts share common spaces as a result of host tissue damage, including undermining (Hass, 1933; MacLennan, 1935b; Buschkiel, 1936; Dogiel et al., 1961; Hoffman, 1978; Ewing and Kocan, 1986). The occurrence of isolated groups of closely packed trophonts within the epidermis may also result from trophont division (Stiles, 1893; Neresheimer, 1908; Suzuki, 1935; Reichenbach-Klinke and Landolt, 1965; Ermolenko, 1985; Ewing et al., 1988).

4. TROPHONT Transformation from theront to trophont is characterized by discharge of mucocysts, disappearance of the organelle of Lieberku¨hn, enlargement of the cytopharynx, and the onset of phagocytosis. These events are completed within 12 h in primary infections of juvenile carp at 201C (Matthews, R.A., et al., 1996).

4.1. Structure and Function 4.1.1. General features of morphology Trophonts removed from host tissue assume a spherical shape, measuring from 30 to 1000 mm in diameter, depending upon age of infection and stage of maturity (Wagner, 1960). Cilia are uniformly arranged over the surface, the kineties conforming to the general pattern for Hymenostomatida (MacLennan, 1935b; Rocque et al., 1967; Lom and Dykova´, 1992). Cytoplasmic inclusions visible by

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light microscopy include mucocysts, food vacuoles, contractile vacuoles, and the macronucleus and micronucleus (Figure 5). The macronucleus becomes greatly enlarged during the parasitic phase, changing from oval to the characteristic horseshoe shape; there is a predictable relationship between the amount of DNA in the ciliate macronucleus serving a physiologically active role and cell size (Shuter et al., 1983). The trophont has many contractile vacuoles located throughout the peripheral region of the body (Mosevitch, 1965). The location of the pores is predictable in relation to kinety in Tetrahymena (see Nanney, 1980) and appears to be fixed within species of ciliates, a feature exploited by taxonomists (Lynn and Corliss, 1991). Such features might prove useful in assessing the taxonomic status of isolates of I. multifiliis, including the putative Ichthyophthiroides browni.

Figure 5 Trophont of Ichthyophthirius multifiliis in the epidermis of juvenile carp. Abbreviations: bl, basal lamina; c, cavity within epidermis; cv, contractile vacuole; fv, food vacuole; ma, macronucleus. Note infolding of pellicle (arrowed).


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4.1.2. Cell structure and function The structural organization of ciliates is conveniently considered under three sections, namely cortex, endoplasm, and nucleoplasm. The relatively few studies undertaken on the nucleoplasm of I. multifiliis have been confined to the free-living stages (Peshkov and Tikhomirova, 1967, 1968b; Hauser, 1972, 1973; Hauser and Van Eys, 1973). (a) Somatic cortex. The somatic cortex of I. multifiliis (Figures 5 and 6) is structurally similar to that of other ciliates (Lynn and Corliss, 1991), with pellicle, ciliary structures, and secretory organelles. Mitochondria, endoplasmic reticulum, Golgi apparatus, and other cell inclusions extend into this outer region (Chapman and Kern, 1983; Lom and Dykova´, 1992; Lobo-da-Cunha and Azevedo, 1994), which is not clearly demarcated from the endoplasm. The pellicle is composed of plasma membrane with glycocalyx, membrane-bound alveolar sacs, and underlying epiplasm. Somatic kineties are meridionally arranged, each separated by an interkinetid ridge, and terminate anteriorly at either pre- or post-oral sutures. Additional kineties occur within the prebuccal region. The somatic kineties and also those within the prebuccal cavity are monokinetids, with single kinetosome, cilium, postciliary microtubular ribbon, and fibrillar associates; the arrangement of the supporting structures has been described by Chapman and Kern (1983). Chapman and Kern (1983) also recorded parasomal sacs at the bases of the cilia in I. multifiliis, although their function remains unknown. Parasomal sacs in Tetrahymena may play a role in nutrient uptake and have been shown in some species to endocytose medium (Lynn and Corliss, 1991). Bouck and Chen (1984) suggested that cilia have considerable control over their own surface composition and in this respect it would be interesting to know whether these structures in I. multifiliis have a similar function to the flagellar pocket of trypanosomes (Borst, 1991), including macromolecule uptake and the insertion of glycoproteins into the plasma membrane. It is possible that the electron-lucent alveoli of I. multifiliis are involved in the secretion of membrane proteins as Lobo-da-Cunha and Azevedo (1990a, 1994) detected thiamine pyrophosphates and

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Figure 6 Cortical region of Ichthyophthirius multifiliis: 5-day-old trophont in the epidermis of juvenile carp. Abbreviations: a, alveola; c, somatic cilium; cm, crystalline mucocyst; f, food vacuole; h, host tissue; l, lipid globule; m, mitochondrion; pl, primary lysosome; s, spongiome; x, xenosome.

nucleoside diphosphatase in them, suggesting a role in glycosyltransferase activities. Matthews, B.F., et al. (1993) have suggested a secretory role for the alveoli in the formation of the cyst wall in the marine ciliate C. irritans. In this species, the somatic alveoli of the


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trophont contained electron-dense material, disappearance of which coincided with encystment. The epiplasm in I. multifiliis is composed of a dense fibrous layer immediately below the alveoli. Cell shape in ciliates is largely attributed to this layer (Peck, 1977) and in I. multifiliis it forms the longitudinal interkinetid ridges, which maintain the characteristic topography of the species. Further support and flexibility are provided by microtubules, up to 24 per ridge being recorded in the trophont (Chapman and Kern, 1983). Whether these are continuations of the post-ciliary microtubular ribbons or homologous with the longitudinal microtubules of Tetrahymena has not been resolved (Chapman and Kern, 1983). The epiplasm appears to remain of uniform thickness throughout the parasite life cycle, a feature that probably accounts for the infolding of the body wall in large trophonts (Figure 5). Such plasticity in cell shape might be of advantage in dislodging host cells before engulfment, particularly when several parasites occupy the same host cavity. Mucocysts occur within the somatic cortex in all stages of the life cycle. These elongated, membrane-bound inclusions (3–4 mm 0.25 mm) are characterized by electron-dense contents with a paracrystalline core (Figure 6). Crystalline mucocysts transform into secretory mucocysts (Figure 7), the contents of which are uniformly electronlucent (Ewing et al., 1985). That the two regions may remain chemically distinct at all stages of development, although appearing morphologically the same, has been indicated in trichocysts of Paramecium by Hausmann et al. (1988) with the aid of indirect immunogold labelling techniques. Mucocyst discharge follows fusion of membrane at the apex with the plasma membrane (Small and Lynn, 1985). In I. multifiliis, mucocysts are secreted by the free-living tomont at encystment and by the theront during host penetration. The origin of mucocysts within the cytoplasm of I. multifiliis has yet to be resolved although secretory products in mammalian cells are processed and packaged in Golgi bodies. In the Golgi complex of I. multifiliis, dicytosomes are shown to be associated with modified cisterna of the endoplasmic reticulum surrounding mitochondria within the cortex (Lobo-da-Cunha and Azevedo, 1990a). Positive

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Figure 7 Ichthyophthirius multifiliis: mucocyst, mc, being secreted by theront during invasion of carp epidermis. Note location of parasite adjacent to basal lamina, bl.

acid phosphatase, thiamine pyrophosphatase, and nucleoside diphosphatase activities identified with thick-membrane cisternae with dilated coated rims provided evidence of a trans-Golgi network (Lobo-da-Cunha and Azevedo, 1994). Lobo-da-Cunha and Azevedo (1990b) confirmed the presence of the first two of these enzymes using cerium as a capture agent. Although in their earlier paper Lobo-daCunha and Azevedo (1990a) suggested that the alveolar sacs might also be included within the Golgi complex this was later repudiated when alkaline phosphatase activity was shown to be an artefact (Lobo-da-Cunha and Azevedo, 1994). The mitochondria with their distinctive tubular cristae are more abundant in the cortex than in the endoplasm. Mosevitch and Mashansky (1965) have described the genesis of these organelles throughout the life cycle. Lobo-daCunha and Azevedo (1993a) identified peroxisomes in I. multifilliis


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following cytochemical investigations and the detection of catalase activity. These organelles are most frequently recorded in association with the mitochondria. (b) Oral cortex. Roque et al. (1967) provided a detailed description of the oral cortex in the trophont of I. multifiliis. Kineties within the buccal cavity include a paroral membrane of four kinetosomes on the left and three kineties to the right, the second of these being composed of dikinetids with two kinetosomes and a single cilium (Lom and Dykova´, 1992). The paroral kinetids are vestigial and without cilia. 4.1.3. Excretory system The excretory system of I. multifiliis is characterized by large numbers of contractile vacuoles distributed throughout the cortex of the trophont. Chapman and Kern (1983) provided a detailed description of the system in this species and usefully brought together previous work (Mosevitch, 1965; Roque et al., 1967; Peshkov and Tikhomirova, 1968a). In general, organization of the excretory system resembles that of other ciliates, comprising the contractile vacuole, spongiome, collecting canals, a discharge canal and pore; the collection canals, however, have no ampullae and lead into the contractile vacuole in an irregular pattern. The term spongiome (Jurand and Selman, 1969) is used here as more representative of current views (Lynn and Corliss, 1991) in describing the cytoplasmic zone immediately surrounding the contractile vacuole. The cytoplasm of this region includes free ribosomes, mitochondria, vesicles, and tubules, the latter entering the collecting canals. The tubules and vesicles are considered to be specializations of the smooth endoplasmic reticulum (Lynn and Corliss, 1991). Chapman and Kern (1983) observed filamentous processes on the cytoplasmic surface of these, which they considered to resemble the permeability modulation coat described in Paramecium by McKanna (1976). The spongiome (Figures 4 and 6) is very well developed in I. multifiliis when compared to other ciliates (Lynn and Corliss, 1991) and varies in thickness from 1.0 to 2.0 mm in young trophonts (Chapman and Kern, 1983) and up to 10 times this in the

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parasite just before encystment (Mosevitch, 1965). Microtubules are present throughout the contractile vacuole system as supportive elements of the injection canals, contractile vacuole, and discharge canal. Chapman and Kern (1983) suggested that microtubular arrays adjacent to the contractile vacuole might represent sites of assembly for collecting canals. The mechanism of operation of contractile vacuoles, in particular how the injection canals are filled, remains unknown (Patterson, 1980; Allen and Fok, 1988; Lynn and Corliss, 1991). Allen and Fok (1988) suggested a role for the filamentous coat in preventing fluid re-entering the tubules as the injection canals approach diastole. Patterson (1980) has discussed possible functions of the contractile vacuole, concluding that it serves primarily to dispel fluid to the exterior of the cell. In I. multifiliis, Ewing and Kocan (1987) have suggested a further function in aiding trophont emergence from the host epithelium (see Section 4.5). 4.1.4. Endoplasm The endoplasm, as the main site of digestion, contains food vacuoles, primary lysosomes, peroxisomes, and lipid globules. Numerous food vacuoles in different stages of the digestive cycle are present in actively feeding trophonts of I. multifiliis (Figure 5). Lobo-da-Cunha and Azevedo (1993b) recognized three stages in the digestive cycle based upon structural and cytochemical observations on the fate of food vacuoles in tomonts, which contained food vacuoles already formed in the trophont before cessation of feeding when the parasite left the host. Recently formed vacuoles, containing undigested food including intact fish cells, and slightly older vacuoles with peripherally arranged dense breakdown products were classified as stage 1. Stage 2 vacuoles were highly dense in appearance, a feature attributed to condensation and loss of membrane. Up to this point the vacuoles have a capability to divide. Finally, in stage 3, the vacuole expands with membrane pulling away from the condensed central mass. Loboda-Cunha and Azevedo (1993b) suggested that this could be the result of fusion between condensed vacuoles and lysosomes. At the end of the cycle, the old vacuoles fuse among themselves before


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the expulsion of waste products at the ectoproct. Cytochemical investigations of food vacuoles by Lobo-da-Cunha and Azevedo (1988a) identified acid phosphatase and two isozymes of aryl sulphatase with activity at pH 4.5 and 6. These enzymes were also detected in primary lysosomes. Although food vacuoles received acid hydrolases during the first stage of the digestion cycle, primary lysosomes continued to fuse with condensed vacuoles. Xenosomes (Figure 6) resembling Gram-negative bacteria have been detected lying free within the cytoplasm of I. multifiliis by Roque et al. (1967) and Lobo-da-Cunha and Azevedo (1988b). Lobo-daCunha and Azevedo (1988b) considered these to be harmless endosymbionts, which might have a permanent relationship with some strains of I. multifiliis. In their isolate the bacteria reproduced, occurred within trophonts, dividing tomonts and theronts, and were transmitted through several generations without apparent effect on parasite viability. In the trophont stage the xenosomes were associated with aggregations of glycogen, suggesting that their feeding activity coincided with that of the ciliate.

4.2. Growth and Development Trophont size is unaffected by species of fish (Wagner, 1960) or location within the fish host (MacLennan, 1942). MacLennan (1942) undertook detailed studies of the growth of I. multifiliis using the speckled dace Apocope oscula carringtoni as an experimental host. Procedures were standardized with respect to isolate, infection, and measurement, trophonts being killed in 1% formalin and measured without distortion in a hanging drop preparation. Mean diameters were related to temperature and duration of the parasitic phase. Although trophonts left the host throughout the course of infection, only those that attained diameters 4100 mm were considered capable of encystment and of further development within the aquatic environment, a claim since confirmed by others (Hines and Spira, 1973a; Ewing et al., 1986). These mature trophonts were produced within 2.5 days at 221C and 2 days at 271C. Individuals at each day of

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infection varied considerably in diameter, those running the full course of infection of 7 days at 221C and 5 days at 271C having ranges of 314–691 and 282–550 mm, respectively. MacLennan (1942) attributed variations in the diameter of trophonts to initial size of theront; however, this could result from trophont division within the fish host (Matthews, R.A., et al., 1996). Nevertheless, overall measurements of I. multifiliis from different isolates and geographical regions (Wagner, 1960; Hines and Spira, 1973a; Ewing et al., 1986) fall well within the size prediction for the species given by MacLennan (1942). Hines and Spira (1973a) have commented upon the remarkable uniformity of this parasite with respect to size and growth rate. Low temperatures inhibit emergence from the host although the parasite might benefit from further growth, trophonts of up to 1 mm in diameter being recorded by Wagner (1960) in over-wintering carp at 2–51C. That parasite emergence can be delayed by reducing water temperature has been exploited in the long-term maintenance of laboratory isolates of I. multifiliis in channel catfish by Noe and Dickerson (1995). Trophonts remained within the host for 204 days at 91C compared to only 5–6 days at normal temperatures of 251C, with no apparent change in viability, antigenicity or infectivity.

4.3. Feeding and Digestion Feeding by I. multifiliis is associated with intense phagocytic activity (Figure 5). Matthews, R.A., et al. (1996) detected food vacuoles containing entire host cells within trophonts as early as 7.5 h after entering the host epidermis. The onset of phagocytosis was associated with theront transformation including cytopharyngeal enlargement and disappearance of the organelle of Lieberku¨hn. Little is known of the nutritional requirements of I. multifiliis or whether nutrients can be taken up directly through the pellicle. That Fouquet (1876) considered all nutrients to be absorbed through the surface of this parasite probably resulted from his failure to detect the oral cavity and cytostome. Transport of nutrients across the plasma membrane has been described in astomous mutants of Tetrahymena by Rasmussen


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and Orias (1975). Ekless and Matthews (1993) have shown increased survival of theronts and trophonts in selected monophasic media compared to controls maintained in water and balanced saline solutions, although the route of uptake was not confirmed and could equally have been via the food vacuoles. Tissue fluids would provide a rich source of nutrients for the trophont, particularly in the early phase of infection when the size of the cytopharynx might restrict the ingestion of particulate material. In free-living oligohymenophoreans the parabasal complex within the cytopharynx serves to filter bacteria from the water (Fenchel, 1986, 1987). This organelle is vestigeal in I. multifiliis; however, it might be significant that parasomal sacs have been suggested as a site of medium endocytosis in some species of Tetrahymena by Lynn and Corliss (1991). Lynn et al. (1991) suggested that strongly developed ridges in the oral cavity, associated with the organelle of Lieberku¨hn, might function in the acquisition and ingestion of host tissues, possibly by assisting the ingestion of host cells in the young trophont. Leucocytes are the most common cells within the cavity between host and parasite (Cross and Matthews, 1993b) and are frequently located within trophont food vacuoles. Parasite-induced cellular infiltration (Figure 1) probably provides a major source of nutrient for the maturing trophont and the constant removal of leucocytes by phagocytosis could explain the apparently low level of localized response associated with older trophonts within the epithelial layer (Ventura and Paperna, 1985). To what extent tissue breakdown adjacent to the trophont (Ventura and Paperna, 1985; Cross and Matthews, 1993b; Matthews, R.A., 1994) can be attributed to histolytic enzymes of host or parasite origin is far from clear. Acid phosphatase and non-specific esterase activity has been detected within the vicinity of young trophonts (Kozel, 1980). Tissue damage might also be amplified by autolysis following trauma (Matthews, R.A., 1994) and as a result of leucocyte degranulation (Cross and Matthews, 1993a; Sigh et al., 2004b). Although the cycle of digestion has not been described for I. multifiliis, the major events are well documented in other ciliates (Lynn and Corliss, 1991) including Tetrahymena (see Nilsson, 1979; Mislan and Smith-Somerville, 1986; Verni and Gualtieri, 1997). Numerous

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food vacuoles in different stages of the digestive cycle are present in actively feeding trophonts of I. multifiliis. These vacuoles, formed from plasma membrane at the cytostome, would be expected to travel a set course within the cytoplasm and be discharged at the cytoproct. Cytochemical investigations by Lobo-da-Cunha and Azevedo (1988a, 1993a, b) show these to contain acid phosphatase and two isozymes of aryl sulphatase with activity at pH 4.5 and 6. These enzymes were also detected in primary lysosomes. Phagocytosis in ciliates has been associated with high rates of food vacuole membrane turnover, with an estimated requirement of 16 000 mm2/20 min in Paramecium (see Allen, 1984). A higher rate of turnover might be expected in I. multifiliis in view of the intense feeding activity within the host tissues. In the ciliates, membrane is thought to be recycled to the cytostome region via the cytoplasm or externally, following egestion at the cytoproct (Allen, 1984). Lobo-da-Cunha and Azevedo (1988a) suggested that evaginations of the food vacuole membrane detected in I. multifiliis may be pinched off and recycled before reaching the cytoproct. Whether or not membrane is also recycled by egestion is unknown; release to the exterior could be significant in pathogenesis and in the establishment of acquired protective immunity against ichthyophthiriosis (Matthews, R.A., 1994). The ingestion of antibody against such antigens could explain the failure of some tomonts to complete development when immunity has been established during the course of an infection (Matthews, R.A., unpublished observations).

4.4. Reproduction within the Fish Host The possibility of isolated groups of trophonts within fish epithelium resulting from reproduction by binary fission was first suspected over 110 years ago (Stiles, 1893; Neresheimer, 1908; Suzuki, 1935). Surprisingly, there have been few further investigations into such an important issue. Reichenbach-Klinke and Landolt (1965) considered that division occurred only under abnormal conditions, including changes in water quality, but provided no evidence in support of


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these views or of parasite reproduction. Ermolenko (1985) and Ewing et al. (1988) have provided convincing evidence of trophont division, the latter on the basis of population density and cluster frequency within experimentally infected channel catfish. Although trophont reproduction within the epithelia of host fish is no longer in doubt, little is known of the nuclear events within the trophont. The use of Eagle’s minimum essential medium (EMEM) in the recovery of premature trophonts from host tissues, described by Matthews, R.A., et al. (1996), provides a valuable approach for future studies. Using this method, Matthews, R.A., et al. (1996) recorded an increase in the number of micronuclei up to four, in trophonts recovered from juvenile carp within 1–5 h after infection. It may be that precocious development precedes asexual division of the trophont or leads to multinucleate tomonts. The possibility that such divisions represent a sexual phase of reproduction cannot be ruled out as Matthews, R.A., et al. (1996) observed some theronts to fuse with established trophonts, from a previous controlled single exposure, within 6 h of entering the host epidermis. They speculated that, if this represented conjugation within the life cycle of I. multifiliis, maturing trophonts might serve as macrogamonts and invading theronts as microgamonts not dissimilar to anisogamy as reported in sedentary and sessile ciliates (Raikov, 1972). Opportunities for conjugation might then be provided within the fish epidermis where trophonts are exposed to viable theronts from within the broad range of the host’s habitat, whereas growth and development would not be compromised should a suitable mating type fail to appear. Sexual reproduction appears to be essential for cell rejuvenation and macronucleus regeneration within other Ciliophora (Nanney, 1980; Lynn and Corliss, 1991) and such processes are well defined in tetrahymenines. Evidence is now required of meiosis, apoptotic macronuclei, and mating types within this species; mating types in Tetrahymena, closely related to Ichthyophthirius within the Oligohymenophorea (Figure 2), were identified over 50 years ago (Elliott and Nanney, 1952). Loss of isolates is widely reported in laboratory-maintained I. multifiliis and could be attributable to senescence (Hauser, 1972; Houghton and Matthews, 1986; Burkart et al., 1990; Ekless and Matthews, 1993;

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Noe and Dickerson, 1995), further supportive of a sexual phase of development in this species. The question concerning sexual reproduction in I. multifiliis is, perhaps, not whether it occurs but where in the life cycle. Pearson (1933) suggested that autogamy might occur within the free-living phase of the life cycle, during the final divisions of the tomites. MacLennan (1935b) and Lom and Dykova´ (1992), however, thought that material extruded from the dividing macronucleus might well have been mistaken for additional micronuclei.

4.5. Exit from the Fish Host Exit of tomonts from the host is asynchronous in I. multifiliis (Figures 2 and 8), although the overall duration of infection at a given temperature is fairly predictable and short at temperatures exceeding 151C. Trophont reproduction could account for variations in both size and maturation time of these stages (Matthews, R.A., et al., 1996). Ewing and Kocan (1987) undertook a detailed investigation of trophont exit from the gill epithelium of channel catfish, following the fate of the parasite over a period of 5 days at 211C. Describing events before parasite emergence, they noted enlargement of the space between trophont and host tissues, general deterioration in the integrity of peripheral host cells, and the overlying epidermis becoming thin and flattened. How the parasite initiates these events is unknown. Ewing and Kocan (1987, 1992) suggested that the contractile vacuole might play a role in one of the two ways. Firstly, the expulsion of fluid could lead to pressure on surrounding tissues and reduce trophont volume, aiding Figure 8 Ichthyophthirius multifiliis: summary of events in the fish host during primary infection (a–e) and following the establishment of protective immunity (f–h). Abbreviations: a, theront invasion of epidermis; b, establishment of trophont; c, trophont growth and maturation; d, emergence of the tomont; e, possible opportunity for conjugation; f, expulsion of theront in response to cutaneous antibodies; g, trophont survival; h, development of trophont following immunosuppression.


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emergence. Secondly, the vacuole could serve as a source of cytotoxic substances. Clark and Dickerson (1997) suggested that escape from host tissues could be aided by trophont hydrolases; excessive host cell disruption was observed at sites vacated by trophonts in channel catfish following passive immunization. It might be expected that damage to surface epithelial cells would also lead to osmotic imbalance, ingress of water, rupture, and trophont escape. This could be enhanced by the excretion of hyperosmotic fluids by the contractile vacuoles before adjustment to life in the aquatic environment. It might be significant that the spongiome region of the contractile vacuole is exceptionally well developed in I. multifiliis compared to other ciliates, particularly in the mature trophont (Mosevitch, 1965). Matthews, R.A., et al. (1996) recovered trophonts of all ages from live carp following immersion of infected epidermis in EMEM. In these instances, interference with osmoregulation may have resulted in increased cell volume forcing trophonts from the epidermis as contractile vacuole activity was arrested at systole, suggesting that the medium was isotonic. It might be that trophonts were unable to distinguish epidermis from medium, a significant number re-entering the latter. Indeed, the highest recoveries of parasites were recorded within 2 h of infection when trophonts would probably retain contact with the exterior via entry routes before entrapment. According to Ewing et al. (1985) gill epithelium regains its normal appearance within 45 min of invasion by I. multifiliis, whereas Iger and Abraham (1990) reported advanced stages of healing in carp epidermis within 3 h of artificially inflicting wounds. Nevertheless, that the percentage of the trophont population recovered using EMEM diminished with time in host suggests that trophonts might become less attracted to high osmolarities with maturation, so that exit is assured in the tomont stage. Modulation of a response to osmolarity with parasite growth could provide a simplistic explanation for the behaviour observed in I. multifiliis. Little is known concerning the exit of the ciliate, C. irritans, from the epidermis of marine fish, although emergence is influenced by circadian rhythms (Burgess and Matthews, 1994). It is probable that a different escape mechanism has evolved in this species, associated


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with its emergence into a hyperosmotic environment and its lack of contractile vacuoles.

4.6. Culture in vitro and Cryopreservation Relatively few attempts have been made to establish the parasitic stage of I. multifiliis in culture in vitro. Hlond (1968) described the survival of tomites for up to 9 d in a medium composed of 0.3 g of pasteurised carp mucus in 100 mL of filtered tap water; however, no detail was provided concerning growth of trophonts. Ekless and Matthews (1993) investigated the survival of theronts in axenic culture, using 12 different monophasic media. Theronts survived longest, up to 120 h, in media containing EMEM with 10% foetal calf serum, although they failed to transform to trophonts. Further studies have aimed at developing systems that best reproduce the feeding environment of the host fish. Xu et al. (2000) investigated the behaviour and development of theronts exposed to freshly excised skin, fin, and gill from channel catfish and also an established fin-cell line of bluegill (Lepomis macrochirus) (BF-2) using medium 199. Theronts showed a positive response to the explants, attaining a 9.6% increase in size within the gill tissue within 4 h after exposure, but did not recognise BF-2 monolayers. That the parasites survived for less than 48 h was attributed to deterioration of the explant tissue following theront invasion. Nevertheless, the method using skin explants has been successfully adapted for short-term investigations of immune response to I. multifiliis and for the recovery of cutaneous antibodies against the parasite (Xu et al., 2000; Xu and Klesius, 2002). Pugovkin et al. (2001) claimed to have cultured I. multifiliis using BF-2 cells from the same source as Xu et al. (2000), although EMEM, rather than medium 119, was used as the basis of their media; no detail was provided concerning growth or survival. They also described a method based on the polymerase chain reaction for the detection of Tetrahymena corlissi, a culture contaminant. Recently, Nielsen and Buchmann (2000) attempted to mimic the fish epidermis by establishing a monolayer of EPC (epithelioma papulosum cyprini) cells in

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a two-compartment system, the monolayer separating water from EMEM culture media. Theronts transformed to trophonts, with significant increase in size (45 mm diameter), and the addition of fish mucus and serum enhanced development. Although the trophonts survived in culture for up to 13 days at 151C they did not develop into tomonts. Culture of I. multifiliis in vitro could provide a valuable approach to the isolation and maintenance of reference serotypes and the supply of antigen for vaccine development. A method for the cryopreservation of this ciliate, however, is urgently required for the establishment of a strain bank. Everett et al. (2002) have claimed some success in formulating a vitrification protocol using Ficolls, 1,2-propanediol and N,N-dimethylacetamide; cryopreserved theronts maintained structural integrity and a few were semi-motile after quick thawing. Until the procedure is perfected, establishing infections in vivo at 91C, extending the trophont stage to 20.4 days, remains the only method of reducing nuclear divisions and maintaining stable strains of I. multifiliis (see Noe and Dickerson, 1995).

5. IMMUNITY TO ICHTHYOPHTHIRIUS MULTIFILIIS 5.1. i-Antigens Dickerson and Clark (1996) provided an excellent review of their pioneering studies of immobilization (i-) antigens in I. multifiliis. Lin and Dickerson (1992) were the first to identify and characterize i-antigens in this parasite using an isolate (G1) from tropical fish purchased in Georgia, USA and maintained through serial passage in Ictalurus punctatus (see Dickerson et al., 1989). They isolated polypeptides of 48 and 60 kDa from a single serotype A, designated G1 (serotype A), with the aid of immobilizing mouse monoclonal antibodies (Dickerson et al., 1986). The epitopes were shown to be conformational in nature, being destroyed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Working with the same serotype, Clark et al. (1992) identified 1.2 kb cDNA


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encoding for the 48 kDa i-antigen. The 1.2 kb cDNA recognized two RNA transcripts, 1.6 and 1.9 kb, under high stringency which they tentatively suggested might correspond to the 48 and 60 kDa antigens, respectively. Their view was based on the strong homology of the RNAs at the sequence level encoding for the same or closely related proteins, thought to arise from different genes, and that both 48 and 60 kDa antigens had common epitopes (Lin and Dickerson, 1992). The immobilization phenotype of serotype A was found to be stable for up to 3 years (Dickerson et al., 1993), by which time there was a drop in viability and the isolate was lost. The appearance of a second serotype B (G1.1), which rapidly replaced A, was thought to be a contaminant, although that it arose by mutation was not ruled out. Dickerson et al. (1993) identified further serotypes, attributing differences in antigenicity to allelic variation in i-antigen genes within the population (drift) rather than to expression of different i-antigens within a multiple gene system as described in free-living representatives of the Hymenostomatida, where a switch of i-antigen may be induced by specific environmental factors. Five different serotypes of I. multifiliis, designated A to E, have been identified (Dickerson and Clark, 1996), these expressing i-antigen polypeptides in the range 40–70 kDa; two different antigens were expressed by each of A, B, C, and E and one by D. Three genes encoding for i-antigens of I. multifiliis have now been cloned, including IAG48[G1] from isolate G1 (serotype A) (Clark et al., 1999) and IAG52A[G5] and IAG52B[G5] from isolate G5 (serotype D) (Lin et al., 2002). The products of these genes are similar in structure, including periodic cysteine residues with the spacing C–X2,3–C and hydrophobic signal peptides at their N- and C-termini, the latter enabling linking to the plasma membrane through a glycosylphospatidyl inositol (GPI) anchor (Clark et al., 2001). Lin et al. (2002) considered the arrangement of cysteine residues to be of critical importance in the structure and hence function of i-antigens in ciliates and speculated that antigenic differences in I. multifiliis reside within the regions between adjacent C–X2–C motifs, particularly in the central repeats. The i-antigens of I. multifiliis are similar in structure to those described in free-living ciliates, including species of

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Paramecium and Tetrahymena, being most closely related to those of Tetrahymena with strongest similarities to T. thermophila (see Lin et al., 2002). The identification of two i-antigen genes from isolate 5 (G5) was unexpected as only serotype D, with a relatively broad band (52/55 kDa) in Western blots (Clark et al., 1995), had previously been detected from this isolate. Further, serotype D-specific immobilizing monoclonal antibody (mAb, G3-61) recognized only the product of IAG52A[G5], although Lin et al. (2002) suspected that IAG52B[G5] would be the dominant gene expressed in this serotype. Lin et al. (2002) suggested that proteins of different specificities with regards to their immobilizing epitopes are coordinately expressed within the same parasite strain, noting that mAbs which target epitopes other than G3-61 are known to immobilize the G5 strain. Clark et al. (1999) found that, although the 1.2 kb cDNA encoding for the 48 kDa antigen from isolate G1 (serotype A) (Clark et al., 1992) corresponded with the gene IAG48[G1], it differed fundamentally in the structure of the 30 end, the encoded product lacking a GPI anchor site. Dickerson et al. (1989) have shown i-antigens to be integral membrane proteins comprising up to 60% of the total ciliary membrane protein of I. multifiliis. That these are GPI-anchored proteins was later demonstrated by Clark et al. (2001); full-length i-antigens expressed in a recombinant form in Tetrahymena thermophila were restricted to the membrane whereas those lacking the C-terminal were secreted. Xu et al. (1995) detected a highly purified form of i-antigen in water surrounding I. multifiliis. They speculated that this resulted from normal protein turnover of the cell surface, being located on the exterior of ciliary and plasma membranes, and that it might be anchored by glycosylinositol phospholipid, as in freeliving ciliates. Clark et al. (1999) suggested that this soluble antigen might be the product of the 1.2 kb cDNA, which would lack the GPI anchor site for membrane inclusion. The i-antigens were also detected in association with membranes of the crystalline mucocysts, although they were not found in secretory mucocysts, the membrane of which becomes confluent with the plasmalemma before discharge (Xu et al., 1995). Kovaćs et al. (1997) have shown that discharged mucocyst material is distributed in an organized manner on the surface of the


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somatic cilia of Tetrahymena pyriformis. As considered earlier, these organelles appear to play an important role at two distinct stages in the life cycle of I. multifiliis, including entry to the fish host and encystment in the aquatic environment. Whether mucocysts are homologous in both theront and trophont is unknown, although their arrangement and relative density within the cell differ in both life cycle stages. Pyle and Dawe (1985) noted protein composition to be stage-dependent in I. multifiliis and it might be significant that Clark et al. (1992, 1995) found genes regulating i-antigens in serotype A to be developmentally regulated, the transcription of the 1.9 kb RNA being substantially elevated in the theront compared to the trophont stage. The high density of mucocysts within the theront (Figure 3) could make a significant contribution to i-antigen-associated cell membrane on entering the host during invasion. Although Clark et al. (1995) demonstrated the involvement of i-antigens in protective immunity against ichthyophthiriosis, the precise function of these antigens in I. multifiliis remains unclear. Clark et al. (1999) have suggested a role in signal transduction as crosslinking, following antibody binding, initiates discharge of the mucocysts (Clark et al., 1987; Cross, 1993; Clark and Dickerson, 1997) and rapid exit of the trophont from the host (Cross and Matthews, 1992; Clark and Dickerson, 1997). That i-antigens of I. multifiliis are GPIanchored proteins strongly supports this view (Clark et al., 2001). Regulatory mechanisms are essential in establishing balanced populations of parasite and host species. In I. multifiliis, this role alone could be attributed to i-antigens, which are highly immunogenic and initiate a robust protective immunity within the teleost host. Such a mechanism would not be dissimilar to host control of Trypanosoma brucei brucei (see Borst and Rudenko, 1994), in that variant-specific surface glycoproteins (VSGs) targeted by humoral antibodies control parasite growth, reflected in undulating parasitaemia. Although structural parallels exist between the i-antigens and the VSGs of African trypanosomes and Giardia lamblia (see Clark and Dickerson, 1997), antigenic variation (shift) does not appear to be a feature of I. multifiliis (see Dickerson et al., 1993) and would be of little advantage as development and transmission are completed before the establishment

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of protective immunity within teleost hosts. i-Antigens may also be involved as virulence factors (Dickerson et al., 1993; Wang and Dickerson, 2002). In support of this view, Wang and Dickerson (2002) noted that vaccination of channel catfish with i-antigen from serotype D purified from a New York isolate of I. multifiliis (NY1) afforded relatively poor protection on challenge with a homologous strain of the parasite.

5.2. Protective Immunity The immune response of fish to I. multifiliis has been discussed in a number of past reviews (Evans and Gratzek, 1989; Matthews, R.A., 1994; Dickerson and Dawe, 1995; Dickerson and Clark, 1996; Buchmann et al., 2001). It suffices here, therefore, to cover only aspects relating to current thinking on the protective response of fish to I. multifiliis. Acquired protective immunity against ichthyophthiriosis is well recognized in freshwater teleosts including carp (Buschkiel, 1910; Hines and Spira, 1973a, 1974c; Houghton and Matthews, 1986), rainbow trout (Wahli and Meier, 1985), channel catfish (Beckert and Allison, 1964; Dickerson et al., 1984), tilapia (Subasinghe and Sommerville, 1986), and tropical ornamental fish (McCallum, 1986). Well-established methods are described for the immunization of fish against the disease, including exposure to theronts (Dickerson et al., 1981; Clark et al., 1987; Houghton and Matthews, 1990) and by intraperitoneal introduction of whole or sonicated parasites (Goven et al., 1980; Clark et al., 1988; Burkart et al., 1990; Dalgaard et al., 2002; Xu et al., 2004) or specific i-antigens (He et al., 1997; Maki and Dickerson, 2003). Xu et al. (2004) have successfully immunized channel catfish against the disease following intraperitoneal injection of sonicated formalin-killed trophonts. Earlier studies (Areerat, 1974; Burkart et al., 1990) recorded only limited success using formolized whole trophonts and cilia, formalin-fixed theronts and theront lysate were ineffective. Wang and Dickerson (2002) and Maki and Dickerson (2003) immunized channel catfish against I. multifiliis following intraperitoneal injection of a purified i-antigen (Lin and


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Dickerson, 1992) administered with Freund’s adjuvant. Although high serum levels of immobilizing antibodies against I. multifiliis are a characteristic feature of the immune response, parasite clearance from the fish host is associated with specific cutaneous antibodies; these occur at significantly low titres (Lin and Dickerson, 1992; Clark et al., 1995; Xu et al., 2004). The degree of protection established depends to some extent upon the method and route of immunization; exposure to live parasites is generally considered the most effective (Dickerson and Clark, 1996) and is associated with higher titres of mucous antibodies (Maki and Dickerson, 2003). Failure to detect cutaneous antibodies in some studies of ichthyophthiriosis (Cross and Matthews, 1993a) or their detection at a relatively low level of activity (Wang and Dickerson, 2002; Maki and Dickerson, 2003) could be partly attributed to technique, mucus being collected from the fish surface with the aid of cotton swabs. Recently, Xu et al. (2002) have developed a valuable approach by recovering these molecules in culture media following short-term maintenance of skin explants from immunized channel catfish. The detection of cutaneous antibodies against I. multifiliis has been further enhanced following the introduction of a modified ELISA by Xu et al. (2003) in which tetramethylbenzidine (TMB) is used as substrate in preference to horseradish peroxidase (HRP). The source of cutaneous antibody in teleosts remains unknown. Dickerson and Clark (1998) and Matthews, R.A. (1994) considered that several anomalies in the protective response to ichthyophthiriosis could be explained on the basis of a cutaneous secretory immune system (Kawai et al., 1981; St Louis-Cormier et al., 1984; Rombout et al., 1986; Lobb, 1987; Cain et al., 2000), including the loss of protection in immune carp following administration of the synthetic corticosteroid triamcinolone when the immobilization activity of humoral antibodies remained high (Houghton and Matthews, 1990, 1993). Recent research into ichthyophthiriosis is providing increasing evidence of separate compartments for cutaneous and serum antibody production in fish (Dickerson and Clark, 1998; Xu et al., 2002; Maki and Dickerson, 2003; Sigh et al., 2004a). Maki and Dickerson (2003) found levels of mucous antibody raised

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against i-antigen in channel catfish to be transient, coinciding with the resolution of infection, but levels were not synchronized with those of serum antibody. This agrees with earlier observations concerning ichthyophthiriosis in rainbow trout (Wahli and Meier, 1985), in which mucous anti-‘ich’ activity decreased significantly shortly after parasite clearance, while serum immobilizing activity developed more slowly and remained at a high level for at least 5 months after recovery from the infection. Cain et al. (2000) characterized the mucosal and systemic immune responses in rainbow trout using surface resonance, which allowed real time detection of antibody– antigen interactions. Interestingly, they found mucosal antibody production to peak earlier than serum responses following intraperitoneal or intra-anal administration of a hapten-carrying antigen; the response was significantly higher when antigen was presented with Freunds’s complete adjuvant. Xu et al. (2002) suggested that anti-‘ich’ antibody may be synthesized within the epidermis as immobilization titres in culture fluids from the excised skin of immune channel catfish were maintained for up to 144 h from the start of culture; preparation of skin explants was designed to eliminate any carry-over of serum antibody. Sigh et al. (2004a) detected an increase in gene expression for IgM and major histocompatibility complex (MHC)-II and complement component C3 in the skin of rainbow trout exposed to I. multifiliis, preceding increased expression of IgM and MHC-II in the head kidney and C3 in the spleen. In the skin, expression levels of MHC-II were raised within 48 h and might have been upregulated by the pro-inflammatory cytokine TNF-a (Sigh et al., 2004b). They were unable to determine the precise location of the IgM-producing cells or whether specific antibody against the parasite was produced. St Louis-Cormier et al. (1984) provided evidence of localized antibody production in the skin of rainbow trout; specific antibody-producing cells were detected in subepidermal locations following immunization and in smear preparations of the epidermis. A better understanding of mucosal immunity in fish is provided from studies of gut-associated lymphoid tissue, GALT. McMillan and Secombes (1997) suggest that T cells with cytotoxic activity occurred within the epithelial compartment of the gut,


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whereas the Ig+ B cells and plasma cells were confined to the lamina propria (Rombout et al., 1993), as in mammals. Such a system in the skin would require an effective mechanism of antibody translocation from dermis to epidermis and mucus. Lobb (1987) has confirmed the presence of lymphocytes in the epidermis of channel catfish, although it is far from clear that they include Ig+ B cell populations. It might be that lymphocytes migrate to the epidermis after activation in subdermal regions as the immune response to I. multifiliis is characterized by a localized cellular infiltration, the response including lymphocytes, granulocytes, and macrophages (Hines and Spira, 1973b; Ventura and Paperna, 1985; Cross and Matthews, 1993b). The possibility that small proteins (o750 kDa) pass directly from serum to epidermis is indicated by studies of passive immunity against I. multifiliis in channel catfish (Clark et al., 1995; Lin et al., 1996). Intraperitoneal administration of IgG class i-mAbs successfully established protection. That a tetrameric serum antibody from naturally immunized channel catfish and an IgM class i-mAb failed to clear trophonts was attributed to the large size of these molecules, and possible failure to breach a physiological barrier between peripheral blood and skin (Lin et al., 1996). Although diffusible monomeric or dimeric forms of IgM have been recorded in a wide range of teleost fish (Rombout et al., 1993; Suzuki et al., 1994; Kaattari and Piganelli, 1996), Xu et al. (2002) have shown that antibodies against I. multifiliis recovered from skin cultures are consistent with the Ig heavy chain of channel catfish with M r ¼ 70 000: Kaattari and Piganelli (1996), while reviewing fish immunoglobulins, hypothesized that structural variation might provide functionally different molecules, those of low molecular weight being effective in eliminating antigens in the absence of complement fixation. That some protective responses against ichthyophthiriosis appear to be the result of antibody neutralizing activity, rather than immobilization or optimization, would not be out of line with such a view. Although Sigh et al. (2004a) detected a 5.3-fold increase in C3 expression levels within the skin of rainbow trout in 4-day infections of I. multifiliis, it is far from clear that complement plays a role in acquired protective immunity against this parasite.

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The localized immune response to I. multifiliis is characterized by cellular infiltration (Ventura and Paperna, 1985; Cross and Matthews, 1993b), granulocytes and neutrophils being detected in the epidermis of carp within 1–2 days in primary infections (Cross and Matthews, 1993b). Recent studies by Sigh et al. (2004b) suggested that these responses parallel the inflammatory reactions of mammals, genes for interleukin (IL-) 1b; tumour necrosis factor a; and IL-8 being expressed in the early stages of the infection in rainbow trout. The possible role of cytokines in the localized response to I. multifiliis is fully discussed by Sigh et al. (2004a). It has long been suspected that protection against ichthyophthiriosis can be transferred from immune to non-immune fish cohabiting in aquatic systems (Hines and Spira, 1974c; Subasinghe and Sommerville, 1989; Ling et al., 1991; Subasinghe, 1993; Sin et al., 1994). The fry of the mouth-brooding fish Oreochromis mossambicus gain protection against ichthyophthiriosis through the immobilization of theronts by mucus secreted from the buccal cavity of the immune mother (Subasinghe, 1993; Sin et al., 1996). Xu and Klesius (2002) detected specific antibodies against I. multifiliis secreted in culture media, containing immune skin explants from channel catfish. These were lethal to theronts in 1:18 dilutions of the media, and at lower concentrations (1:35) over extended periods in excess of 30 minutes. Xu and Klesius (2003) have further shown that catfish, naive to I. multifiliis, gain some protection against ichthyophthiriosis when cohabiting with immune fish, at a stocking density of 15 non-immune (8 cm in length) and 1 or 2 immune fish (27 cm in length) in 50 L aquaria. Whether secreted antibodies against I. multifiliis control theront levels in wild fish populations, other than during mouth brooding, seems unlikely in view of dilution in large water bodies. Nevertheless, some protection might be afforded within fish shoals and within the confines of aquaria (Ling et al., 1991), where immune fish and fish susceptible to ichthyophthiriosis are in close proximity. Protection against ichthyophthiriosis is achieved following both active and passive immunization. It is possible, however, that different triggers are involved in initiating the expulsion of parasites. Passive immunization of channel catfish using i-mAbs was effective only


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against homologous serotypes of I. multifiliis (see Dickerson et al., 1991). Vaccination by exposure to live parasites, however, provided cross immunity against different serotypes although serum antibody prepared from fish within 7 days of challenge immobilized only the serotype with which they had been immunized (Leff et al., 1994). Leff et al. (1994) suggested that antibody may be directed against non-immobilizing epitopes of the same proteins as immobilization antigens sharing common determinants (Dickerson et al., 1993). Although the function of these proteins in ciliates remains uncertain it is possible that non-immobilizing epitopes are conserved within Hymenostomatida, being of survival value in evading immune attack in parasitic representatives. Such a system might not be of mutual advantage to individual parasitic species, however, as they might be denied a susceptible host immunized by another species. Sigh and Buchmann (2002) recorded a low degree of protection against ichthyophthiriosis following immunization with three species of Tetrahymena: T. pyriformis, T. pigmentosa, and T. thermophila. They suggested that protection may be afforded by low-specificity antibody directed at common epitopes, possibly mannosyl epitopes, as these were detected in all species including I. multifiliis; the involvement of non-specific factors was also considered. That immobilization by serum antibodies is not a requirement of the protective response to I. multifiliis is an important finding and questions some of the evidence presented against cross immunity of this parasite with Tetrahymena pyriformis (see Goven et al., 1981a, b). ELISA has now superceded the immobilization assays used in these early studies for detection of specific antibody against I. multifiliis. Sigh and Buchmann (2001), while comparing the two methods, emphasized the need for caution in attributing immobilization to specific antibodies in heat-inactivated fish serum (441C for 20 min) as factors other than complement may play a part in immobilization. Non-specific immune factors are also associated with low degrees of protection against ichthyophthiriosis, the early studies having been reviewed by Matthews, R.A. (1994). It is becoming increasingly evident that the involvement of non-specific immunity has been underestimated in protection against ichthyophthiriosis (Graves et al., 1985; Buchmann

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et al., 1999, 2001; Sigh and Buchmann, 2000, 2001, 2002; Sigh et al., 2004a, b). Buchmann et al. (1999) have demonstrated that rainbow trout immunized against the monogenetic fluke Gyrodactylus derjavini can also show partial cross protection against I. multifiliis. Advances in our knowledge of host–parasite interactions provide a clearer understanding of how protection might operate against ichthyophthiriosis (Cross and Matthews, 1992; Clark et al., 1996; Wahli and Matthews, 1999; Maki and Dickerson, 2003). Cross and Matthews (1992) were the first to investigate the fate of I. multifiliis within immune fish, noting that theronts entered immune carp at similar levels to non-immune controls. In the immune host, however, parasites actively re-emerged, some immediately on entering the epidermis, and all had been expelled within 2 h of exposure. Wahli and Matthews (1999) have confirmed these findings in juvenile carp and have shown that some expelled parasites retain a capability to re-infect a further fish host. Clark et al. (1996) have elegantly demonstrated that immobilizing antibodies can trigger expulsion of I. multifiliis from the immune host; trophonts 3 days old were induced to emerge from channel catfish following passive immunization with IgG class i-mAbs against i-antigens. They concluded that forced exit is dependent upon mAb binding to the ciliary surface of the trophont and occurs at sublethal levels, as parasite development proceeded normally with the production of theronts on entering water. That antibodies are similarly involved in protection of naturally immunized fish is no longer in doubt. Such activity is consistent with the rapid clearance of parasites from the immune host (Cross and Matthews, 1992; Wahli and Matthews, 1999) and the fact that dead parasites are rarely recorded in fish recovering from ichthyophthiriosis. Wahli and Matthews (1999) noted that the infectivity of trophonts actively leaving immune carp fell more rapidly with time after infection compared with those recovered from non-immune controls. Nevertheless, that parasite clearance is affected following exposure to sublethal levels of specific antibodies could explain the enigma of low titres of cutaneous antibodies being associated with protective immunity against ichthyophthiriosis.


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5.3. Parasite Evasion of the Host Immune Response Ways of evading the immune response of vertebrate hosts are a characteristic feature of eukaryotic parasites and are typically associated with prolonged active infection within the immune host (Parkhouse, 1984). The strategy adopted by I. multifiliis appears so far to be unique among eukaryotic parasites. The salient features for success can be attributed to the superficial site of infection in skin and gills, an ability to leave rapidly in response to sublethal levels of specific antibody, and a potential to survive within the aquatic environment throughout the parasitic phase of the life cycle, while retaining opportunities to complete transmission (Figure 8). It is possible that similar strategies will be discovered in ectoparasitic ciliates of fish, including species of Tetrahymena, Chilodonella, and Trichodina. In considering how I. multifiliis might escape from the immune host, Matthews, R.A. (1994) drew attention to the capability of some ciliates to respond to subtle changes in their environment, behaviour being mediated through membrane-bound receptors (Machemer and Dietmer, 1987; Lukas et al., 1989). Sudden increases in ciliary activity including reversal of ciliary beat are recognized as avoidance reactions of free-living species (Naitoh and Sugino, 1984). Clark and Dickerson (1997) considered the possibility of trophonts actively moving away from low concentrations of antibodies, pointing out that classical avoidance responses to antibodies at subthreshold levels for immobilization had been demonstrated in Paramecium almost 40 years earlier (Preer, 1989). Such avoidance behaviour could explain the rapid re-emergence of theronts after entering immune carp (Cross and Matthews, 1992; Wahli and Matthews, 1999) and might be triggered by antibody binding to receptors or occlusion of these by immune complexes. The speed of exit suggests that theronts remain in direct contact with the aquatic environment and simply retrace their route of entry. Mucocyst secretions, discharged during the infection process, may also serve to reduce immune damage by concealment of the parasite surface (Cross, 1993) and by antigen saturation of surrounding tissues (Matthews, R.A., 1994). The development of natural protective immunity against I. multifiliis during

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the course of an infection is likely to lead to the expulsion of trophonts of all ages. It may be that different mechanisms are involved in the exit of established trophonts from immune hosts. Clearance of 3-day-old trophonts from channel catfish, following passive immunization, led Clark and Dickerson (1997) to speculate that i-antigen cross linking at the cell surface may trigger a development switch signalling the parasite to leave the host. This is an attractive theory as emergence from the host is a normal event in the life cycle of I. multifiliis associated with trophont maturation, as considered earlier (see Section 4.5). Clark and Dickerson (1997) further discussed the possibility of antibody binding stimulating uncontrolled release of substances, such as tissue hydrolases, which are thought to be associated with trophont emergence from non-immune hosts. The involvement of such enzymes was supported by observation of excessive host cell disruption at sites vacated by trophonts in channel catfish following passive immunization (Clark and Dickerson, 1997). Trophont expulsion from immune hosts might, therefore, result from changes in osmoregulatory activity triggered by an alteration of surface membrane permeability, possibly induced by antibody binding at subthreshold levels for immobilization. Mechanisms for the evasion of host immune responses other than premature escape from the host may also be involved in I. multifiliis. Houghton and Matthews (1993) described trophont survival in immune carp for up to 3 days, the parasites being revealed by the administration of the synthetic corticosteroid, triamcinolone acetonide, when infections developed to completion in the same host. Similarly, Wahli and Matthews (1999) recovered live trophonts from immune carp for up to 2 days after infection, following EMEM treatment (Matthews, R.A., et al., 1996), although within this period the parasite had not reached a sufficient stage of maturity for further development in water. That extended survival of small numbers of trophonts within the immune host is also frequently recorded in laboratory studies of ichthyophthiriosis could partially be explained on the basis of the high-challenge exposures leading to antigen saturation of the epidermis and localized antibody depletion. Houghton and Matthews (1993) and Wahli and Matthews (1999) detected


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dwindling numbers of parasites with time in immune carp, consistent with increasing antibody activity. Low-level infections of I. multifiliis reported in immunized fish are probably indicative of a decline in the protective response as in these instances trophonts are typically reported within the peripheral regions of fins (Houghton and Matthews, 1993). Evasion of host response might also be attributed to the trophont’s ability to modify its immediate environment by destruction and phagocytosis of host cells, including leucocytes (Cross and Matthews, 1993a; Matthews, R.A., 1994). Although surviving parasites do not appear to represent latent stages they could be significant in the transmission of ichthyophthiriosis in stressed fish populations (Houghton and Matthews, 1993). Expulsion of trophonts rather than killing them within tissues brings additional benefits to the host in reducing immune damage, including granulomata.

6. ICHTHYOPHTHIRIOSIS Hines and Spira (1973a, b, 1974a, b), in their seminal works on I. multifiliis, provided a comprehensive account of the pathogenesis of ichthyophthiriosis in carp covering aspects of immunopathology and pathophysiology. Ventura and Paperna (1985) have undertaken a detailed histopathological investigation of the disease in the skin and gills in a range of fish hosts, including carp. Further studies have also focused on events within the skin and gills during the course of infection. Significant tissue damage results from theront invasion as a result of histolysis and trauma (Ventura and Paperna, 1985; Matthews, R.A., 1994). Tumbol et al. (2001) measured whole body ionic effluxes of goldfish exposed to theronts, noting a significant decrease in Na+ and Cl 2 days following exposure to theronts. By day 3, however, the fish showed positive compensation of these ions, which was associated with regeneration of gill epithelia and an increase in chloride cells and mucous cells. Although exposure to large numbers of theronts can cause death within 8–12 h (Ewing et al., 1985), it is probable that most mortalities result during the chronic phase, associated with intense feeding activity of the trophont and

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disruption of the epidermis and gill epithelia caused by the emerging tomonts (Ewing and Kocan, 1992). Ichthyophthiriosis is characterized by a marked localized cellular response (Hines and Spira, 1973b, 1974a, b; Ventura and Paperna, 1985; Cross and Matthews, 1993b; Cross, 1994); however, the full extent of infiltration is probably masked by continual removal of leucocytes by actively feeding trophonts. Cross and Matthews (1993b) detected a variety of leucocytes, including neutrophils, eosinophils, basophils, and remnants of filament cells within cavities containing trophonts, neutrophils being present in carp within 2 days of a primary infection (Matthews, R.A. and Matthews, B.F., 1984; Hines and Spira, 1974b). The disruption of these cells within the parasite cavity in non-immune carp with the release of cytotoxins (Cross and Matthews, 1993b; Sigh et al., 2004b) may be a further factor in the lytic necrosis associated with this disease. Release of toxic non-oxidative neutrophil constituents enhances parasite-mediated destruction in human amoebiasis (Ravdin, 1990). Whether the cellular immune response contributes to protection or pathogenesis has been further discussed by Cross (1994). Ichthyophthiriosis induces more general effects on fish hosts leading to significant changes in physiological, and biochemical characteristics (Hines and Spira, 1973a, b, 1974a, b). Ieshko et al. (1991) described changes in enzyme activity in the muscle of infected Carassius carassius with inhibition of nucleases and b-glucosidase. Kurovskaya and Osadchaya (1993) investigated 35 morphophysiological, haematological, and biochemical indicators in yearling carp infected with I. multifiliis and noted 15 distinctive changes in fish exposed to high levels of infection. In terminal ichthyophthiriosis, rapid decline in fish health is associated with depletion of energy reserves, impaired haemopoiesis and failure of gill epithelia and epidermis to regenerate, resulting in ingress of water, ion imbalance and sensitivity to oxygen tension and uptake (Hines and Spira, 1973a, b, 1974a, b). Although viruses (Lobo-da-Cunha and Azevedo, 1992) and bacteria (Antychowicz et al., 1992) have been reported in trophonts of I. multifiliis, there is no evidence that these include pathogens of fish. Nevertheless, Lobo-da-Cunha and Azevedo (1992) indicated that


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viral particles may be taken up from fish tissues by the trophont during feeding. The possibility of these being transmitted to further fish is considered as the particles were also found within food vacuoles of tomites originating from the same isolate. This view was supported by Moewus-Kobb (1965), who demonstrated the ability of the parasitic ciliate, Miamiensis avidus, to act as a reservoir for fish viruses taken up from fish cell cultures.

7. CONTROL AND TREATMENT Control of ichthyophthiriosis, like that of other parasitic diseases, requires an integrated approach based upon knowledge of aetiology and epidemiology. In temperate regions, disease risk forecasting by monitoring water temperatures in spring and early summer would provide some warning of impending epizootics and ensure a more targeted approach in the application of control measures. The prevalence of I. multifiliis is known to fluctuate with season (Dickerson and Dawe, 1995). In central Finland, Valtonen and Koskivaara (1994) noted that salmon smolts under culture were at greatest risk of developing ichthyophthiriosis during June and July when I. multifiliis was most prevalent in the wild fish population. In recording the seasonal occurrence of diseases in cage-reared channel catfish in the south-eastern United States, Duarte et al. (1993) observed infections from January to June with peaks in February and May. No outbreak occurred in July and August when water temperatures reached 30–321C, optimal for fish growth but lethal for I. multifiliis. Other factors, including good husbandry, influence disease outbreaks and require consideration when designing control strategies. Recently, Davis et al. (2002) demonstrated increased susceptibility of channel catfish to ichthyophthiriosis following confinement stress. Elevated serum cortisol levels detected within 2 h of confinement were associated with suppression of the protective mechanism against the parasite, similar results being induced on administration of cortisol in food at 200 mg/kg (Davis et al., 2003). The inclusion of the immuneenhancer ascorbic acid into fish food is known to reduce the severity

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of ichthyophthiriosis (Halver, 1972; Wahli et al., 1985, 1986, 1995), improved health status being attributed to the stimulation of nonspecific immunity (Wahli et al., 1995). Kakuta (1998) also reported a reduction of stress in carp held under adverse environmental conditions following the oral administration of bovine lactoferrin. Environmental stressors are also known to affect the susceptibility of fish to ichthyophthiriosis. Ewing et al. (1982) recorded slight but significant increases in numbers of I. multifiliis in channel catfish exposed to a sublethal level of dissolved copper (1030 mg/L). Environmental pollutants may also influence the survival of I. multifiliis. Everett and Dickerson (2003) selected the free-living stages of I. multifiliis and T. thermophila to study toxicity of glyphosate, the active ingredient of the herbicide Roundups, used in controlling aquatic weeds. Roundup was found to be 100% more lethal to tomonts and theronts than glyphosate alone, suggesting that the surfactant in the commercial herbicide was the probable toxic factor. Although not directly related to methods of control, their studies have been of value in furthering our understanding of metabolic pathways in these ciliates, fundamental in drug design. Although I. multifiliis shows no apparent host specificity within teleosts, increased resistance to the parasite has been experimentally demonstrated among different strains of aquarium-maintained Xiphophorus maculates Gunther (see Clayton and Price, 1992, 1994) and between natural populations of Melanotaenia eachamensis Allen and Cross (see Gleeson et al., 2000). Price and Clayton (1999) also showed a relationship between scale pattern in mirror carp and susceptibility to I. multifiliis, fully scaled varieties being most resistant. That the increased resistance appears to be under genetic control indicates some scope for selective breeding against ichthyophthiriosis (Price, 1985). Chemical agents aimed at interrupting the life cycle by killing the free-living stages of the parasite play the major role in current strategies for controlling ichthyophthiriosis in food fish, although in some situations water management can also be effective by reducing exposure to theronts. These methods can usefully exploit the capability of fish to acquire protective immunity against I. multifiliis following a


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low threshold of infection. At present there is no acceptable curative drug for the disease in food fish, although the screening of antiprotozoal drugs used in veterinary medicine for anti-‘ich’ activity provides some promise for the future (see Section 7.2.2). Significant advances have been made in vaccine development against ichthyophthiriosis (see Section 7.3).

7.1. Water Management Manipulation of water quality, including changes in temperature, salinity, and pH, has proved an effective approach to the elimination of I. multifiliis from closed systems where the conditions for survival favour the host rather than the parasite. Increasing salinity, by addition of NaCl, was one of the first methods prescribed for the control of ichthyophthiriosis (Stiles, 1893; Butcher, 1947; Wagner, 1960; Hickling, 1962). Cross (1972) reviewed many of the early studies of salt treatment, concluding that although it is not an effective curative technique it might aid recovery from the disease by redressing the osmotic stress resulting from parasite damage to the fish epithelium. Aihua and Buchmann (2001), however, showed that NaCl at 5 g/L significantly reduced theront production and survival. Selosse and Rowland (1990) recommended its use at the same concentration for the control of ichthyophthiriosis in selected species of Australian warm water fishes. Miron et al. (2004) have also reported successful control of the disease in silver catfish Rhamdia quelin in Brazil using 4 g/L of NaCl. Fingerlings infected with I. multifiliis and held at this concentration for 45 days showed a gradual reduction in ‘white spots’ with 100% survival. It is possible that NaCl could find wider use in the control of the parasite in euryhaline species of fish, notably eels and those tolerant of brackish water conditions including channel catfish (Allen and Avault, 1970) and tilapia. Changes in pH are also known to affect the survival of freeliving stages of I. multifiliis. Rychlicki (1968) successfully controlled ichthyophthiriosis in Polish carp farms by the addition of quicklime, raising the pH from 7–7.5 to 8.5. Cross (1972), however, advised that

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such treatment is effective only in soft water. According to Amlacher (1970) ichthyophthiriosis is not a problem in acid-loving fish occurring in waters of pHo5. The short duration of the free-living phase of the life cycle (Figure 2) provides a further opportunity to eliminate the parasite in closed situations, including aquaria, where fish can easily be removed to new containers. Further transfers can prevent reinfection provided that these are undertaken at intervals within the minimum period for theront production over the expected duration of infection (Houghton and Matthews, 1990). Raising water temperature to 321C for 5 days has limited application, but could be suitable for treating tropical species of fish (Van Duijn, 1967; Hoffman, 1978). Ultraviolet light has also been used for the eradication of theronts in closed recirculating systems (Gratzek et al., 1983). Bodensteiner et al. (2000) described a novel approach to the control of ichthyophthiriosis based on water flow rate which is applicable to raceways. A velocity rate of 20.3 cm/min and turnover rate of 2.5/h in raceways 5 m long reduced the mortality rate of channel catfish fingerlings to 10% compared to 40–70% achieved using modified standard formalin treatment. Increasing the velocity to provide a turnover rate of 4.5/h led to the elimination of the infection.

7.2. Use of Chemicals The application of chemical treatments should be approached with caution, as toxicity to fish will be influenced by many factors, including species, age, condition, and water quality, including temperature. Further, the application of drugs in aquatic systems poses major problems concerning safety, environmental pollution and conservation, and areas covered by legislation. In the USA the Food and Drug Administration (FDA) (Schnick et al., 1986) is responsible for approving the use of all chemical agents used in the treatment of diseases in food fish. Legislation within the European Community (now European Union) (directive no. 90/676/EEC; article 14, regulation 2377/90/EEC), introduced in 1996, also provides safeguards for the use of fish medicines with the establishment of maximum


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permitted residue levels in food fish; malachite green, an effective and widely used chemotherapeutant against I. multifiliis and Saprolegnia infections in fish, is no longer permitted for the treatment of food fish. The search for an effective treatment for ichthyophthiriosis has led to a large number of investigations on a wide range of chemicals. These are conveniently considered in two groups (below), those that kill the free-living stages of the parasite and are effective in controlling transmission only and curative drugs which kill the trophont in situ. 7.2.1. Chemicals effective against free-living stages of the parasite Formalin is the only chemical permitted for use by the FDA. Brown and Gratzek (1980) recommended the use of formalin at 25 p.p.m. in three treatments on alternate days each followed with a 75% water change after 4–8 h. Short-term exposures of up to 1 h at higher concentrations of 100–250 p.p.m. are still recommended for raceways. In treating ichthyophthiriosis in Atlantic salmon at temperatures of 151C, Valtonen and Kera¨nen (1981) extended each exposure to 12 h, adding formalin to ponds on nine occasions at 3-day intervals. That less than half of the fish recovered was probably due to the advanced stage of the disease at the start of treatment, with poor prognosis. Bodensteiner et al. (2000) also failed to prevent 40–70% mortality in channel catfish using formalin at concentrations of 25 mg/L, the chemical being dispensed in raceways for 4 h on four consecutive days during winter when temperatures ranged from 9 to 181C. At these levels, however, Smith and Piper (1972) have demonstrated severe pathological changes in gill epithelium of rainbow trout, death being associated with inability to maintain osmotic and acid–base balance. Bills et al. (1977) demonstrated that 4 days continuous exposure to formalin at levels as low as 11.8 p.p.m. at 121C could be lethal to rainbow trout. The safety of formalin treatment is also dependent on water temperature (Bills et al., 1977), as oxygen depletion may occur in warm weather, notably through the death of algae (Hoffman, 1970a). According to Van Duijn (1967), fish are more predisposed to fungal infections following treatment with formalin below 181C due to detrimental effects on the mucus. A decrease in numbers of

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mucous cells in rainbow trout fin epithelia was demonstrated by Buchmann et al. (2004) following 1 h exposure to formalin at levels of 200–300 p.p.m. for 1 h or at 50–100 p.p.m. for 24 h, although shortterm exposure (1 h) to the low concentrations stimulated mucus production. Nevertheless, formalin is useful in preventing outbreaks of disease by reducing parasite transmission but it has limited applications as a cure and is unlikely to be successful in treating severe cases of ichthyophthiriosis. Formalin does not appear to accumulate in salmonid tissues following exposure to high concentrations (300 p.p.m. for 1 h); however, whether this applies to other fish species is questionable. Subasinghe and Yusoff (1993) detected formalin within the muscle of Chlarias batrachus (L.) and Puntius gonionotus (Bleeker) following 24 h exposure to levels exceeding 25 and 50 p.p.m., respectively. They recommended that formalin treatment be withdrawn 24 h before harvesting these two warm water species. Most of the agents reviewed by Cross (1972) and Hoffman and Meyer (1974) find use today, including formalin, potassium permanganate, and chloramine-T. All are effective in eliminating free-living stages of the parasite and have found global application within control programmes against I. multifiliis. Other chemicals used to kill the free-living stages of I. multifiliis, together with more recent studies, are as follows: bronopol (Shinn et al., 2001), potassium permanganate (Straus and Griffin, 2002), copper sulphate (Straus, 1993; Klesius and Rogers, 1995; Schlenk et al., 1998), sodium chloride (Selosse and Rowland, 1990), silver nitrate (Farley and Heckemann, 1980), chloramine-T (Cross and Hursey, 1973; Shinn et al., 2001), sodium percarbonate (Buchmann et al., 2003), garlic (Buchmann et al., 2003), methylene blue (Van Duijn, 1967), and ronidizole (Farley and Heckemann, 1980). Shinn et al. (2001) have shown a new veterinary product, bronopol (Pycezes), to be of value in the control of ichthyophthiriosis in rainbow trout. In comparative tests, bronopol was less effective than chloramine-T in reducing trophont intensities (17 to 95% reduction); however, any reduction in the severity of infection would be beneficial in aiding recovery by affording a better opportunity to establish protective immunity against the disease. Bronopol is licensed within


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the European Union for the treatment of Saprolegnia infections in food fish and is available for the control of ichthyophthiriosis. 7.2.2. Drugs which kill the trophont in situ (a) Current status. At present, no licensed chemotherapeutant is available for the treatment of ichthyophthiriosis in food fish. Malachite green has in the past provided the most effective chemical treatment against the disease and was used extensively as a fish chemotherapeutic agent since its introduction as a fungicide in 1936 (Alderman, 1985). Reports of teratogenic properties (Meyer and Jorgenson, 1983) led to the withdrawal of this drug for the treatment of food fish in the USA and recently within the member states of the European Union, although it remains freely available as a fish medicament in most other countries and for use within the ornamental fish trade (Schmahl et al., 1996). Application of the chemical was by addition to water with uptake through skin and gills. Schmahl et al. (1992), however, tested a medicated food, containing a non-watersoluble formulation of malachite green, which provided a less hazardous approach for the treatment of ornamental fishes by aquarists. The product was found to be effective in killing the trophont in situ within 4 days using daily treatment. No toxic effect was recorded in fish even when fed the drug continuously for two months, in contrast to the application of malachite green by water bath. Further studies by Schmidt et al. (1993) confirmed the product’s efficacy against a variety of ornamental fish, including species sensitive to water-soluble malachite green. Schmahl et al. (1996) developed a safer approach for the treatment of ichthyophthiriosis in tropical ornamental fish, substituting quinine for malachite green in medicated food. This, administered at 5 g quinine/kg food daily, was shown to kill trophonts within the epidermis. Inappetance was noted in some fish when maintained on the diet for 12 weeks. Quinine has not found application in the treatment of food fish. (b) New drugs against ichthyophthiriosis. Although a safe replacement for malachite green is urgently required, it seems unlikely

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that the drug industry will be prepared to invest in the development of products targeted specifically at fish parasites, particularly ciliates, a predominantly free-living group, with no major pathogen of importance in higher vertebrates. Many veterinary-approved products have shown anti-‘ich’ activity killing trophonts in situ, but have also proved toxic to some species of fish host. Toltrazuril, for example, proved an effective treatment for ichthyophthiriosis in eels and sticklebacks (Schmahl et al., 1989) but was too toxic for use with rainbow trout (From et al., 1992). Three recent studies have aimed to find an effective cure for the disease in rainbow trout. Tojo Rodriguez and Santamarina Fernandez (2001) investigated the efficacy of 16 veterinary and medically available drugs for the treatment of ichthyophthiriosis in this species. Each drug was orally administered at 40 g/kg daily for 10 days and parasite intensities assessed following counts of trophonts removed in mucus scrapings from one side of the test fish. Four of the drugs, namely secnidazole, diethylcarbamazine, metronidazole, and niridazole, showed some activity against the parasite and secnidazole, in particular, might warrant further investigation (Shinn et al., 2003). Shinn et al. (2003) tested six coccidiostats, developed for use in the poultry industry, for activity against I. multifiliis. Three of these, amprolium, salinomycin sodium, and clopidol, when administered in feed over a 10-day period significantly reduced the number of trophonts established in rainbow trout fingerlings. Salinomycin was the most effective compound tested, reducing the number of surviving trophonts by 72 to 93.4%. Luzardo-A´lvarez et al. (2003) have investigated efficacy of the veterinary drug triclabendazole, in an inclusion complex with b-cyclodextrin, against I. multifiliis in rainbow trout. Results showed a significant reduction in parasite intensities and trophont size following administration of the complex in food at 20 g of complex/kg feed daily for 10 days. Although these were only preliminary studies, the authors were optimistic that the treatment could find application in the control of ichthyophthiriosis in rainbow trout, and could meet environmental and public health safety requirements. Assessing drug efficacy against ichthyophthiriosis would benefit from the adoption of a standard procedure for estimating parasite


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intensity. Examination of fish on death enables a more realistic estimate of parasite intensity (Shinn et al., 2003). Screening live fish for infection is unlikely to detect trophonts within the gills. Clayton and Price (1988) described a novel approach suitable for small tropical aquaria fish. The procedure adopted by Tojo Rodriguez and Santamarina Fernandez (2001) and Luzardo-A´lvarez et al. (2003) is more appropriate for large fish and relies on the detection of trophonts in mucus scrapes of skin and fin, although samples were taken from only one side of their test fish.

7.3. Vaccine Development 7.3.1. Vaccines based on whole cells and sonicates The provision of sufficient antigen has been one of the major problems in the commercial development of vaccines against eukaryotic parasites. Although acquired protective immunity against ichthyophthiriosis is readily established in fish following administration of whole parasites and sonicates (see Section 5.2), these approaches are not a feasible way forward for a commercial vaccine as a suitable method for culture of the parasite in vitro has yet to be developed. The possibility of a Tetrahymena vaccine against ichthyophthiriosis has received much attention in the past (Goven et al., 1981a, b; Wolf and Markiw, 1982; Dickerson et al., 1984; Houghton et al., 1992; Ling et al., 1993; Sigh and Buchmann, 2002), particularly as wellestablished methods are available for the culture of this species in vitro. The weight of evidence, however, is not supportive of solid cross immunity between these species (Dickerson and Clark, 1994; Matthews, R.A., 1994). Nevertheless, more recent studies (Sigh and Buchmann, 2002) have demonstrated partial cross protection against I. multifiliis infections in fingerling Salmo trutta (4 g) following intraperitoneal introduction of high numbers (68 000) of Tetrahymena. Such protection is generally attributed to the enhancement of non-specific immune factors; however, it would provide a better

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opportunity for the establishment of acquired protective immunity before a severe disease outbreak. 7.3.2. Recombinant vaccines The use of Tetrahymena thermophila as a delivery system for recombinant vaccines against I. multifiliis (see Brunk, 1999) provides an exciting and promising approach to the control of ichthyophthiriosis. This species is closely related to I. multifiliis (see Van den Bussche et al., 2000) (Figure 3), common in freshwater ecosystems and readily cultured in vitro using simple media based on peptone broth, thus providing a basis for commercial production. Gaertig et al. (1999) have successfully inserted the i-antigen gene IAG52B[G5] (Lin et al., 2002) into T. thermophila using a cadmium-inducible metallothionein gene promoter to achieve high-level expression of the i-antigen at the cell surface. Protection against the specific serotype of I. multifiliis was recorded in channel catfish following parenteral administration of the live vaccine (1 106 cells). Bisharyan et al. (2003) confirmed that the protection can be attributed to acquired immunity, eliminating the possibility that toxic residues of cadmium originating from the gene promoter killed the trophonts; introduction of non-transformed Tetrahymena, grown in the presence or absence of CdCl2, showed no significant difference in trophont intensities when exposed to primary infection of I. multifiliis. Nevertheless, cadmium was detected in fish epidermis at levels lethal to the parasite. That trophonts survived is attributed to the probable sequestration of these heavy metal ions by metal-chelating proteins within the fish tissue. Cadmium accumulation in fish tissues, including the epidermis, associated with the recombinant vaccine raises serious health and environmental issues and warrants investigation of zinc as a substitute for heavy metals in regulating foreign gene expression (Bisharyan et al., 2003). The selection of genes expressing suitable parasite epitopes has been based on studies of affinity-purified i-antigens of I. multifiliis. Although these antigens are potent immunogens, protection is directed only against homologous serotypes of the parasite (Wang and Dickerson, 2002; Wang et al., 2002). As five different serotypes have


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already been identified (see Section 5.1), it is likely that vaccine design will incorporate multiple i-antigens, selecting those prevalent within the targeted aquatic environment. According to Wang et al. (2002), serotypes A and D are the most prevalent within the United States. He et al. (1997) reported some success in the immunization of goldfish against ichthyophthiriosis using a recombinant fusion vaccine (GST–iAgI) based on the epitope of 48 kDa i-antigen inserted into Escherichia coli. Although they claimed that 95% of immunized fish survived, compared with 55% of control fish, no further detail was given concerning the possible application of their vaccine. Presumably, protection would not be afforded against heterologous serotypes of I. multifiliis. That exposure to live theronts and intraperitoneal injection of whole and sonicated parasites provides solid protection against all serotypes of I. multifiliis indicates that other antigens, possibly non-immobilizing epitopes, are also involved in initiating protective immunity. Lin et al. (1997) discussed the administration of such vaccines in promoting protective antibody response.

8. CONCLUSION Significant advances have been made in our knowledge of I. multifiliis since reviewing the parasite in 1994 (Matthews, R.A., 1994). Notable achievements include the characterization of serotypes, the isolation of genes expressing i-antigens, and the successful insertion of one of these genes into Tetrahymena thermophila, affording a way forward for the development of recombinant vaccines against ichthyophthiriosis. Immunological studies have led to a better understanding of the protective response to I. multifiliis, in particular the involvement of cutaneous antibodies and the role of non-specific mechanisms in partial protection against the disease. Nevertheless, many questions concerning fundamental aspects of the biology of this parasite remain unanswered; relatively little is known of the parasitic phase within the fish host. Trophont reproduction and nutrition might more easily be investigated within a system in vitro when a suitable method becomes available. Whether sexual processes are involved in the life cycle has

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yet to be resolved, although strain loss has been attributed to senescence in laboratory-maintained isolates. A strain reference bank for I. multifiliis, essential for the support of epidemiological studies and for the development of multi i-antigen recombinant vaccines, still awaits the perfection of a method for cryopreservation. A wide range of approaches is available for controlling ichthyophthiriosis in specific situations and under careful management. The majority of these aim to reduce trophont intensities by limiting exposure to infection or by immune enhancement, thus capitalizing on the ability of fish hosts to acquire protective immunity. The need for an effective and safe curative drug against the disease in food fish still remains a priority.

ACKNOWLEDGEMENT This review would not have been possible without the support of my wife, Dr Ben Matthews. I thank her for her patience and valuable advice throughout its preparation, and for providing the electron micrographs.

REFERENCES Aihua, L. and Buchmann, K. (2001). Temperature- and salinity-dependent development of a Nordic strain of Ichthyophthirius multifiliis from rainbow trout. Journal of Applied Ichthyology 17, 273–276. Alderman, D.J. (1985). Malachite green: a review. Journal of Fish Diseases 8, 289–298. Allen, K.O. and Avault, J.W., Jr. (1970). Effects of brackish water on ichthyophthiriasis of channel catfish. The Progressive Fish-Culturist 32, 227–230. Allen, R.D. (1984). Paramecium phagosome membrane; oral region to ectoproct and back again. Journal of Protozoology 31, 1–6. Allen, R.D. and Fok, A.K. (1988). Membrane dynamics of the contractile vacuole complex in Paramecium. Journal of Protozoology 35, 63–71. Allison, R. and Kelly, H.D. (1963). An epizootic of Ichthyophthirius multifiliis in a river fish population. The Progressive Fish-Culturist 25, 149–150.


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Amlacher, E. (1970). Textbook of Fish Diseases. Neptune City, New Jersey: TFH Publications. Antychowicz, J., Rogulska, A., Zelazny, J. and Wrobel, M. (1992). Scanning electron microscopic studies on Ichthyophthirius multifiliis infection in the skin of carp (Cyprinus carpio). Bulletin of the Veterinary Institute of Pulawy 35, 1–6. Areerat, S. (1974). The Immune Response of Channel Catfish, Ictalurus punctatus (Rafinesque), to Ichthyophthirius multifiliis. Master’s thesis, Auburn University, Alabama, USA. Bauer, O.N. (1958). Biologie und Beka¨mpfung von Ichthyophthirius multifiliis Fouquet. Zeitschrift fu¨r Fischerei und deren Hilfswissenschaften 7, 575–581. Bauer, O.N. and Yunchis, O.N. (2001). A new genus of parasitic ciliata from tropical fishes. Parazitologiya 35, 142–144. Bauer, O.N., Musselius, V.A. and Strelkov, Y.A. (1973). Diseases of Pond Fishes. Jerusalem: Keter Press (Israel Program for Scientific Translations). Beckert, H. and Allison, R. (1964). Some host response of white catfish to Ichthyophthirius multifiliis (Fouquet). Proceedings of the South-eastern Association of Game and Fish Commissioners 18, 438–441. Bills, T.D., Marking, L.L. and Chandler, J.H., Jr. (1977). Malachite green: its toxicity to aquatic organisms, persistence and removal with activated carbon. Washington, DC: US Department of the Interior, Fish and Wildlife Service, Investigations in Fish Control no. 75, 6 pp. Bisharyan, Y., Chen, Q., Hossain, M.M., Papoyan, A. and Clark, T.G. (2003). Cadmium effects on Ichthyophthirius: evidence for metal-sequestration of fish tissues following administration of recombinant vaccines. Parasitology 126, S87–S94. Bodensteiner, L.R., Sheehan, R.J., Wills, P.S., Brandenburg, A.M. and Lewis, W.M. (2000). Flowing water: an effective treatment for ichthyophthiriasis. Journal of Aquatic Animal Health 12, 209–219. Borst, P. (1991). Molecular genetics of antigenic variation. Immunoparasitology Today 7, A29–A33. Borst, P. and Rudenko, G. (1994). Antigenic variation in African trypanosomes. Science 264, 1872–1873. Bouck, G.B. and Chen, S.-J. (1984). Synthesis and assembly of flagellar surface. Journal of Protozoology 31, 21–24. Brown, E.M. and Gratzek, J.B. (1980). Fish Farming Handbook. Food, Bait, Tropicals and Goldfish. Westport, Connecticut: AVI Publishing Company. Brunk, C.F. (1999). Ciliates display promise for foreign gene expression. Nature Biotechnology 16, 424–425.

222

R.A. MATTHEWS

Buchmann, K. and Nielsen, M.E. (1999). Chemoattraction of Ichthyophthirius multifiliis (Ciliophora) theronts to host molecules. International Journal for Parasitology 29, 1415–1423. Buchmann, K., Lindenstrom, T. and Sigh, J. (1999). Partial cross protection against Ichthyophthirius multifiliis in Gyrodactylus derjavina immunised rainbow trout. Journal of Helminthology 73, 189–195. Buchmann, K., Sigh, J., Nielsen, C.V. and Dalgaard, M. (2001). Host responses against the fish parasitizing ciliate Ichthyophthirius multifiliis. Veterinary Parasitology 100, 105–116. Buchmann, K., Jensen, P.B. and Kruse, K.D. (2003). Effects of sodium percarbonate and garlic extract on Ichthyophthirius multifiliis theronts and tomocysts: in vitro experiments. North American Journal of Aquaculture 65, 21–24. Buchmann, K., Bresciani, J. and Jappe, C. (2004). Effects of formalin treatment on epithelial structure and mucous cell densities in rainbow trout, Oncorhynchus mykiss (Walbaum), skin. Journal of Fish Diseases 27, 99–104. Burgess, P.J. and Matthews, R.A. (1994). Cryptocaryon irritans (Ciliophora): photoperiod and transmission in marine fish. Journal of the Marine Biological Association of the UK 74, 535–542. Burkart, M.A., Clark, T.G. and Dickerson, H.W. (1990). Immunization of channel catfish, Ictalurus punctatus Rafinesque, against Ichthyophthirius multifiliis (Fouquet): killed versus live vaccines. Journal of Fish Diseases 13, 401–410. Buschkiel, A.L. (1910). Beitra¨ge zur Kenntnis des Ichthyophthirius multifiliis Fouquet. Archiv fu¨r Protistenkunde 21, 61–102. Buschkiel, A.L. (1936). Neue Beitra¨ge zur Kenntnis des Ichthyophthirius multifiliis Fouquet. Archives Neérlandaises de Zoologie 1, 178–224. Butcher, A.D. (1943). Observations on some phases of the life cycle of Ichthyophthirius multifiliis Fouquet, 1876, a ciliate protozoan parasite of fresh-water fish. Australian Zoology 10, 125–137. Butcher, A.D. (1947). Ichthyophthiriasis in Australian trout hatchery. The Progressive Fish-Culturist 9, 21–26. Cain, K.D., Johns, D.R. and Raison, R.L. (2000). Characterization of mucosal and systematic immune responses in rainbow trout (Oncorhynchus mykiss) using surface plasmon resonance. Fish and Shellfish Immunology 10, 651–660. Canella, M.F. (1964). L’apparato vestibolo–bucco–faringeo di Ophryoglena. Pubblicazione del Civico Museo di Storia Naturale di Ferraro 6, 1–21. Canella, M.F. and Rocchi-Canella, I. (1976). Contributions a` la connaissance des Cilie´s. VII Biologie des Ophryoglenina (Cilie´s Hymeńostomes Histophages). ‘L’e´poque des ultrastructures’ de la ciliatologie: pourra-t-elle aboutir a` une syste´matique phylogeńe´tique ou s’agit-il d’une


223

flatteuse illusion? Annali Dell’Universita Di Ferrera, Seg: II Biologia Animale 3, 1–510. Chapman, G.B. (1984). Ultrastructural aspects of the host–parasite relationship in ichthyophthiriasis. Transactions of the American Microscopical Society 103, 364–375. Chapman, G.B. and Kern, R. (1983). Ultrastructure aspects of the somatic cortex and contractile vacuole of the ciliate Ichthyophthirius multifiliis Fouquet. Journal of Protozoology 30, 481–490. Clark, T.G. and Dickerson, H.W. (1997). Antibody-mediated effects on parasite behavior: evidence of a novel mechanism of immunity against a parasitic protist. Parasitology Today 13, 477–480. Clark, T.G., Dickerson, H.W., Gratzek, J.B. and Findly, R.C., (1987). In vitro response of Ichthyophthirius multifiliis to sera from immune channel catfish. Journal of Fish Biology 31, supplement, A203–A208. Clark, T.G., Dickerson, H.W. and Findly, R.G. (1988). Immune response of channel catfish to ciliary antigens of Ichthyophthirius multifiliis. Developmental and Comparative Immunology 12, 581–594. Clark, T.G., McGraw, R.A. and Dickerson, H.W. (1992). Developmental expression of surface antigen genes in the parasitic ciliate Ichthyophthirius multifiliis. Proceedings of the National Academy of Sciences of the USA 86, 6363–6367. Clark, T.G., Lin, T.-L. and Dickerson, H.W. (1995). Surface immobilization antigens of Ichthyophthirius multifiliis: their role in protective immunity. Annual Review of Fish Diseases 5, 113–131. Clark, T.G., Lin, T.-L. and Dickerson, H.W. (1996). Surface antigen crosslinking triggers forced exit of a protozoan parasite from its host. Proceedings of the National Academy of Sciences of the USA 93, 6825–6829. Clark, T.G., Lin, T.-L., Jackwood, D.A., Sherrill, J., Lin, Y. and Dickerson, H.W. (1999). The gene for an abundant parasite coat protein predicts tandemly repetitive metal binding domains. Gene 229, 91–100. Clark, T.G., Gao, Y., Wang, J. and Cheng, G. (2001). The I-antigens of Ichthyophthirius multifiliis are GPI-anchor proteins. Journal of Eukaryotic Microbiology 48, 332–337. Clayton, G.M. and Price, D.J. (1988). Ichthyophthirius multifiliis: standardization of the infection–response model in Ameca splendens (Miller & Fitzsimmons). Journal of Fish Diseases 11, 371–377. Clayton, G.M. and Price, D.J. (1992). Interspecific and intraspecific variation in resistance to ichthyophthiriasis among poeciliid and goodeid fishes. Journal of Fish Biology 40, 445–453. Clayton, G.M. and Price, D.J. (1994). Heterosis in resistance to Ichthyophthirius multifiliis infections in poeciliid fish. Journal of Fish Biology 44, 59–66.

224

R.A. MATTHEWS

Crilley, R.V. and Buckelew, T.B. (1977). The ultrastructure of Ichthyophthirius multifiliis. Proceedings of the Pennsylvania Academy of Science 51, 98. Cross, D.G. (1972). A review of methods to control ichthyophthiriasis. The Progressive Fish-Culturist 34, 165–170. Cross, D.G. and Hursey, P.A. (1973). Chloramine T for the control of Ichthyophthirius multifiliis. Journal of Fish Biology 5, 789–798. Cross, M.L. (1993). Antibody binding following exposure of live Ichthyophthirius multifiliis (Ciliophora) to serum from immune carp, Cyprinus carpio. Diseases of Aquatic Organisms 17, 159–164. Cross, M.L. (1994). Localised cellular responses to Ichthyophthirius multifiliis: protection or pathogenesis? Parasitology Today 10, 364–368. Cross, M.L. and Matthews, R.A. (1992). Ichthyophthiriasis in carp, Cyprinus carpio L.: fate of parasites in immunized fish. Journal of Fish Diseases 15, 497–507. Cross, M.L. and Matthews, R.A. (1993a). Ichthyophthirius multifiliis (Ciliophora): the location of sites immunogenic to the host Cyprinus carpio (L.). Fish and Shellfish Immunology 3, 13–24. Cross, M.L. and Matthews, R.A. (1993b). Localized leucocyte response to Ichthyophthirius multifiliis establishment in immune carp, Cyprinus carpio L. Veterinary Immunology and Immunopathology 38, 341–358. Dalgaard, M., Buchmann, K. and Li, A. (2002). Immunization of rainbow trout fry with Ichthyophthirius multifiliis sonicate: protection of host and immunological changes. Bulletin of the European Association of Fish Pathologists 22, 287–297. Dashu, N. and Lien-Siang, L. (1960). Studies on the morphology and life cycle of Ichthyophthirius multifiliis and its control, with a description of a new species. Acta Hydrobiologica Sinica 2, 213–215. Davis, K.B., Griffin, B.R. and Gray, W.L. (2002). Effect of handling stress on susceptibility of channel catfish Ictalurus punctatus to Ichthyophthirius multifiliis and channel catfish virus infection. Aquaculture 214, 55–66. Davis, K.B., Griffin, B.R. and Gray, W.L. (2003). Effect of dietary cortisol on resistance of channel catfish to infection by Ichthyophthirius multifiliis and channel catfish virus disease. Aquaculture 218, 121–130. De Moor, I.J. and Bruton, M.N. (1988). Atlas of alien and translocated indigenous aquatic animals in South Africa. Pretoria: Council for Scientific and Industrial Research, South African National Scientific Programmes Report no. 144. Dickerson, H.W., Brown, J., Dawe, D.L. and Gratzek, J.B. (1984). Tetrahymena pyriformis as a protective antigen against Ichthyophthirius multifiliis infection: comparisons between isolates and ciliary preparation. Journal of Fish Biology 24, 523–529.


225

Dickerson, H.W. and Clark, T.G. (1994). Vaccination against Ich. Aquaculture 127, 277–282. Dickerson, H.W. and Clark, T.G. (1996). Immune response of fishes to ciliates. Annual Review of Fish Diseases 6, 107–120. Dickerson, H.W. and Clark, T.G. (1998). Ichthyophthirius multifiliis: a model of cutaneous infection and immunity in fishes. Immunological Reviews 166, 337–384. Dickerson, H.W. and Dawe, D.L. (1995). Ichthyophthirius multifiliis and Cryptocaryon irritans (Phylum Ciliophora). In: Fish Diseases and Disorders (P.T.K. Woo, ed.), Vol. 1, pp. 181–226. Cambridge: Cambridge University Press. Dickerson, H.W., Dawe, D.L., Gratzek, J.B., Brown, J. and Pyle, S.W. (1981). Induction of Ichthyophthirius multifiliis Fouquet infections in channel catfish, Ictalurus punctatus Rafinesque: standardization of the procedure. Development of Biological Standards 49, 331–336. Dickerson, H.W., Lohr, A.L. and Gratzek, J.B. (1985). Experimental intraperitoneal infection of channel catfish, Ictalurus punctatus (Rafinesque), with Ichthyophthirius multifiliis (Fouquet). Journal of Fish Diseases 8, 139–142. Dickerson, H.W., Evans, D.L. and Gratzek, J.B. (1986). Production and preliminary characterization of murine monoclonal antibodies to Ichthyophthirius multifiliis, a protozoan parasite of fish. American Journal of Veterinary Research 47, 2400–2404. Dickerson, H.W., Clark, T.G. and Findly, R.G. (1989). Ichthyophthirius multifiliis has membrane-associated immobilization antigens. Journal of Protozoology 36, 159–164. Dickerson, H.W., Lin, T.L., Burkart, M.A. and Clark, T.G. (1991). Surface immobilization antigens of Ichthyophthirius multifiliis: identification and passive protection by monoclonal antibodies. Proceedings of the 16th Annual Eastern Fish Health Workshop, Martinsburg, West Virginia. Dickerson, H.W., Clark, T.G. and Leff, A.A. (1993). Serotypic variation among isolates of Ichthyophthirius multifiliis based on immobilization. Journal of Eukaryotic Microbiology 40, 816–820. Dickerson, H.W., Lin, T.L., Chunhui, Xu and Clark, T.G. (1997). Ichthyophthirius multifiliis as an experimental system for the study of cutaneous immunity in fish. In: Fish Vaccinology. Developments in Biological Standardization (R. Gudding, A. Lillehaug, P.J. Midtlyng and F. Brown, eds), Vol. 90, p. 428. Basel: Karger. Diggles, B.K. and Adlard, R.D. (1995). Taxonomic affinities of Cryptocaryon irritans and Ichthyophthirius multifiliis inferred from ribosomal RNA sequence data. Diseases of Aquatic Organisms 22, 39–43. Dogiel, V.A., Petrushevski, G.K. and Polyanski, Y.F. (1961). Parasitology of Fishes. London: Oliver and Boyd.

226

R.A. MATTHEWS

Duarte, S.A., Masser, M.P. and Plumb, J.A. (1993). Seasonal occurrence of diseases in cage-reared channel catfish, 1987–1991. Journal of Aquatic Fish Health 5, 223–229. Egusa, S., Ahmed, A.T.A. and Kubota, M. (1970). Ichthyophthiriasis of elvers imported from France. Fish Pathology 4, 176–179. Ekless, L. and Matthews, R.A. (1993). In vitro maintenance of Ichthyophthirius multifiliis: axenic isolation and survival in selected media. Journal of Fish Diseases 16, 437–447. Elliott, A.M. and Nanney, D.L. (1952). Conjugation in Tetrahymena. Science 116, 33–34. Elser, H.J. (1955). An epizootic of ichthyophthiriasis in a large reservoir. The Progressive Fish-Culturist 17, 132–133. Ermolenko, A.V. (1985). On the reproduction of Ichthyophthirius multifiliis (Ciliata, Ophryoglenidae). Parazitologiya 19, 470–471. Evans, D.L. and Gratzek, J.B. (1989). Immune defense mechanisms in fish to protozoan and helminth infections. American Zoologist 29, 409–418. Everett, K.D.E. and Dickerson, H.W. (2003). Ichthyophthirius multifiliis and Tetrahymena thermophila tolerate glyphosate but not a commercial herbicidal formulation. Bulletin of Environmental Contamination and Toxicology 70, 731–738. Everett, K.D.E., Knight, J.R. and Dickerson, H.W. (2002). Comparing tolerance of Ichthyophthirius multifiliis and Tetrahymena thermophila for new cryopreservation methods. Journal of Parasitology 88, 41–46. Ewing, M.S. and Kocan, K.M. (1986). Ichthyophthirius multifiliis (Ciliophora) development in gill epithelium. Journal of Protozoology 33, 369–374. Ewing, M.S. and Kocan, K.M. (1987). Ichthyophthirius multifiliis (Ciliophora) exit from gill epithelium. Journal of Protozoology 34, 309–312. Ewing, M.S. and Kocan, K.M. (1992). Invasion and development strategies of Ichthyophthirius multifiliis, a parasitic ciliate of fish. Parasitology Today 8, 204–208. Ewing, M.S., Ewing, S.A. and Zimmer, M.A. (1982). Sub lethal copper stress and susceptibility of channel catfish Ictalurus punctatus to experimental infections with Ichthyophthirius multifiliis. Bulletin of Environmental Contamination and Toxicology 28, 674–681. Ewing, M.S., Kocan, K.M. and Ewing, S.A. (1983). Ichthyophthirius multifiliis: morphology of the cyst wall. Transactions of the American Microscopical Society 102, 122–128. Ewing, M.S., Kocan, K.M. and Ewing, S.A. (1985). Ichthyophthirius multifiliis (Ciliophora) invasion of the gill epithelium. Journal of Protozoology 32, 305–310.


227

Ewing, M.S., Lynn, M.E. and Ewing, S.A. (1986). Critical periods in development of Ichthyophthirius multifiliis (Ciliophora) populations. Journal of Protozoology 33, 388–391. Ewing, M.S., Ewing, S.A. and Kocan, K.M. (1988). Ichthyophthirius (Ciliophora): population studies suggest reproduction in host epithelium. Journal of Protozoology 35, 549–552. Ewing, M.S., Hefler, C., Kocan, K.M. and Stebler, E. (1991). Extracellular calcium concentration affects infectivity of Ichthyophthirius (Ciliophora) theronts. Journal of Protozoology 38, 178–181. Farley, O.G. and Heckemann, R. (1980). Attempts to control Ichthyophthirius multifiliis Fouquet (Ciliophora: Ophryoglenidae) by chemotherapy and electrotherapy. Journal of Fish Diseases 3, 203–212. Fenchel, T. (1986). Protozoan filter feeding. In: Progress in Protistology (J.O. Corliss and D.J. Patterson, eds), Vol. 1, pp. 65–113. Bristol: Biopress. Fenchel, T. (1987). Ecology of Protozoa: The Biology of Free-living Phagotrophic Protists. Berlin: Springer-Verlag. Fouquet, D. (1876). Note sur une espe`ce d’infusoires parasites des poissons d’eau douce. Archives de Zoologie Expe´rimentale et Geńe´rale 5, 159–165. From, D.J., Karas, N. and Vordermeier, T. (1992). Trials with toltrazuril against Ichthyophthirius multifiliis Fouquet (1876). Bulletin of the European Association of Fish Pathologists 12, 137–138. Gaertig, J., Gao, Y., Tishgarten, T., Clark, T.G. and Dickerson, H.W. (1999). Surface display of a parasite antigen in the ciliate Tetrahymena thermophila. Nature Biotechnology 17, 462–465. Geisslinger, M. (1987). Observations on the caudal cilium of the tomite of Ichthyophthirius multifiliis Fouquet, 1876. Journal of Protozoology 34, 180–182. Gleeson, D.J. (1999). Experimental infection of striped marshfrog tadpoles (Limnodynastes peronii) by Ichthyophthirius multifiliis. Journal of Parasitology 85, 568–570. Gleeson, D.J., McCallum, H.I. and Owens, I.P.F. (2000). Differences in initial and acquired resistance to Ichthyophthirius multifiliis between populations of rainbow fish. Journal of Fish Biology 57, 466–475. Goven, B.A., Dawe, D.L. and Gratzek, J.B. (1980). Protection of channel catfish Ictalurus punctatus against Ichthyophthirius multifiliis by immunization. Journal of Fish Biology 17, 311–316. Goven, B.A., Dawe, D.L. and Gratzek, J.B. (1981a). Protection of channel catfish (Ictalurus punctatus) against Ichthyophthirius multifiliis (Fouquet) by immunization with varying doses of Tetrahymena pyriformis (Lwoff) cilia. Aquaculture 23, 269–273. Goven, B.A., Dawe, D.L. and Gratzek, J.B. (1981b). In vitro demonstration of serological cross reactivity between Ichthyophthirius multifiliis and

228

R.A. MATTHEWS

Tetrahymena pyriformis. Developmental and Comparative Immunology 5, 283–290. Gratzek, J.B., Gilbert, J.P., Lohr, A.L., Shotts, E.B., Jr. and Brown, J. (1983). Ultraviolet light control of Ichthyophthirius multifiliis Fouquet in a closed fish culture recirculation system. Journal of Fish Diseases 6, 145–153. Graves, S.S., Evans, D.L. and Dawe, D.L. (1985). Antiprotozoan activity of nonspecific cytotoxic cells (NCC) from the channel catfish (Ictalurus punctatus). Journal of Immunology 134, 78–85. Haas, W., Haberl, B., Hofmann, M., Kerschensteiner, S. and Ketzer, U. (1998). Theronts of Ichthyophthirius multifiliis find their fish hosts with complex behaviour patterns and in response to different chemical signals. Tokai Journal of Experimental and Clinical Medicine 23, 329–331. Haas, W., Haberl, B., Hofmann, M., Kerschensteiner, S. and Ketzer, U. (1999). Ichthyophthirius multifiliis invasive stages find their fish hosts with complex behavior patterns and in response to different chemical signals. European Journal of Protistology 35, 129–135. Halver, J.E. (1972). The role of ascorbic acid in fish disease and tissue repair. Bulletin of the Japanese Society of Scientific Fisheries 38, 79–92. Hammerschmidt, B., Schlegel, M., Lynn, D.H., Leipe, D.D., Sogin, M.L. and Raikov, I.B. (1996). Insights into the evolution of nuclear dualism in the ciliates revealed by phylogenetic analysis of rRNA sequences. Journal of Eukaryotic Microbiology 43, 225–230. Hass, G. (1933). Beitra¨ge zur Kenntnis der Cytologie von Ichthyophthirius multifiliis Fouq. Archiv fu¨r Protistenkunde 81, 88–137. Hauser, M. (1972). The intranuclear mitosis of the ciliates Paracineta limbata and Ichthyophthirius multifiliis. Part 1. Electron microscope observations on the pre-metaphase stages. Chromosoma ðBerlinÞ 36, 158–175. Hauser, M. (1973). Aktomyosinartige Filamente im Teilungsmakronukleus des Ciliaten Ichthyophthirius multifiliis. Chromosoma ðBerlinÞ 44, 49–71. Hauser, M. and Van Eys, H. (1973). Intranuklea¨re Wa¨hrend der Mikronukleusmitose von Ichthyophthirius multifiliis. Cytobiologie 7, 215–220. Hausmann, K., Fok, A.K. and Allen, R.D. (1988). Immunocytochemical analysis of the trichocyst structure and development in Paramecium. Journal of Ultrastructure and Molecular Structure Research 99, 213–225. He, J., Yin, Z., Xu, G., Gong, Z., Lam, T.J. and Sin, Y.M. (1997). Protection of goldfish against Ichthyophthirius multifiliis by immunization with a recombinant vaccine. Aquaculture 158, 1–10. Hickling, C.F. (1962). Fish Culture. London: Faber & Faber.


229

Hines, R.S. and Spira, D.T. (1973a). Ichthyophthirius multifiliis (Fouquet) in the mirror carp Cyprinus carpio L. I. Course of infection. Journal of Fish Biology 5, 385–392. Hines, R.S. and Spira, D.T. (1973b). Ichthyophthiriasis in the mirror carp II. Leukocyte response. Journal of Fish Biology 5, 527–534. Hines, R.S. and Spira, D.T. (1974a). Ichthyophthiriasis in the mirror carp Cyprinus carpio (L.) III. Pathology. Journal of Fish Biology 6, 189–196. Hines, R.S. and Spira, D.T. (1974b). Ichthyophthiriasis in the mirror carp Cyprinus carpio (L.) IV. Physiological dysfunction. Journal of Fish Biology 6, 365–371. Hines, R.S. and Spira, D.T. (1974c). Ichthyophthiriasis in the mirror carp Cyprinus carpio (L.) V. Acquired immunity. Journal of Fish Biology 6, 373–378. Hlond, S. (1968). Experiments in in vitro culture of Ichthyophthirius multifiliis. Food and Agriculture Organization Fisheries Report 44, 365–368. Hoffman, G.L. (1967). Parasites of North American Freshwater Fishes. Berkeley: University of California Press. Hoffman, G.L. (1970a). Parasites of North American Fishes. Berkeley: University of California Press. Hoffman, G.L. (1970b). Intercontinental and transcontinental dissemination and transfaunation of fish parasites with emphasis on whirling disease (Myxosoma cerebralis). In: A Symposium on Diseases of Fish and Shellfish (S.F. Snieszko, ed.), pp. 69–81. Washington, DC: American Fisheries Society Special Publication no. 5. Hoffman, G.L. (1978). Ciliates of freshwater fishes. In: Parasitic Protozoa (J.P. Kreier, ed.), Vol. 2, pp. 583–632. New York: Academic Press. Hoffman, G.L. and Meyer, F.P. (1974). Parasites of Freshwater Fishes. Neptune City, New Jersey: TFH Publications. Houghton, G. and Matthews, R.A. (1986). Immunosuppression of carp (Cyprinus carpio L.) to ichthyophthiriasis using the corticosteroid triamcinolone acetonide. Veterinary Immunology and Immunopathology 12, 413–419. Houghton, G. and Matthews, R.A. (1990). Immunosuppression in juvenile carp, Cyprinus carpio L. The effects of the corticosteroids triamcinolone acetonide and hydrocortisone 21-hemisuccinate cortisol on acquired immunity and humoral antibody response to Ichthyophthirius multifiliis Fouquet. Journal of Fish Diseases 13, 269–280. Houghton, G. and Matthews, R.A. (1993). Ichthyophthirius multifiliis (Fouquet): survival within immune juvenile carp, Cyprinus carpio L. Fish and Shellfish Immunology 3, 157–166. Houghton, G., Healey, L.J. and Matthews, R.A. (1992). The cellular proliferative response, humoral antibody response and cross reactivity studies of Tetrahymena pyriformis with Ichthyophthirius multifiliis Fouquet in

230

R.A. MATTHEWS

juvenile carp (Cyprinus carpio L.). Developmental and Comparative Immunology 16, 301–312. Huchzermeyer, K.D.A. (1994). Risks of new fish diseases in ornamental fish industry. In: The South African Ornamental Fish Trade (P.J. Britz and S. De Kock, eds), pp. 30–35. Rand Afrikaans University: Proceedings of the Aquaculture Association of Southern Africa. Ieshko, E.P., Vysotskaya, R.U. and Serezhenko, L.P. (1991). Host–parasite relationships seen as a non-specific adaptive syndrome. In: E´kologopopulyatsionnyı˘ Analiz Parazitov i Krovososushchikh Chlenistonogikh, pp. 103–109. Petrozavodsk: Karelian Scientific Centre. Iger, Y. and Abraham, M. (1990). The process of skin healing in experimentally wounded carp. Journal of Fish Biology 36, 421–437. Jurand, A. and Selman, G.G. (1969). The Anatomy of Paramecium aurelia. New York: St Martin’s Press. Kaattari, S.L. and Piganelli, J.D. (1996). The specific immune system: humoral defense. In: The Fish Immune System: Organism, Pathogen, and Environment (G. Iwama and T. Nakanishi, eds), pp. 207–254. London: Academic Press. Kakuta, I. (1998). Reduction of stress responses in carp, Cyprinus carpio L., held under deteriorating environmental conditions, by oral administration of bovine lactoferrin. Journal of Fish Diseases 21, 161–167. Kawai, K., Kusuda, R. and Hami, T. (1981). Mechanisms of protection in ayu orally vaccinated for vibriosis. Fish Pathology 15, 257–262. Klesius, P. and Rogers, W. (1995). Parasitism of catfish and other farmraised food fish. Journal of the American Veterinary Medical Association 207, 1473–1478. Ko¨rting, W. (1984). Important parasite infections of carp in northern Germany. Zentralblatt fu¨r Bakteriologie, Mikrobiologie und Hygiene, A 258, 387. Kovaćs, P., Mu¨ller, W.E.G. and Csaba, G. (1997). A lectin-like molecule is discharged from the mucocysts of Tetrahymena pyriformis in the presence of insulin. Journal of Eukaryotic Microbiology 44, 487–491. Kozel, T.R. (1976). The occurrence of Ichthyophthirius multifiliis: Ciliata; Hymenostomatidae in Kentucky waters. Transactions of the Kentucky Academy of Science 37, 41–44. Kozel, T.R. (1980). Contact and penetration of Ictalurus punctatus by Ichthyophthirius multifiliis. Proceedings of the American Society of Parasitologists 55, 201. Kurovskaya, L.N. and Osadchaya, S.A. (1993). The influence of Ichthyophthirius multifiliis on under yearling carp, Cyprinus carpio. Journal of Ichthyology 33, 81–92. Leff, A.A., Yoshinaga, T. and Dickerson, H.W. (1994). Cross immunity in channel catfish, Ictalurus punctatus (Rafinesque), against two


231

immobilization serotypes of Ichthyophthirius multifiliis (Fouquet). Journal of Fish Diseases 17, 429–432. Lin, T.L. and Dickerson, H.W. (1992). Purification and partial characterization of immobilization antigens from Ichthyophthirius multifiliis. Journal of Protozoology 39, 457–463. Lin, T.L., Clark, T.L. and Dickerson, H.W. (1996). Passive immunization of channel catfish (Ictalurus punctatus) against the ciliated protozoan parasite Ichthyophthirius multifiliis by the use of murine monoclonal antibodies. Infection and Immunology 6, 4085–4090. Lin, T.L., Clark, T.G. and Dickerson, H.W. (1997). Towards a subunit vaccine against Ichthyophthirius multifiliis. In: Fish Vaccinology. Developments in Biological Standardization (R. Gudding, A. Lillehaug, P.J. Midtlyng and F. Brown, eds), Vol. 90, p. 461. Basel: Karger. Lin, Y., Lin, T.L., Wang, C.-C., Wang, X., Steiger, K., Klopfleisch, R. and Clark, T.G. (2002). Variation in primary sequence and tandem repeat copy number among i-antigens of Ichthyophthirius multifiliis. Molecular and Biochemical Parasitology 120, 93–106. Ling, K.H., Sin, Y.M. and Lam, T.J. (1991). A new approach to controlling ichthyophthiriasis in a closed culture system of freshwater ornamental fish. Journal of Fish Diseases 14, 595–598. Ling, K.H., Sin, Y.M. and Lam, T.J. (1993). Protection of goldfish against some common ectoparasitic protozoans using Ichthyophthirius multifiliis and Tetrahymena pyriformis for vaccination. Aquaculture 116, 303–314. Lobb, C.J. (1987). Secretory immunity in catfish, Ictalurus punctatus, following bath immunization. Developmental and Comparative Immunology 11, 727–738. Lobo-da-Cunha, A. and Azevedo, C. (1988a). Ultrastructural and cytochemical study of food vacuoles and primary lysosomes in Ichthyophthirius multifiliis (Ciliophora). European Journal of Protistology 24, 60–63. Lobo-da-Cunha, A. and Azevedo, C. (1988b). Association between xenosomes and glycogen in the cytoplasm of the ciliate Ichthyophthirius multifiliis. Endocytobiosis and Cell Research 5, 225–231. Lobo-da-Cunha, A. and Azevedo, C. (1990a). Enzyme cytochemistry of the alveolar sacs and golgian-like cisternae in the ciliate Ichthyophthirius multifiliis. Journal of Protozoology 37, 206–211. Lobo-da-Cunha, A. and Azevedo, C. (1990b). Ultrastructural localization of phosphatases with cerium in a ciliated protozoan. Acta Histochemica et Cytochemica 23, 467–474. Lobo-da-Cunha, A. and Azevedo, C. (1992). Virus-like particles in the fish parasite Ichthyophthirius multifiliis (Ciliophora). Journal of Fish Diseases 15, 273–277.

232

R.A. MATTHEWS

Lobo-da-Cunha, A. and Azevedo, C. (1993a). Ultrastructural and cytochemical identification of peroxisomes in the ciliate Ichthyophthirius multifiliis. Journal of Eukaryotic Microbiology 40, 169–171. Lobo-da-Cunha, A. and Azevedo, C. (1993b). Processing of food vacuoles in the parasitic ciliate Ichthyophthirius multifiliis after exit from the host. Parasitology Research 79, 272–278. Lobo-da-Cunha, A. and Azevedo, C. (1994). The golgi apparatus of the ciliated protozoan Ichthyophthirius multifiliis: a cytological study. European Journal of Protistology 30, 97–103. Lom, J. and Cˇerkasovova´, A. (1974). Host finding in invasive stages of Ichthyophthirius multifiliis. Journal of Protozoology 21, 457. Lom, J. and Dykova´, I. (1992). Protozoan Parasites of Fishes. Developments in Aquaculture and Fisheries Science, Vol. 26. London: Elsevier. Lukas, T.J., Wallen-Friedman, M., Kung, C. and Watterson, D.M. (1989). In vivo mutations of calmodulin: a mutant Paramecium with altered ion current regulation has an isoleucine-to-threonine change at residue 136 and altered methylation state at lysine residue 115. Proceedings of the National Academy of Sciences of the USA 86, 7331–7335. Luzardo-A´lvarez, A., Martı´ nez-Mazagatos, J., Santamarina-Fernańdez, M.T., Otero-Espinar, F.J. and Blanco-Meńdez, J. (2003). Oral pharmacological treatments for ichthyophthiriosis of rainbow trout (Oncorhyncus mykiss). Aquaculture 220, 15–25. Lynn, D.H. and Corliss, J.O. (1991). Ciliophora. In: Microscopic Anatomy of Invertebrates (F.W. Harrison and J.O. Corliss, eds), Vol. 1, pp. 333–469. New York: Wiley–Liss. Lynn, D.H., Frombach, S., Ewing, M.S. and Kocan, K.M. (1991). The organelle of Lieberku¨hn as a synapomorphy for the Ophryoglenina (Ciliophora: Hymenostomatida). Transactions of the American Microscopical Society 110, 1–11. Machemer, H. and Dietmer, J.W. (1987). From structure to behaviour: Stylonichia as a model system for cellular physiology. In: Progress in Protistology (J.O. Corliss and D.J. Patterson, eds), Vol. 2, pp. 213–330. Bristol: Biopress. MacLennan, R.F. (1935a). Observations on the life cycle of Ichthyophthirius, a ciliate parasitic on fish. North-West Science 9, 12–14. MacLennan, R.F. (1935b). Dedifferentiation and redifferentiation in Ichthyophthirius. I. Neuromotor system. Archiv fu¨r Protistenkunde 86, 191–210. MacLennan, R.F. (1936). Dedifferentiation and redifferentiation in Ichthyophthirius multifiliis. II. The origin and function of cytoplasmic granules. Archiv fu¨r Protistenkunde 86, 404–426. MacLennan, R.F. (1937). Growth in the ciliate Ichthyophthirius multifiliis. I. Maturity and encystment. Journal of Experimental Zoology 76, 423–440.


233

MacLennan, R.F. (1942). Growth in the ciliate Ichthyophthirius multifiliis. II. Volume. Journal of Experimental Zoology 91, 1–13. Maki, J.L., Brown, C.C. and Dickerson, H.W. (2001). Occurrence of Ichthyophthirius multifiliis within the peritoneal cavities of infected channel catfish Ictalurus punctatus. Diseases of Aquatic Organisms 44, 41–45. Maki, J.L. and Dickerson, H.W. (2003). Systematic and cutaneous mucus antibody responses of channel catfish immunized against the protozoan parasite Ichthyophthirius multifiliis. Clinical and Diagnostic Laboratory Immunology 10, 876–881. Matthews, B.F., Matthews, R.A. and Burgess, P. (1993). Cryptocaryon irritans Brown, 1951 (Ichthyophthiriidae): the ultrastructure of the somatic cortex throughout the life cycle. Journal of Fish Diseases 85, 339–349. Matthews, R.A. (1994). Ichthyophthirius multifiliis Fouquet, 1876: infection and protective response in the fish host. In: Parasitic Diseases of Fish (A.W. Pike and J.W. Lewis, eds), pp. 17–42. Tresaith, UK: Samara Publishing. Matthews, R.A. and Matthews, B.F. (1984). Ichthyophthirius multifiliis Fouquet in juvenile carp: the infection process. Parasitology 89, supplement, S33. Matthews, R.A., Matthews, B.F. and Ekless, L. (1996). Ichthyophthirius multifiliis (Fouquet): observations on the life cycle and indications of a possible sexual phase. Folia Parasitologica 43, 203–208. McCallum, H.I. (1982). Infection dynamics of Ichthyophthirius multifiliis. Parasitology 85, 475–488. McCallum, H.I. (1986). Acquired resistance of black mollies Poecilia latipinna to infection by Ichthyophthirius multifiliis. Parasitology 93, 251–261. McCartney, J.B., Fortner, G.W. and Hansen, M.F. (1985). Scanning electron microscopic studies of the life cycle of Ichthyophthirius multifiliis. Journal of Parasitology 71, 218–226. McClaren, D.J. (1980). Schistosoma mansoni: the Parasite Surface in Relation to Host Immunity. Letchworth: Research Studies Press. McKanna, J.A. (1976). Fine structure of fluid segregation organelles of Paramecium contractile vacuole. Journal of Ultrastructure Research 54, 1–10. McMillan, D.N. and Secombes, C.J. (1997). Isolation of rainbow trout (Oncorhynchus mykiss) intestinal intraepithelial lymphocytes (IEL) and measurement of their cytotoxic activity. Fish and Shellfish Immunology 7, 527–541. Meyer, F.P. and Jorgenson, T.A. (1983). Teratological and other effects of malachite green on the development of rainbow trout and rabbits. Transactions of the American Fisheries Society 112, 818–824.

234

R.A. MATTHEWS

Milinski, M. and Bakker, T.C.M. (1990). Female sticklebacks use male coloration in mate choice and hence avoid parasitized males. Nature 344, 330–332. Miron, D.S., Silva, L.V.F., Golombieski, J.I. and Baldisserotto, B. (2004). Efficacy of different salt (NaCl) concentrations in the treatment of Ichthyophthirius multifiliis-infected silver catfish, Rhamdia quelen, fingerlings. Journal of Applied Aquaculture 14, 155–161. Mislan, T.W. and Smith-Somerville, H.E. (1986). Food vacuole morphology and membrane retrieval in the microstomal form of Tetrahymena vorax. Journal of Protozoology 33, 172–179. Moewus-Kobb, L. (1965). Studies with IPN virus in marine hosts. Annals of the New York Academy of Sciences 126, 328–342. Mosevitch, T.N. (1965). Electron microscopic study of the structure of the contractile vacuole in the ciliate Ichthyophthirius multifiliis (Fouquet). Acta Protozoologica ðWarsawÞ 3, 61–67. Mosevitch, T.N. and Mashansky, V.F. (1965). On the genesis of mitochondria in the parasitic ciliate Ichthyophthirius multifiliis. Protist 1, 37–41. Mouton, A., Basson, L. and Impson, D. (2001). Health status of ornamental freshwater fishes imported to South Africa: a pilot study. Aquarium Sciences and Conservation 3, 327–333. Naitoh, Y. and Sugino, K. (1984). Ciliary movement and its control in Paramecium. Journal of Protozoology 31, 31–40. Nanney, D.L. (1980). Experimental Ciliatology. New York: Wiley. Neresheimer, E. (1908). Der Zeugungskreis des Ichthyophthirius multifiliis. Berichtaus der K. Bayerischen biologischen Versuchsstation, Mu¨nchen 1, 165–184. Nickell, T.A. and Ewing, M.S. (1989). Dispersal of Ichthyophthirius multifiliis (Ciliophora). Proceedings of the Oklahoma Academy of Science 69, 23–26. Nielsen, C.V. and Buchmann, K. (2000). Prolonged in vitro cultivation of Ichthyophthirius multifiliis using EPC cell line as substrate. Diseases of Aquatic Organisms 42, 215–219. Nigrelli, R.F., Pokorny, K.S. and Ruggieri, G.D. (1976). Notes on Ichthyophthirius multifiliis, a ciliate parasitic on fresh-water fishes, with some remarks on possible physiological races and species. Transactions of the American Microscopical Society 95, 607–613. Nilsson, J.R. (1979). Phagotrophy in Tetrahymena: In: Biochemistry and Physiology of Protozoa (M. Levandowsky and S.H. Hutner, eds), Vol. 2, pp. 339–379. New York: Academic Press. Noe, J.G. and Dickerson, H.W. (1995). Sustained growth of Ichthyophthirius multifiliis at low temperature in the laboratory. Journal of Parasitology 81, 1022–1024.


235

Paperna, I. (1972). Infection by Ichthyophthirius multifiliis of fish in Uganda. The Progressive Fish-Culturist 34, 162–164. Paperna, I. (1991). Diseases caused by parasites in the aquaculture of warm water fish. Annual Review of Fish Diseases 1, 155–194. Parkhouse, R.M.E., ed. (1984). Parasite Evasion of the Immune Response. Symposia of the British Society for Parasitology, Vol. 21 (published in Parasitology 88, 571–682). Patterson, D.J. (1980). Contractile vacuoles and associated structures: their organization and function. Biological Reviews 55, 1–46. Pearson, N.E. (1933). Some observations on the reproductive cycle of the infusorian, Ichthyophthirius multifiliis. Proceedings of the Indian Academy of Science 42, 251–255. Peck, R.K. (1977). The ultrastructure of the somatic cortex of Pseudomicrothorax dubius: structure and function of the epiplasm in ciliated protozoa. Journal of Cell Science 25, 367–385. Peshkov, M.A. and Tikhomirova, L.A. (1967). [Structure of the macronucleus of the parasitic infusoria Ichthyophthirius multifiliis]. Doklady Akademii Nauk SSSR 175, 211–212 [in Russian]. Peshkov, M.A. and Tikhomirova, L.A. (1968a). [Structure and function of the excretory apparatus of the parasitic infusian Ichthyophthirius multifiliis]. Doklady Akademii Nauk SSSR 183, 697–699 [in Russian]. Peshkov, M.A. and Tikhomirova, L.A. (1968b). [Ultrastructure of the nuclear apparatus of Ichthyophthirius multifiliis at cyst stage of protomites]. Doklady Akademii Nauk SSSR 183, 451–452 [in Russian]. Preer, J.R. (1989). Update on the molecular genetics of Paramecium. Journal of Protozoology 36, 182–184. Price, D.J. (1985). Genetics of susceptibility and resistance to diseases in fishes. Journal of Fish Biology 26, 509–519. Price, D.J. and Clayton, G.M. (1999). Genotype–environment interactions in the susceptibility of common carp, Cyprinus carpio, to Ichthyophthirius multifiliis infections. Aquaculture 173, 149–160. Pugovkin, D., Felleisen, R. and Wahli, T. (2001). A PCR-based method for the detection of Tetrahymena corlissi contaminations of Ichthyophthirius multifiliis in vitro cultures. Bulletin of the European Association of Fish Pathologists 21, 222–227. Pyle, S.W. and Dawe, D.L. (1985). Stage-dependent protein composition in the life cycle of synchronous Ichthyophthirius multifiliis, a ciliate fish parasite. Journal of Protozoology 32, 355–357. Raikov, I.B. (1972). Nuclear phenomena during conjugation and autogamy in ciliates. In: Research in Protozoology (T.T. Chen, ed.), Vol. 4, pp. 147–289. London: Pergamon. Rasmussen, L. and Orias, E. (1975). Tetrahymena: growth without phagocytosis. Science 190, 464–465.

236

R.A. MATTHEWS

Ravdin, J.I. (1990). Cell biology of Entamoeba hystolytica and immunology of amoebiasis. In: Modern Parasite Biology: Cellular, Immunological and Molecular Aspects (D.J. Wyler, ed.), pp. 126–150. New York: W.H. Freeman and Company. Reichenbach-Klinke, H. and Landolt, M. (1965). The Principal Diseases of Lower Vertebrates. Book 1: Diseases of Fishes. Neptune City, New Jersey: TFH Publications. Rogers, W.A. and Gaines, J.L. (1975). Lesions of protozoan diseases in fish. In: The Pathology of Fishes (W.E. Ribelin and G. Migaki, eds), pp. 117–141. Madison, Wisconsin: University of Wisconsin Press. Rombout, J.H.W.M., Block, L.J., Lamers, C.H.J. and Egberts, E. (1986). Immunization of carp (Cyprinus carpio) with Vibrio anguillarum bacterium: indications for a common mucosal immune system. Developmental and Comparative Immunology 10, 341–351. Rombout, J.H.W.M., Taverne, N., Van de Kampe, M. and Taverne-Thiel, A.J. (1993). Differences in mucus and serum immunoglobulin of carp (Cyprinus carpio L.). Developmental and Comparative Immunology 17, 309–317. Roque, M. and de Puytorac, P. (1968). Infraciliature d’un nouvel Ophryoglenidae: Ichthyophthiroides browni n.g., n.sp. Protistologica 3, 465–473. Roque, M., de Puytorac, P. and Lom, J.L. (1967). L’architecture buccale et la stomatogeńe`se d’Ichthyophthirius multifiliis Fouquet, 1876. Protistologica 3, 77–90. Rychlicki, Z. (1968). Eradication of Ichthyophthirius multifiliis in carp. Food and Agriculture Organization Fisheries Reports 44, 361–364. Schlenk, D., Gollon, J.L. and Griffin, B.R. (1998). Efficacy of copper sulfate for the treatment of ichthyophthiriasis in channel catfish. Journal of Aquatic Animal Health 10, 390–396. Schmahl, G., Mehlhorn, H. and Taraschewski, H. (1989). Treatment of fish parasites. 5. The effects of sym. triazinone (toltrazuril) on fish parasitic Ciliophora (Ichthyophthirius multifiliis Fouquet, 1876, Apiosoma amoebea Grenfell, 1884, Trichodina sp. Ehrenberg, 1831). European Journal of Protistology 24, 152–161. Schmahl, G., Ruider, S., Mehlhorn, H., Schmidt, H. and Ritter, G. (1992). Treatment of fish parasites: 9. Effects of a medicated food containing malachite green on Ichthyophthirius multifiliis Fouquet, 1876 (Hymenostomatida, Ciliophora) in ornamental fish. Parasitology Research 78, 183–192. Schmahl, G., Schmidt, H. and Ritter, G. (1996). The control of ichthyophthiriasis by a medicated food containing quinine: efficacy tests and ultrastructural investigations. Parasitology Research 82, 697–705.


237

Schmidt, M., Ritter, G., Ruider, S., Schmahl, G. and Mehlhorn, H. (1993). Administration of a medicated food containing malachite green as active ingredient for the control of Ichthyophthirius multifiliis and other ectoparasitic diseases of fish. In: Fish Ecotoxicology and Ecophysiology (T. Braunbeck, W. Hanke and H. Segner, eds), pp. 173–178. Weinheim: VCH Verlagsgesellschaft. Schnick, R.A., Meyer, F.P. and Walsh, D.F. (1986). Status of fishery chemicals in 1985. The Progressive Fish-Culturist 48, 1–3. Selosse, P.M. and Rowland, S.J. (1990). Use of common salt to treat ichthyophthiriasis in Australian warm water fishes. The Progressive FishCulturist 52, 124–127. Shinn, A.P., Wootten, R., Somerville, C. and Conway, D. (2001). Putting the squeeze on whitespot. Trout News 32, 20–25. Shinn, A.P., Wootten, R., Coˆte´, I. and Sommerville, C. (2003). Efficacy of selected oral chemotherapeutants against Ichthyophthirius multifiliis (Ciliophora: Ophyroglenidae) infecting rainbow trout Oncorhynchus mykiss. Diseases of Aquatic Organisms 55, 17–22. Shuter, B., Thomas, J.E., Taylor, W.D. and Zimmerman, A.M. (1983). Phenotypic correlates of genomic DNA content in unicellular eukaryotes and other cells. American Naturalist 122, 26–44. Sigh, J. and Buchmann, K. (2000). Associations between epidermal thioninpositive cells and skin parasitic infections in brown trout Salmo trutta. Diseases of Aquatic Organisms 41, 135–139. Sigh, J. and Buchmann, K. (2001). Comparison of immobilization assays and enzyme-linked immunosorbent assays for detection of rainbow trout antibody-titers against Ichthyophthirius multifiliis Fouquet, 1876. Journal of Fish Diseases 24, 49–51. Sigh, J. and Buchmann, K. (2002). Comparative analysis of cross-reactivity between Ichthyophthirius multifiliis and Tetrahymena. Bulletin of the European Association of Fish Pathologists 22, 37–44. Sigh, J., Lindenstrøm, T. and Buckmann, K. (2004a). The parasite ciliate Ichthyophthirius multifiliis induces expression of immune relevant genes in rainbow trout, Oncorhynchus mykiss (Walbaum). Journal of Fish Diseases 27, 409–417. Sigh, J., Lindenstrøm, T. and Buckmann, K. (2004b). Expression of proinflammatory cytokines in rainbow trout (Oncorhynchus mykiss) during an infection with Ichthyophthirius multifiliis. Fish and Shellfish Immunology 17, 75–86. Sin, Y.M., Ling, K.H. and Lam, T.J. (1994). Passive transfer of protective immunity against ichthyophthiriasis from vaccinated mother to fry in tilapias, Oreochromis aureus. Aquaculture 120, 229–237.

238

R.A. MATTHEWS

Sin, Y.M., Ling, K.H. and Lam, T.J. (1996). Cell-mediated immune responses of goldfish, Carassius auratus (L.), to Ichthyophthirius multifiliis. Journal of Fish Diseases 19, 1–7. Skotarczak, B. (1999). Cytochemical identification of mucocysts in Balantidium coli trophozoites. Folia Biologica ðKrakowÞ 47, 61–65. Small, E.B. and Lynn, D.H. (1985). Phylum Ciliophora Doflein, 1901. In: An Illustrated Guide to the Protozoa (J.J. Lee, S.H. Hutner and E.C. Bovee, eds), pp. 393–575. Lawrence, Kansas: Allen Press for the Society of Protozoologists. Smith, C.E. and Piper, R.G. (1972). Pathological effects in formalin-treated rainbow trout (Salmo gairdneri). Journal of the Fisheries Research Board of Canada 29, 328–329. Stiles, C.W. (1893). Report on a parasitic protozoan observed on fish in aquarium. Bulletin of the United States Fish Commission 13, 173–190. St Louis-Cormier, E.A., Osterland, C.K. and Anderson, P.D. (1984). Evidence for a cutaneous secretory immune system in rainbow trout (Salmo gairdneri). Developmental and Comparative Immunology 8, 71–80. Straus, D.L. (1993). Prevention of Ichthyophthirius multifiliis infestation in channel catfish fingerlings by copper sulphate treatment. Journal of Aquatic Animal Health 5, 152–154. Straus, D.L. and Griffin, B.R. (2002). Efficacy of potassium permanganate in treating ichthyophthiriasis in channel catfish. Journal of Aquatic Animal Health 14, 145–148. Subasinghe, R.P. (1993). Effects of immunosuppression on protection of fry from Ichthyophthirius multifiliis Fouquet 1876 during mouthbrooding of Oreochromis mossambicus (Peters). Fish and Shellfish Immunology 3, 97–107. Subasinghe, R.P. and Sommerville, C. (1986). Acquired immunity of Oreochromis mossambicus to the ciliate ectoparasite Ichthyophthirius multifiliis (Fouquet). In: The First Asian Fisheries Forum (J.L. Maclean, L.B. Dizon and I.V. Hosillos, eds), pp. 279–282. Manila, Philippines: Asian Fisheries Society. Subasinghe, R.P. and Sommerville, C. (1989). An experimental study into the possible protection of fry from Ichthyophthirius multifiliis infections during mouth brooding of Oreochromus mossambicus. Journal of Fish Diseases 12, 143–149. Subasinghe, R.P. and Yusoff, F.M. (1993). Retention of formaldehyde in the tissues of two tropical fish species following exposure to therapeutic levels. Aquaculture and Fisheries Management 24, 693–697. Suzuki, J. (1935). On the reproduction of Ichthyophthirius multifiliis Fouquet in relation to water temperature. Bulletin of the Japanese Society of Scientific Fisheries 3, 265–272.


239

Suzuki, Y., Orito, M., Furukawa, K. and Aida, K. (1994). Existence of low molecular weight immunoglobulin M in carp eggs. Fisheries Science 60, 159–162. Tojo Rodriguez, J.L. and Santamarina Fernandez, M.T. (2001). Attempts at oral pharmacological treatment of Ichthyophthirius multifiliis in rainbow trout, Oncorhynchus mykiss (Walbaum). Journal of Fish Diseases 24, 249–252. Traxler, G.S., Richard, J. and McDonald, T.E. (1998). Ichthyophthirius multifiliis (Ich) epizootics in spawning sockeye salmon in British Columbia, Canada. Journal of Aquatic Animal Health 10, 143–151. Tumbol, R.A., Powell, M.D. and Nowak, B.F. (2001). Ionic effects of infection of Ichthyophthirius multifiliis in goldfish. Journal of Aquatic Health 13, 20–26. Uspenskaja, A.V. (1963). [Hyaluronidase from different developmental stages of Ichthyophthirius multifiliis]. Doklady Akademii Nauk SSSR 151, 1476–1478 [in Russian]. Uspenskaja, A.V. and Ovchinnikova, L.P. (1966). [Quantitative changes of DNA and RNA during the life cycle of Ichthyophthirius multifiliis]. Acta Protozoologica ðWarsawÞ 4, 127–141 [in Russian, with English summary]. Valtonen, E.T. and Kera¨nen, A. (1981). Ichthyophthiriasis of Atlantic salmon, Salmo salar L., at the Montta hatchery in northern Finland in 1978–1979. Journal of Fish Diseases 4, 405–411. Valtonen, E.T. and Koskivaara, M. (1994). Relationships between the parasites of some wild fish and cultured fishes in two lakes and a fish farm in central Finland. International Journal for Parasitology 24, 109–118. Van Den Bussche, R.A., Hoofer, S.R., Drew, C.P. and Ewing, M.S. (2000). Characterization of histone H3/H4 gene region and phylogenetic affinity of Ichthyophthirius multifiliis based on H4 DNA sequence variation. Molecular Phylogenetics and Evolution 4, 461–468. Van Duijn, C., Jr. (1967). Diseases of Fishes, edition 2. London: Illiffe Books. Ventura, M.T. and Paperna, I. (1985). Histopathology of Ichthyophthirius multifiliis infections in fishes. Journal of Fish Biology 27, 185–203. Verni, F. and Gualtieri, P. (1997). Feeding behaviour in ciliated protists. Micron 28, 487–504. Wagner, G. (1960). Der Entwicklungszyklus von Ichthyophthirius multifiliis Fouquet und der EinfluX physikalischer und chemischer AuXenfaktoren. Zeitschrift fu¨r Fischerei und deren Hilfswissenschaften 9(NF), 425–443. Wahli, T. and Matthews, R.A. (1999). Ichthyophthiriasis in carp Cyprinus carpio: infectivity of trophonts prematurely exiting both the immune and non-immune host. Diseases of Aquatic Organisms 36, 201–207.

240

R.A. MATTHEWS

Wahli, T. and Meier, W. (1985). Ichthyophthiriasis in trout: investigations of natural defence mechanisms. In: Fish and Shellfish Pathology (A. Ellis, ed.), pp. 347–352. London: Academic Press. Wahli, T. and Meier, W. (1987). The occurrence and significance of Ichthyophthirius multifiliis in Switzerland. Schweizer Archiv fu¨r Tierheilkunde 129, 205–213. Wahli, T., Steiff, K. and Meier, W. (1985). Influence of ascorbic acid on Ichthyophthirius multifiliis infections in trout. Bulletin of the European Association of Fish Pathologists 5, 86–87. Wahli, T., Meier, W. and Pfister, K. (1986). Ascorbic acid induced immunemediated decrease in mortality in Ichthyophthirius multifiliis infected rainbow-trout (Salmo gairdneri). Acta Tropica 43, 287–289. Wahli, T., Meier, W. and Schmitt, M. (1991). Affinity of Ichthyophthirius multifiliis theronts to light and/or fish. Journal of Applied Ichthyology 7, 244–248. Wahli, T., Frischknecht, M., Schmitt, M., Gabaudan, J., Verlhac, V. and Meier, W. (1995). A comparison of the effect of silicone coated ascorbic acid and ascorbyl phosphate on the course of ichthyophthiriosis in rainbow trout, Oncorhynchus mykiss (Walbaum). Journal of Fish Diseases 18, 347–355. Wang, X. and Dickerson, H.W. (2002). Surface immobilization antigen of the parasitic ciliate Ichthyophthirius multifiliis elicits protective immunity in channel catfish (Ictalurus punctatus). Clinical and Diagnostic Laboratory Immunology 9, 176–181. Wang, X., Clark, T.G., Noe, J. and Dickerson, H.W. (2002). Immunization of channel catfish, Ictalurus punctatus, with Ichthyophthirius multifiliis immobilization antigens elicits serotype-specific protection. Fish and Shellfish Immunology 13, 337–350. Wolf, K. and Markiw, M.E. (1982). Ichthyophthiriasis: immersion of rainbow trout (Salmo gairdneri) using Tetrahymena thermophila as protective immunogen. Canadian Journal of Fisheries and Aquatic Sciences 39, 1722–1725. Wright, A.-D.G. and Colorni, A. (2002). Taxonomic re-assignment of Cryptocaryon irritans, a fish parasite. European Journal of Protistology 37, 375–378. Wright, A.-D. and Lynn, H.L. (1995). Phylogeny of the fish parasite Ichthyophthirius multifiliis and its relatives Ophryoglena and Tetrahymena (Ciliophora, Hymenostomatia) inferred from 18S ribosomal RNA sequences. Molecular Biology and Evolution 12, 285–290. Wright, A.-D. and Lynn, H.L. (1997). Maximum ages of ciliate lineages estimated using a small subunit rRNA molecular clock: crown eukaryotes date back to the paleoproterozoic. Archiv fu¨r Protistenkunde 148, 329–341.


241

Wurtsbaugh, W.A. and Tapia, R.A. (1988). Mass mortality in Lake Titicaca (Peru–Bolivia) associated with the protozoan parasite Ichthyophthirius multifiliis. Transactions of the American Fisheries Society 117, 213–217. Xu, C., Clark, T.G., Leff, A.A. and Dickerson, H.W. (1995). Analysis of the soluble and membrane-bound immobilization antigens of Ichthyophthirius multifiliis. Journal of Eukaryotic Microbiology 42, 558–564. Xu, D.-H. and Klesius, P.H. (2002). Antibody mediated response against Ichthyophthirius multifiliis using excised skin from channel catfish, Ictalurus punctatus (Rafinesque), immune to Ichthyophthirius. Journal of Fish Diseases 25, 299–306. Xu, D.-H. and Klesius, P.H. (2003). Protective effect of cutaneous antibody produced by channel catfish, Ictalurus punctatus (Rafinesque), immune to Ichthyophthirius multifiliis Fouquet on cohabited non-immune catfish. Journal of Fish Diseases 26, 287–292. Xu, D.-H., Klesius, P.H., Shoemaker, C.A. and Evans, J.J. (2000). The early development of Ichthyophthirius multifiliis in channel catfish in vitro. Journal of Aquatic Animal Health 12, 290–296. Xu, D.-H., Klesius, P.H. and Shoemaker, C.A. (2001). Effect of lectins on the invasion of Ichthyophthirius multifiliis theront to channel catfish tissue. Diseases of Aquatic Organisms 45, 115–120. Xu, D.-H., Klesius, P.H. and Shelby, R.A. (2002). Cutaneous antibodies in excised skin from channel catfish, Ictalurus punctatus Rafinesque, immune to Ichthyophthirius multifiliis. Journal of Fish Diseases 25, 45–52. Xu, D.-H., Klesius, P.H. and Shelby, R.A. (2003). A modified enzymelinked immunosorbent assay for the detection of cutaneous antibody against Ichthyophthirius multifiliis. Bulletin of the European Association of Fish Pathologists 23, 228. Xu, D.-H., Klesius, P.H. and Shelby, R.A. (2004). Immune responses and host protection of channel catfish, Ictalurus punctatus (Rafinesque), against Ichthyophthirius multifiliis after immunization with theronts and sonicated trophonts. Journal of Fish Diseases 27, 135–141.

Biology of the Phylum Nematomorpha B. Hanelt1, F. Thomas2 and A. Schmidt-Rhaesa3 1

Department of Biology, 167 Castetter Hall, University of New Mexico, Albuquerque, NM 87131-1091, USA 2 GEMI/UMR CNRS-IRD 2724, Equipe: ‘‘Evolution des Syste`mes Symbiotiques’’ IRD, 911 Avenue Agropolis, B.P. 5045, 34032 Montpellier Cedex 1, France 3 Evolutionary Biology, Universita¨t Bielefeld, Postfach 100131, D-33501 Bielefeld, Germany

Abstract. . . . . . . . . . . . . . . . . . . . . 1. Morphology . . . . . . . . . . . . . . . . . . 1.1. Morphology of Gordiida . . . . . 1.2. Morphology of Nectonema . . . 1.3. The Larva . . . . . . . . . . . . . . . 1.4. Development within the Host . 2. Taxonomy and Systematics . . . . . . 2.1. Biodiversity of Nematomorpha 2.2. Intraspecific Variation . . . . . . . 2.3. Biogeography . . . . . . . . . . . . 2.4. Phylogeny . . . . . . . . . . . . . . . 3. Life Cycle and Ecology . . . . . . . . . . 3.1. General Life Cycle . . . . . . . . . 3.2. Paratenic Hosts . . . . . . . . . . . 3.3. Experimental Life Cycles . . . . 3.4. Sex Ratio . . . . . . . . . . . . . . . 3.5. Distribution . . . . . . . . . . . . . . 3.6. Humans and Gordiids. . . . . . . 4. Host Behavioural Alterations . . . . . . 4.1. Introduction . . . . . . . . . . . . . . 4.2. Study System . . . . . . . . . . . . 4.3. Host Behaviour . . . . . . . . . . .


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4.4. Host Changes Caused by Manipulation . 4.5. Manipulation or Collaboration? . . . . . . . 4.6. Conclusion and Future Studies . . . . . . . 5. General Conclusion . . . . . . . . . . . . . . . . . . . Acknowledgements. . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . .

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ABSTRACT Compared with most animal phyla, the Nematomorpha, also known as hair worms, is a relatively understudied metazoan phylum. Although nematomorphs make up only 1 of 3 animal phyla specializing solely on a parasitic life style, little attention has been focused on this enigmatic group scientifically. The phylum contains two main groups. The nectonematids are parasites of marine invertebrates such as hermit crabs. The gordiids are parasites of terrestrial arthropods, such as mantids, beetles, and crickets. Members of both of these groups are free-living as adults in marine and freshwaters respectively. In recent years, large strides have been made to understand this group more fully. New information has come from collection efforts, new approaches in organismal biology, modern techniques in microscopy and molecular biology. This review will focus on the advances made in four main areas of research: (1) morphology, (2) taxonomy and systematics, (3) life cycle and ecology and (4) host behavioural alterations. Recent research focus on the structure of both nectonematids and gordiids has added new insights on the morphology of adult worms and juveniles. The nervous system of gordiids is now well described, including the documentation of sensory cells. In addition, the availability of material from the juvenile of several species of gordiids has made it possible to document the development of the parasitic stage. New collections and reinvestigations of museum specimens have allowed for a critical reevaluation of the validity of established genera and species. However, traditional taxonomic work on this group continues to be hampered by two impeding factors: first is the lack of

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species-specific characters; and second is the problem of intraspecific variation, which has likely led to the description of numerous synonyms. Modern molecular techniques have been used recently to support independently the broad relationships among gordiids. During the turn of the millennium, the study of the life cycle and general ecology of gordiids enjoyed a revival. The pivotal outcome of this research was the domestication of a common American gordiid species, Paragordius varius. This species was the first of this phylum to be laboratory-reared. Through this research, the life cycle of several distantly related gordiid species was investigated. Other work showed that gordiids persist in the environment in the cyst stage by moving through different hosts by paratenesis. These cysts have been shown to retain infectivity for up to a year. These factors have likely contributed to the finding that gordiid cysts are one of the most common metazoans in some aquatic environments. Finally, recent work has focused on elucidating the mechanism of how gordiids make the transition from terrestrially based definitive hosts to a free-living aquatic environment. It has been shown that hosts are manipulated by the parasites to enter water. Using this study system, and using histology and proteomic tools, the method of manipulation used by these parasites is being further investigated. This manipulation, and the reaction of the cricket to this manipulation, has been postulated to benefit both the parasite and the host. Although large strides have been made within the last 10 years in the understanding of nematomorphs, we make the case that a lot of basic information remains to be uncovered. Although seemingly a daunting task, the recent advances in information and techniques lay a solid foundation for the future study of this unique group of parasites.

1. MORPHOLOGY When Bresciani (1991) summarized the knowledge on the microscopical and especially ultrastructural anatomy of nematomorphs, he could refer to only a handful of previous investigations. Since then, the number of ultrastructural studies has increased significantly,

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although the number of investigations is still scarce in comparison to several other animal taxa. In general, few species within the phylum Nematomorpha have been thoroughly investigated, but it appears that the freshwater species (taxon Gordiida) hardly vary in their internal organization. This also applies to the marine genus Nectonema. Most data come from cross sections in the central region of the body, while detailed investigations of the anterior or posterior end are rare. Very little is known about the development of the juvenile parasite inside the hosts. However, recent studies have investigated these developmental stages ultrastructurally (see Lanzavecchia et al. (1995) for Gordius sp. and Schmidt-Rhaesa (2005) for P. varius).

1.1. Morphology of Gordiida The body of all nematomorphs is covered in the adult stage with a thick and rigid cuticle which is secreted by epidermal cells. This cuticle contains a thick layer of large fibres, which are organized in sheets with fibres of adjacent sheets being arranged in an angle of about 601. The number of sheets varies between species, but also vary within a single animal with the highest number of sheets being present in the central body region (Protasoni et al., 2003). Therefore, the number of sheets can only be used cautiously as a taxonomic character. The large fibres are proteinaceous, but not collageneous in nature (Brivio et al., 2000; Protasoni et al., 2003), and contain dityrosine compounds which indicate tyrosine cross-linking in a hardening process (Brivio et al., 2000). The cuticles of Pseudochordodes bedriagae (de Villalobos and Restelli, 2001) and P. varius (SchmidtRhaesa, 2005; Zapotosky, 1971) include a further cuticular layer distal of the fibres. This layer is lacking in Gordius aquaticus. During development, the cuticle described above, which is sometimes called adult cuticle, is formed in the last third of the intraparasitic phase under a thin larval cuticle (Schmidt-Rhaesa, 2005). This larval cuticle is moulted. It appears to be continuous with the cuticle covering the larva and therefore capable of enormous growth.


247

Epidermal cells under the larval cuticle of P. varius contain abundant endoplasmatic reticulum and mitochondria, which are signs of physiological activity. This is certainly due to the uptake of nutrients from the body cavity of the host. When the larval cuticle is replaced by the adult cuticle, the function of the cuticle appears to be protective, which is supported by its thickness, evidence for hardening processes and peroxidase activity within the cuticle (Brivio et al., 2000). The epidermis is very thin in adults, but remains cellular in structure. The nervous system consists of a peripheral system of basiepidermal nerves, which appear to innervate the underlying musculature (Restelli et al., 2002; Schmidt-Rhaesa, 1996a). On the ventral side, the nervous system is organized in a single nerve strand that originates (at least in P. varius) from intraepidermal nerves in a ventral thickening of the epidermis and shifts into a subepidermal position during further development (Schmidt-Rhaesa, 2005). In the anterior end, there is a brain which is circumpharyngeal, but the region ventral of the pharynx is dominant and from this region small connectives run dorsally around the pharynx (Schmidt-Rhaesa, 1996a). Knowledge about sensory cells remains extremely scarce, and only Schmidt-Rhaesa (2004c) reported probable ciliary receptors passing through the cuticle in P. varius (Figure 1). The musculature is formed as a thick sheet of longitudinal muscle cells (see Restelli et al. (2002) for a detailed description; Schmidt-Rhaesa, 1998a). The sheet is a monolayer, but the abundant cells are distinctly flattened in the proximodistal direction. The intestine is comparatively small and joins the gonads in the posterior end in a cloacal opening. Intestinal cells appear to be active in nutrient uptake in early stages but not in later stages (SchmidtRhaesa, 2005). The structure of the intestinal system in the anterior end remains incompletely known. In the centre of the nematomorph body is a primary body cavity (i.e. lined by extracellular matrix and not by an epithelium) in early developmental stages (Lanzavecchia et al., 1995; Schmidt-Rhaesa, 2005). This body cavity is progressively reduced during further development by growth of gonads and parenchyma. The exact development of the gonads remains incompletely known, but from investigations on Gordius sp. (Lanzavecchia et al., 1995) and

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Figure 1 Transmission electron micrograph of the transcuticular structure of Paragordius varius with proposed sensory function from 25 days post-infection.

P. varius (Schmidt-Rhaesa, 2005) the following picture emerges. In both sexes, gonads originate as dorsolateral strands of solid tissue. These extend in males and become surrounded by abundant parenchyma cells. Cells within the testes differentiate into epithelial cells and gametes. The structure of spermatozoa (aflagellate, rod-shaped


249

cells) is unique among metazoans, but appears to be uniform among freshwater species (Donin and Cotelli, 1977; Poinar, 2001; SchmidtRhaesa, 1997c). In females, fewer parenchyma cells are found. It appears that maturing oocytes proliferate from the dorsolateral strands until they almost completely fill the large body cavity.

1.2. Morphology of Nectonema Besides few details from the Nearctic species Nectonema agile (Skaling and MacKinnon, 1988), all fine structural investigations dealt with the Norwegian species Nectonema munidae (Bresciani, 1991; Schmidt-Rhaesa, 1996b, 1997a,b, 1998a). The morphology of the cuticle and epidermis appears broadly comparable to that of freshwater Gordiida (Bresciani, 1991), but there are differences in the other organ systems. The muscle cells in males of N. munidae contain a large proximal portion which is free of myofilaments and which almost completely fills the body cavity of the animal (SchmidtRhaesa, 1998a). The ventral nerve cord remains within an epidermal thickening and an additional dorsal nerve cord is present (SchmidtRhaesa, 1996a). The intestine ends blindly; it is composed of two types of cells (Schmidt-Rhaesa, 1996b). The reproductive system differs remarkably between freshwater and marine nematomorphs. In males, there is an unpaired ‘‘sperm-sac’’ which is attached to the dorsal epidermal cord (Schmidt-Rhaesa, 1999). Gametes found within this sac were not mature and can therefore not be compared with the ones of freshwater species. In females, Feyel (1936) has reported a unique mode of proliferation of oocytes from a tissue proximal of the musculature. It could be confirmed by an ultrastructural reinvestigation (Schmidt-Rhaesa, 1997b) that there is a tissue consisting of cells extremely rich in vesicles and that circular parts, probably the oocytes, of this tissue are given off to the central body cavity. In the anterior end of N. agile and N. munidae are four conspicuous giant cells. An ultrastructural investigation showed that these cells are connected to the nervous system and form a region rich in microvilli with the epidermis of the lateral body wall (Schmidt-Rhaesa, 1996b).

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This suggests a sensory function, but the exact nature remains mysterious.

1.3. The Larva The ultrastructural investigation of the larva of P. varius by Zapotosky (1974, 1975) has been complemented only by an ultrastructural and cytochemical investigation dealing with the larval musculature (Mu¨ller et al., 2004) and two scanning electron microscopy (SEM) investigations (Adrianov et al., 1998; Bohall et al., 1997). Mu¨ller et al. (2004) confirm observations made by Zapotosky (1974, 1975) and add further details such as the presence of isolated muscles on the spines of the outermost ring of spines and differences in the arrangement of musculature in the posterior part of the body (postseptum) between the larvae of P. varius and G. aquaticus. The larva of Nectonema has not been investigated ultrastructurally.

1.4. Development within the Host The whole morphogenesis from the microscopic larva to the macroscopic adult takes place within the host. Whereas in the laboratory system (Hanelt and Janovy, 1999, 2004b) the development of P. varius is rapid and takes about 30 days, while Gordius robustus needs about 3 months to be ready to leave the host. Ten days after experimental infection, P. varius had reached a length of about 1 mm; after about 25 days, the final length was reached (Figure 2). Young stages are white in colour, but late in development, beginning from the posterior end, a brown colouration proceeds. The rapid growth within the host is caused by epidermal and probably also intestinal uptake of nutrients (see Section 1.1). After the adult cuticle is formed (around day 20 after infection) and the larval cuticle is moulted, epidermis and intestine degenerate to a certain degree and decrease in size (Schmidt-Rhaesa, 2005). Other tissues, such as the musculature, develop continuously. Gonads are recognizable in their anlagen very


251

Figure 2 Developmental stages of Paragordius varius at various days post infection (DPI) of host Gryllus firmus. Adult (30 DPI), far right; note dark coloration. Juveniles: 10 DPI, immediately adjacent to adult; 15 DPI, top left; 25 DPI bottom left.

early, extend massively in size, and carry gametes during the last third of the development within the host (Figure 3 is Plate 4.3 in the Separate Color Plate section).

2. TAXONOMY AND SYSTEMATICS 2.1. Biodiversity of Nematomorpha To date, approximately 300 species of horsehair worms have been described. The majority occur in freshwater and five species live in the sea. Since 1990, a total of 49 new species have been described (Table 1). Most new species were described from Argentina, the nematomorph fauna of which was already comparatively well studied (see de Miralles and de Villalobos, 1993b and references therein). New species have also been found in Europe and North America, which are densely sampled regions. This shows that even in the better sampled regions, the biodiversity of Nematomorpha is not completely known. Between 1758 (genus Gordius) and 1965 (genus Dacochordodes), 20 genera of freshwater Nematomorpha (Gordiida) and one marine

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Table 1

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Species described since 1990

Collected from

Number of new species

North America

4

South America

21

Central America Europe

3 5

Africa

3

Asia

8

Australia

4

South Pacific Ocean

1

Author

de Miralles and de Villalobos (1995), Schmidt-Rhaesa (2004a), Schmidt-Rhaesa et al. (2003b) de Miralles and de Villalobos (1996a–e, 1997, 1998, 2000), de Villalobos (1995), de Villalobos and Camino (1999), de Villalobos and de Miralles (1997), de Villalobos et al. (2003), de Villalobos and Voglino (2000) Schmidt-Rhaesa and Menzel (2005) de Villalobos et al. (1999), Degrange and Martinot (1996), Schmidt-Rhaesa (2000), Spiridonov (1998) Schmidt-Rhaesa and de Villalobos (2001), Spiridonov (2001) Baek (1993), Baek and Noh (1992), Schmidt-Rhaesa et al. (2003a), Spiridonov (1993, 1998, 2000) Schmidt-Rhaesa (2002), Schmidt-Rhaesa and Bryant (2004) Poinar and Brockerhoff (2001)

genus, Nectonema, have been described. One further genus, Noteochordodes, was added in 2000 (de Miralles and de Villalobos, 2000). Besides the description of new species, several species were reinvestigated during the past years, in combination with a critical evaluation of the validity of genera. A reinvestigation of Chordodiolus echinatus revealed that this species, the only representative of the genus Chordodiolus, belongs to the genus Beatogordius (SchmidtRhaesa, 2001d; Schmidt-Rhaesa and Ehrmann, 2001). SchmidtRhaesa (2001a) suspected that several other genera including only one or very few species are not valid, but synonyms to other genera. Unpublished results confirm this for the genera Pantacordodes (with the only species P. europaeus (Heinze, 1952)) and Dacochordodes (with D. bacescui (Capuse, 1966)), which both belong to the genus Spinochordodes (Zanca and Schmidt-Rhaesa, unpublished results). Crucial for the justification of genera is that they represent monophyletic taxa which can be recognized by autapomorphies (evolutionary novelties). From the 15 genera including more than one species, such autapomorphies could be found only for five genera


253

(Chordodes, Nectonema, Beatogordius, Acutogordius and Noteochordodes) (Schmidt-Rhaesa, 2001a). For some other genera, the evaluation for being monophyletic requires further investigation. For example, when introducing the genus Gordionus, Mu¨ller (1927) named adhesive warts (‘‘Greifwarzen’’) as a characteristic feature. But since then, adhesive warts were documented only for a fraction of all known Gordionus species, which is better explained by lack of observation instead of confirmed absence. Additionally, such structures have also been recently found in species of Beatogordius (de Villalobos et al., 2003; Schmidt-Rhaesa and Bryant, 2004). Further problems in taxonomy arise from difficulties in clear characterizations of genera. Interestingly, a comparable problem exists for the genus pair Gordionus/Parachordodes and for Neochordodes/ Pseudochordodes. From each pair of genera, one genus (Parachordodes and Pseudochordodes) is characterized by the presence of two types of cuticular elevations, the so-called areoles (for examples of the diversity of cuticular structures see Figure 4). The other genus (Gordionus/Neochordodes) has only one type of areoles. For this typification, attention is only paid to the size and elevation of the areoles and not to their arrangement. The second type of areole, which is larger and more elevated, often arranged in characteristic clusters which may appear also when only one type of areole is present. Little attention has been focused on this type of arrangement of areoles, named the megareolar pattern (Schmidt-Rhaesa, 2001a). Nematomorphs are relatively poor in characters which are important for species determination. Macroscopic characters are the shape of the posterior end (i.e. bilobed or round in males, trilobed or round in females) and the presence of cuticular structures such as a crescentlike fold close to the cloacal opening in the genera Gordius and Acutogordius. All other characters are found on the cuticle and many of them are so small that reliable detection with light microscopy is extremely difficult. Therefore, SEM has become the standard tool in recent species descriptions and reinvestigations (Schmidt-Rhaesa, 2001c) and has been included in the majority of recent species descriptions. One particular problem concerns the variability of the cuticle in different regions of the body. Especially in representatives

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Figure 4 SEM image of the cuticle of Chordodes queenslandi from Australia. Note the diversity of cuticular structures.

of the genus Chordodes, it is essential to gain information not only on the types of cuticular structures, but also about their distribution (Schmidt-Rhaesa, 2002). For example, several species of Chordodes have two different types of the so-called crowned areoles. These are conspicuous elevated areoles with a basic ‘‘stem’’ and a ‘‘crown’’ of apical bristle-like filaments. In some species, crowned areoles with short apical filaments are present on the lateral sides of the body, while crowned areoles with very long filaments border both sides of the ventral and sometimes additionally the dorsal midline (see e.g. Schmidt-Rhaesa, 2002). Species with two types of crowned areoles appear to be sexually dimorphic because these differences in crowned areoles are distinctly elaborated in females but absent or insignificant


255

in males (see Schmidt-Rhaesa (2002), for Chordodes queenslandi and further unpublished results).

2.2. Intraspecific Variation Many reports on nematomorphs are based on findings of single or few species and a number of species are described on the basis of only one specimen. As some species are distinguished from others often by very delicate differences in shape or size of areoles, it is important to know whether these characters are specific for certain species. It seems, however, to be the case that several characters are quite variable. This has been stated previously by some authors whenever they could investigate larger numbers of specimens (see e.g. Montgomery (1898) for Parachordodes tolosanus and Neochordodes occidentalis; Vejdovsky (1888) for P. tolosanus) and was also confirmed in more recent investigations (see e.g. de Villalobos and Zanca (2001) for Chordodes festae; Schmidt-Rhaesa et al. (2003b) for Chordodes morgani). In investigating museum specimens of the genus Gordionus from Great Britain, Schmidt-Rhaesa (2001e) found that there is a continuous transition between the cuticular characters of Gordionus violaceus and of G. wolterstorffii. If further confirmed, this indicates one species with a wide range of morphological variability in cuticular characters. This in turn would have a great impact on taxonomy of the genus Gordionus because many species would fall within this range of morphological variation.

2.3. Biogeography The recognition of biogeographical patterns of distribution rests on a representative sampling, and on a good quality of taxonomic assignments. Both are still in progress for nematomorphs, but first patterns have emerged. North America appears to be a comparatively well-investigated region (summarized in Schmidt-Rhaesa et al., 2003b), but with only

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15 species the diversity appears to be low. In South America, nematomorph species are well known from Argentina (see Table 1 and de Miralles and de Villalobos, 1993b), but species from the remaining countries are much less known (de Miralles and de Villalobos, 1993a). The same applies to the Central American region, from which 22 species are known (Schmidt-Rhaesa and Menzel, 2005). With 99 species, which represent about one-third of all species described, Europe (including the Caucasian states Georgia, Armenia and Azerbeijan) appears to be a densely sampled region (Schmidt-Rhaesa, 1997a). This is, however, still a sketchy picture. From some countries, e.g. Denmark, Norway, Netherlands, Spain, Portugal, Greece, Albania and Turkey, there are no or very few reports. There are a number of species known from Africa, but no country has received special attention. African representatives of the genus Beatogordius were recently reviewed (Schmidt-Rhaesa and de Villalobos, 2001). However, nematomorph diversity in Africa and Asia has received comparatively little attention. Probably, the best known nematomorph fauna is found in Japan (see Inoue, 1994; Schmidt-Rhaesa, 2004b). From Australia, 11 species are known (Schmidt-Rhaesa, 2002; Schmidt-Rhaesa and Bryant, 2004) and from New Zealand, 5 (Poinar, 1991a; Schmidt-Rhaesa et al., 2000). The marine genus Nectonema is known from several locations worldwide (including both coasts of the Northern Atlantic, the Mediterranean, Indonesia, Japan and two questionable reports from the Southern Atlantic), with the latest report being from New Zealand (Poinar and Brockerhoff, 2001). With two exceptions, reports are single findings. The exceptions are the Bay of Fundy and some fjords close to Bergen in Norway. In most cases, Nectonema was found inside its hosts and rarely free-living. Representatives of the genera Gordius and Paragordius occur worldwide while the distribution of other genera is more restricted. Among these, Chordodes, Beatogordius, Neochordodes and Pseudochordodes deserve further attention. With few exceptions, Chordodes species occur only in tropical and subtropical regions. This might be connected to the distribution of their main host group, praying mantids (Schmidt-Rhaesa and Ehrmann, 2001).


257

Species of the genus Beatogordius have been reported from Africa (Schmidt-Rhaesa and de Villalobos, 2001), South America (de Villalobos et al., 2003) and recently also from Madagascar and Australia (Schmidt-Rhaesa and Bryant, 2004). This distribution can best be explained by the presence of the genus on the Gondwana continent before its break up and implies that Beatogordius is at least 180 million years old (MYO). This indirect measure of minimal age is of importance, because with the exception of two young reports (Dominican amber, see Poinar, 1999; Eocene brown coal, see Voigt, 1938), no fossils of nematomorphs have been found. The genera Neochordodes and Pseudochordodes occur predominantly in South and Central America (de Miralles and de Villalobos, 1993b; Schmidt-Rhaesa and Menzel, 2005), but few species are found in the Southwest of the United States (Schmidt-Rhaesa et al., 2003b). This might indicate that Neochordodes and Pseudochordodes originate in South America and spread to North America after the establishment of the Panama land bridge, which is about 45 MYO.

2.4. Phylogeny Traditionally, nematomorphs have been classified into two taxonomic groups: the marine Nectonematoidea and the freshwater Gordiida (or Gordioidea). Among Gordiida, four families and 21 genera are recognized. Following the broad use of phylogenetic systematics during the past decades, this classification was translated into a tree, i.e. a hypothesis of phylogenetic relationships (Bleidorn et al., 2002; Schmidt-Rhaesa, 2001a). The first step was to test if the taxa used in the traditional classification are monophyletic. This is evident only for five genera; 10 genera are either paraphyletic or their monophyly remains to be shown (Schmidt-Rhaesa, 2001a). A combination of morphological and molecular (18S rRNA gene) data supports a sister-group relationship between the marine genus Nectonema and the freshwater Gordiida (Bleidorn et al., 2002). Among these, the Gordiidae (including the genera Gordius and Acutogordius) and Chordodidae (including all other genera) are sister taxa. It appears

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that the bifurcation of the male posterior end is an ancestral character in Gordiida and the posterior end became secondarily undivided in Chordodinae, a subtaxon of Chordodidae. Three hypotheses of relationships of Nematomorpha have been tested, a closer relationship to Nematoda, to the nematode taxon Mermithida and to Scalidophora (Kinorhyncha, Priapulida and Loricifera). Among these, Schmidt-Rhaesa (1998b) assumed a sister–group relationship between Nematomorpha and Nematoda to be well supported, while both other hypotheses are based on fewer potential synapomorphies and are mainly due to convergences. This lach of work has been supported by 18S rRNA sequence analysis (see e.g. Bleidorn et al., 2002; Garey and Schmidt-Rhaesa, 1998) and combined morphological and molecular analyses (Giribet et al., 2000; Peterson and Eernisse, 2001; Sørensen et al., 2000; Zrzavy´ et al., 1998).

3. LIFE CYCLE AND ECOLOGY In the last 10 years, large strides have been made in describing and experimentally determining the life cycle and ecology of freshwater nematomorphs or gordiids. Comparatively, virtually nothing is known about the general biology of marine nematomorphs or nectonematids. No life cycles of taxa within this group have been described, and to our knowledge, no laboratory work has been done on the life cycle of nectonematids. This lack of work has left large gaps in our knowledge of the basic biology of this group, such as the number of hosts involved in their life cycle, generation time, etc. Thus, this section will only deal with the advances made in the study of gordiids.

3.1. General Life Cycle Gordiids are parasites as juveniles in terrestrial arthropods. Adults are free-living in aquatic environments such as rivers, streams and lakes. Here, worms mate and produce eggs. Eggs develop over 7–14 days


259

into semi-sessile larvae (Figure 5), which are able to survive for a maximum of 2 weeks. Gordiids must thus make two critical transitions during their life cycle. The first transition is that from the terrestrial definitive host to water (see Section 4). The second transition is that from aquatic larvae to the definitive host. This latter transition has been the topic of much speculation, but has only recently been intensively studied. Three transmission mechanisms have been proposed for gordiid larvae to reach their definitive hosts (see reviews by Hanelt and Janovy, 2003; Schmidt-Rhaesa, 2001b): (1) larvae are directly transmitted to definitive host without going through additional developmental stages or hosts; (2) larvae form cysts in the environment and infect definitive hosts when accidentally ingested with vegetation or water; (3) larvae enter and encyst within a paratenic host, which are preyed or scavenged upon by the definitive host.

Figure 5 Natural life cycle of Paragordius varius (modified from Hanelt and Janovy, 2004b).

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The first two transmission mechanisms are unlikely to figure prominently in the natural life cycle of gordiids. Dorier (1930, 1935a, b) reported that gordiid larvae were able to encyst freely in the absence of a host, for example on aquatic vegetation. Terrestrial arthropods could be infected by eating or drinking in the periphery of a water source. At that time, it was not elucidated whether these life cycle stages were actually infective. Since these reports by Dorier, free-living cysts have not been reported again. The second method of transmission has been supported by several studies, indicating the infection of definitive hosts by feeding of gordiids larvae (Hanelt and Janovy, 2004b; Inoue, 1962). Both of these transmission mechanisms rely on agile and highly mobile larvae to allow for their positioning in areas maximizing host encounters. However, gordiid larvae have often been described as being immobile, certainly not able to travel distances required to navigate from the bottom of a lake or stream to the edge’s surface (Meissner, 1856; Poinar and Doelman, 1974; Villot, 1874a). Other hosts must therefore be responsible for bridging the gap between the free-living aquatic environment and parasitic terrestrial environment.

3.2. Paratenic Hosts The paratenic host was originally described as one that bridges a trophic gap between the intermediate and the definitive host (Baer, 1951). Since the sole role of this type of host is transportation, the parasite does not undergo development while within this host. Paratenic hosts have been divided into different categories; spurious and true. Those able to become infected but unable to transmit this infection have been called ‘‘spurious’’ paratenic host (Schultz and Davtian, 1954). Only those animals able to become infected and able to transmit this infection are referred to as ‘‘true’’ paratenic hosts, or simply paratenic hosts. The idea of the involvement of a paratenic host within the gordiid life cycle is an old one, starting with Meissner (1856). He suggested that aquatic insects could be a natural host able to carry gordiids


261

from water to terrestrial hosts. The gordiid life cycle stage capable of surviving for an extended time in a paratenic host, the cyst (Figure 5), was first thoroughly researched about 20 years later (Villot, 1874a, b). This discovery, however, did not clear up the mystery of the life cycle since the cysts were first reported in hosts unlikely to play a role in the natural gordiid life cycle: annelids, snails and fish. Subsequent data did show cysts present in aquatic insects, such as mayflies and midges (see e.g. White, 1966, 1969). Although the life cycle of gordiids was able to be pieced together by these findings and subsequent infection trials (Hanelt and Janovy, 1999, 2004b; Inoue, 1960b, 1962), studies have only recently focused their attention on the host specificity and significance of gordiid cysts within spurious and true paratenic hosts. 3.2.1. The identity of paratenic hosts The paratenic host specificity of the cyst stage of various species of gordiids has been difficult to quantify. Although studies have reported on naturally and laboratory-exposed paratenic hosts, the results of these studies remain difficult to interpret. Studies investigating the infection with cysts in natural populations (see e.g. White, 1969) have been able to provide data on the kinds of animals carrying gordiid cysts. However, these studies were not able to provide information on the host specificity of individual gordiid species since infection with multiple species of gordiids could not be ruled out. Only recently have morphometric methods been described allowing discrimination of species of gordiids based on non-adult characters (Hanelt and Janovy, 2002). Laboratory infection experiments have similar problems, in which most of these experiments did not involve laboratory-reared hosts and did not contain control groups. It was recently found in a survey of 1000 snails that almost 40% were naturally infected with gordiids (Hanelt et al., 2001). Thus, infection experiments require carefully monitored control groups to avoid false-positive samples. Recently, a study of the host specificity of gordiid cysts was conducted considering these factors. Hanelt and Janovy (2003) studied the paratenic host specificity of three Nearctic gordiid species. Freshly hatched gordiid larvae were

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used to expose laboratory-reared putative paratenic hosts. These host groups were chosen due to their likelihood of coming into contact naturally with gordiid larvae at the bottom of a river or stream. Putative paratenic hosts included a range of organisms from annelids, mollusks and arthropods to fish. Incredibly, of the 12 species exposed, all but two became infected with gordiid cysts (Table 2). All three gordiid species showed an identical infection pattern. Two putative paratenic hosts did not become infected, the water mite and the triclad flatworm. Failure of infection was most likely caused by behavioural traits rather than incompatibility. Both of these animals require the presence of prey or detritus material to initiate feeding behaviours. All other groups became infected with cysts within the gut lining or the haemocoel. Of all paratenic hosts able to become infected with cysts, only those that could act to bridge the gap between aquatic and Table 2 Prevalence of infection of putative paratenic hosts exposed to cysts of three gordiid species Putative host species

Platyhelminthes: Turbellaria Dugesia tigrina Oligochaeta: Tubificidae Limnodrilus hoffmeisteri Mollusca: Gastropoda Physa sp. Crustacea Hyalella azteca (Amphipoda) Cypris sp. (Ostracoda) Arachnoidea: Hydracarina Oxus sp. Insecta Tenebrio molitor (Coleoptera) Tanitarsis sp. (Diptera) Caenis sp. (Ephemeroptera) Neotrichia sp. (Trichoptera) Culex tarsalis (Diptera) Vertebrata: Osteichthyes Notropis ludibundus

Gordius robustus

Paragordius varius

0

0

0

85

80

100

75

95

95

60 50

70 60

95 65

0

0

0

60 45 100 100

55 40 95 100

85 70 100 100

a

a

a

100

100

100

Modified from Hanelt and Janovy (2003). Denotes destruction of cysts by immune reaction of host species.

a

Chordodes morgani


263

terrestrial environments were considered a true paratenic host, while the others were considered spurious paratenic hosts. 3.2.2. Natural paratenic hosts For Meissner’s (1856) hypothesis (that aquatic insects that metamorphose into flying adults are the most likely natural paratenic hosts involved in the gordiid life cycle) to be correct, three conditions must be met by this host (Hanelt and Janovy, 2004a). First, the cysts must survive the metamorphosis of this host. Inoue (1960b, 1962) tested the survival of Chordodes japonensis cysts through the metamorphosis in midges, mayflies, and mosquitoes. Infection experiments of cysts from adult insects resulted in infections in the definitive hosts, mantids. Hanelt and Janovy (2004a) tested the survival of three common Nearctic species of gordiids, and found survival in laboratory-infected midges. This study also confirmed survival in mayflies infected naturally with Gordius difficilis. Second, the natural paratenic host should not be able to mount an immune reaction efficient enough to kill cysts. Two different types of immune reactions to cysts have been described to cysts of C. japonensis (Inoue, 1960b). The reaction by mayflies was to surround the cyst in a thin spherical envelope of haemocytes. The more severe reaction was by midges, which not only consisted of a layer of haemocytes but also included a melanization reaction. This latter immune reaction caused a marked decrease in the viability of the cysts. This kind of melanization has also been reported in mosquito larvae leading to the destruction of cysts by N. occidentalis (Poinar and Doelman, 1974) and by P. varius, G. robustus and C. morgani (Hanelt and Janovy, 2003). However, experiments with the latter three gordiid species using midges, mayflies and trichoperans showed haemocyte encasement only in a few instances without melanization (Hanelt and Janovy, 2003). The viability of P. varius cysts was found to be unaffected by this mild type of host immune reaction (Hanelt and Janovy, 2004a). The third condition necessary for aquatic insects to act naturally as gordiid paratenic hosts is that cysts must not alter the morphology or

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development of the paratenic host. White (1969), studying natural infection in aquatic insect larvae in Wisconsin, USA, found that individuals infected with cysts varied morphologically. The wing pads of mayflies were found to be smaller compared with uninfected individuals. More important, it was also reported that some species of Diptera and Trichoptera failed to pupate, making their role as true paratenic host impossible. However, multiple studies investigating metamorphosis of infected insects exposed in the laboratory did not report developmental irregularities of hosts (Hanelt and Janovy, 2004a; Inoue, 1960a). Additionally, Hanelt and Janovy (2004a), studying naturally infected mayflies, found no morphological differences between infected and uninfected hosts. Ultimately, since the three conditions necessary to have aquatic insects act as natural paratenic hosts in the gordiid life cycle, it is clear that insects play an integral part in the life cycle of gordiids. However, further studies are necessary to study whether organisms raised and exposed in the laboratory act and develop differently than in nature. Furthermore, research needs to focus on whether changes in micro- and macrohabitat through the geographic range of gordiids may require local adaptations to different paratenic hosts. 3.2.3. Paratenesis Paratenesis is defined as a parasite being transferred from one paratenic host to another (Beaver, 1969). Paratenesis has been documented in species of trematodes (Shoop, 1988), cestodes, (Halvorsen and Wissler, 1973) and nematodes (Daengsvang, 1968). Recent evidence appears to indicate that gordiid cysts can also be transmitted between paratenic hosts through paratenesis. Hanelt and Janovy (2003) found that although the free-living aquatic flatworm Dugesia tigrina did not become infected when exposed to larvae of P. varius, G. robustus, and C. morgani, flatworms could become infected by keeping them in tanks with infected snails. Thus, cysts were transferred from snails to the flatworms feeding on them. de Villalobos et al. (2003) found that P. varius cysts from snail tissues fed to fish established within the musculature and intestinal wall of the fish.


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Although in the latter study it was assumed that some cysts may have been recruited from unencysted larvae within the snail tissue, it is clear that many cysts were transferred from the snail to the fish. Hanelt and Janovy (2004a) found that P. varius cysts could also be transferred from snails to species of beetles, and in one case to an unsuitable (one which could not serve as a definitive host) cricket host. Since gordiid cysts appear to lack host specificity, mechanisms must exist allowing the recovery of cysts in spurious paratenic hosts, which are considered lost infective stages. Examples of these hosts include copepods, fish, amphipods, etc. However, through paratenesis these lost infected stages can be recovered and can therefore reenter the normal gordiid life cycle. Scavenger or predatory mayflies could easily pick up cysts from food items. It appears that the transmission of gordiids from aquatic environments to the terrestrial environment is not as random as it first appears. It has often been assumed that parasites with high fecundity transmit their progeny by pure chance (or luck!), like randomly shooting a shotgun; eventually pellets will hit a target. Gordiids, through the mechanism of paratenesis, however, appear to get several attempts at getting to a paratenic host: this process is much more like a pinball game, in which cysts ‘‘bounce’’ around the environment until successfully transmitted. Future studies are needed to investigate the impact of long-lasting cysts that are able to persist for extended periods and are able to move through multiple hosts during their life cycle. Due to the persistence of multiple generations of cysts at any point in time, this system is ideal for population and selection studies. 3.2.4. The role of the paratenic host The evolution of parasite life cycle complexity continues to be a hot topic of discussion (Parker et al., 2003). Although life cycle diagrams are usually drawn as static representations (Olsen, 1986), some have argued that life cycles are only fixed when perceived over the lifetime of human investigators (Combes, 2001). Over evolutionary time,

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most life cycles are probably astonishingly fluid. Many explanations have been given for the mechanisms leading to the change in complex life cycles. These can be broadly placed into two categories: accident and adaptation (Poulin, 1998b). Recent evidence has suggested that adaptation might be involved in some systems during the addition of hosts (Brown et al., 2001; Choisy et al., 2003; Morand et al., 1996; Robert et al., 1988), although the proximate mechanism for such an addition is unclear. Dogiel (1966) contended that the acquisition of intermediate hosts often began with the addition of a host as a paratenic host. Furthermore, Ryzhikov (Barus, 1964) believed that paratenic hosts were an intermediate step in the addition or deletion of an intermediate host. Although the involvement of a paratenic host in the evolution of complex life cycles could be critical, surprisingly few studies have been conducted to document the role of this type of host (but see Choisy et al., 2003). Especially important are the benefits a parasite gains by using a paratenic host. These benefits could ultimately lead natural selection to include permanently this host in the parasite life cycle. Gordiids can provide a good model with which to study the role of paratenic hosts, beyond the definition of this host as capable of bridging a trophic gap. Ryzhikov (Barus, 1964) provides an outline of additional roles that should be investigated. The summary of these roles provided below are presented in an expanded context of the suggestions provided by Rhzhikov. (a) Increasing infectivity of parasites. Inoue (1962) found that infection of definitive hosts with C. japonensis cysts increased prevalence by up to 65% over infection with larvae. Hanelt and Janovy (2004b) showed that the infectivity of P. varius increased dramatically when exposed to cysts compared with larvae. For G. robustus, infection of the definitive host was found to be possible with cysts, not larvae (Hanelt and Janovy, 1999). These findings suggest that infection by larvae is much less effective than infection by cysts and has become impossible in some gordiid species. Although the mechanisms leading to higher infection ability of cysts is unknown, it could be caused by benefits from being surrounded by a cyst wall


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(e.g. protection from mechanical damage) or physiological benefits of going through the cyst stage. (b) Increasing parasite longevity. Gordiid larvae have been reported to be viable from 1 to 4 weeks (Meissner, 1856; Mu¨hldorf, 1914). P. varius larvae remained active for only 10 days posthatching. The rate of cyst formation of this species decreased to zero after 30 days, indicating complete loss of viability of the larvae (Hanelt and Janovy, 2004b). Similar rates of larval survival were found in C. morgani (Hanelt, personal observation). Cysts, however, can remain viable for much longer. Cysts of P. varius remained infective for at least 6 months (Hanelt and Janovy, 2003). In addition, G. robustus cysts, which were mechanically excysted 12 months postinfection were found to move in an identical manner to freshly hatched larvae (Hanelt, personal observation). Data of field-collected physid snails during the spring indicated that infections occurring during the late summer and early fall persist in snails through May, 8–10 months following the formation of the cysts (Hanelt et al., 2001). It is clear that the cyst stage greatly increases the window of time gordiids have to be transmitted to their definitive hosts. (c) Recovery of lost infective stages. Evidence suggests that cysts can be transferred between paratenic hosts (see Section 3.2.3). This passage may allow cysts being formed within spurious paratenic hosts to be transmitted to a definitive host. Thus, larvae encysting within hosts, which are incapable of transferring the parasite to the definitive host, could be transmitted to a paratenic host capable of such transition. In addition, this type of paratenesis could cause cysts to be transmitted following the death of a host to a scavenger animal. Transmission between paratenic hosts may thus allow cysts to remain viable for an extended period by being passed from one host to another. (d) Dispersal/maintenance at a site. Aquatic insect paratenic hosts may also serve a role in the dispersal of cysts. Aquatic insects harbouring cysts that have metamorphosed into flying adults are

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capable of distributing parasites over wide geographic areas. For example, using mitochondrial DNA markers, it has been established that trichoptera species can regularly disperse up to about 20 km (Myers et al., 2001). This distance could likely increase based on weather factors such as temperature, relative humidity, wind and atmospheric pressure (McManus, 1988). Thus, the paratenic host could be an important factor in determining the genetic structure of populations of gordiids. Furthermore, it has been suggested that the innate behaviour of insects might be responsible for maintaining gordiids at a particular site (Hanelt and Janovy, 2004a). Gordiids, as well as their paratenic hosts, commonly inhabit rivers and streams. Within this environment, inhabitants are constantly being displaced unidirectionally (i.e. downstream). Calculations of several macroinvertebrate taxa suggested that downstream displacement can be as high as 10 km during a single generation (Hemsworth and Brooker, 1979). Displacement can occur through two types of drifts (Brittain and Eikeland, 1988). Constant drift, which is the background drift, normally occurs in flowing waters; and catastrophic drift, which is rare and occurs when high discharge physically disturbs the substrate. These drift events have been shown to affect most aquatic insects capable of serving as gordiid paratenic hosts, as well as gordiid adults (Thorne, 1940; Hanelt, personal observation). Drift also possibly affects other life cycle stages such as the egg strings, eggs, and larvae. Only C. morgani may mitigate drift of eggs by attaching them to the substrate (Hanelt and Janovy, 2002). Despite the possibility of drift, a recent study showed that gordiids are found in 70% of isolated first- and second-order streams in Lancaster County, NE, USA (Hanelt et al., 2001). This finding suggests that a mechanism exists to compensate for drift displacement. The persistence of upstream populations of invertebrates despite stream drift has been termed the ‘‘stream drift paradox’’ (Hershey et al., 1993). One hypothesis to explain this paradox is by the ‘‘colonization cycle’’ (Mottram, 1932; Mu¨ller, 1982). This hypothesis states that the upstream-directed flight behaviour exhibited by many freshwater aquatic insects compensates for the gradual downstream movement


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of insect larvae (Mu¨ller, 1982). More recently, this same compensational effect has also been shown for active upstream movement under water by aquatic insect larvae (So¨derstro¨m, 1987). Gordiids may ultimately benefit from the colonization cycle by being transported by their paratenic hosts upstream, compensating for drift and allowing gordiids to remain in the headwaters of many rivers and streams. (e) The significance of the paratenic host in the evolution of the parasites. This is certainly the most important question raised by Ryzhikov (Barus, 1964). Regrettably, with the current paucity of data, this question cannot be addressed. Future studies on the life cycles of gordiids, especially nectonematids, must be conducted and combined with existing phylogenies to uncover the evolutionary history of the paratenic host within this phylum. (f) Conclusion. Studies on the life cycle and ecology of gordiids over the last 10 years have allowed us a glimpse into the role of a paratenic host beyond the definition of a transport host. Ultimately, attempting to extend the above-described role of gordiid paratenic hosts to other groups of parasites must be made with caution. As Dogiel (1966) has pointed out for the role and origin of intermediate hosts, ‘‘ythe hypotheses of the origin of the intermediate hostsysuffer from the attempt to place all existing types of host–parasite relationships in one scheme, true for all parasitesy It is the task of investigators to study the origin of the case in hand individually’’.

3.3. Experimental Life Cycles The domestication of gordiid has proven extremely difficult. Many attempts made over the last 100 years at completing the life cycle experimentally have failed. These studies usually ended prematurely yielding adult or juvenile worms from experimentally infected hosts (Hanelt and Janovy, 1999; Inoue, 1962; Sva´benı´ k, 1925), but failed at successfully mating worms. The main reason for making culturing difficult is the long developmental time within the definitive hosts.

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For example, Hanelt and Janovy (1999) isolated G. robustus using Mormon crickets as definitive hosts naturally. To make this system more amenable to laboratory culture, Gryllus firmus was used as a host. Maturation time for G. robustus within the definitive host is up to 3 months, although the average adult life span of adult G. firmus is about 30–40 days. Several years later, Hanelt and Janovy (2004b) reported the successful domestication of P. varius (Figure 6). This species was found to be much more amenable to lab rearing. The most important feature is that the worms complete development within definitive hosts in as few as 27 days. Thus, crickets with short life spans can be used as hosts. This includes the easily available ‘‘feeder cricket’’, Acheta domestica, which are available at most pet stores for feeding pet reptiles. In this laboratory-maintained life cycle, Physa sp. is used as a paratenic host. This aquatic snail is long-lived, can maintain cysts for more than a year, and can get infected with hundreds of cysts. In

Figure 6 Laboratory life cycle of Paragordius varius (modified from Hanelt and Janovy, 2004b). Note the use of snails as paratenic hosts rather than insects.


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addition, Physa sp. can easily be reared in the laboratory. The P. varius life cycle can be completed in the laboratory in as few as 45 days with minimal effort at rearing hosts. This experimental life cycle can now be used to investigate novel facets of gordiid biology (such as their behaviour), or to investigate field observations in a very controlled environment (see e.g. Section 3.5). In addition, since material of various life cycle stages is available from this worm species on a consistent basis and in a controlled manner, the study of nematomorphs has been made more tractable, especially using molecular techniques. Ultimately, we hope that the P. varius life cycle will become a model system to investigate gordiid biology as well as parasite–host interactions.

3.4. Sex Ratio The literature is full of reports indicating that the sex ratio of gordiid adults collected in nature is skewed. However, the direction of the skew is not consistent. In some collections, females are predominant. Watermolen and Haen (1994) report collecting 67 G. robustus in Wisconsin, 66 of which were females. More often, however, a predominance of males is found. Cochran et al. (2004), enumerating G. difficilis worms within Gordian knots collected during a 32-year period in six Midwestern states of the United States, reported that of the 1391 worms, 1205 were males. Similarly, Thomas et al. (1999), studying Euchordodes nigromaculatus in New Zealand, collected only 61 males and no females. Other studies have shown that males and females were collected in equal ratios (de Villalobos and Camino, 1999; Valvassori et al., 1988). The discrepancies in these studies have recently been clarified by two investigations involving multiple samples from a site at different times of the season. Bolek and Coggins (2002) studied the seasonal occurrence of G. difficilis in Wisconsin by conducting a worm removal experiment over 3 years. The samples revealed that the same population of worms can change from being female bias early in the mating season, to being male bias late in the season. In total, all years

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of data showed an overall male bias. Poulin (1996) collected Gordius dimorphus from New Zealand’s South Island at two time points during the same year. During the spring, the sex ratio was female-biased; whereas during the summer, the sex ratio was highly male-biased. These two studies suggest that rather than a real difference in sexes being produced during meiosis, observed differences are instead due to ecological factors. Hanelt and Janovy (2004b), studying a laboratory maintained isolate of P. varius, supported this contention. Of worms produced during several generations, there was no statistically significant difference between the number of males and females produced. Ecological causes that may be contributing to the often-reported skew in nature have never been empirically investigated. However, the literature offers several suggested explanations. Poulin (1996) proposes that variation in life spans causes the accumulation of males towards the end of the mating season. In addition, he suggests that males and females may have different developmental times, leading to insects hosting the larger worms (in this case males) to release these parasites later in the season. In order to accept these explanations, additional field and laboratory experiments will be necessary. Ultimately, a genetic test able to discriminate sexes should be used to determine sex ratio of various life cycle stages, such as eggs, larvae, cysts, and juvenile worms. Rapid molecular methods have, for example, been employed to identify the sex of nonadult life cycle stages of schistosomes (Walker et al., 1989).

3.5. Distribution 3.5.1. Introduction Historically, the manner in which gordiid worms is distributed in space and time has been difficult to quantify. Data of their spatial distribution have largely come from random or semi-random collections of adult worms. These collections either have been chance


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encounters or were made in areas where worms happen to overlap with human activity (see Section 3.7). Distribution data have been further biased by limiting collection to a single time point. Recently, several studies have been conducted on gordiids to address their distribution both at small scales and temporally. 3.5.2. Distribution at fine scale Recently, new methods of quantifying gordiids have led to very different perceptions of how these worms are distributed in limited geographic areas. Several studies have used meta-analysis to determine distribution within individual states in the United States. These studies used collection data of adults to identify sites. Chandler (1985) compiled 18 years worth of data for the state of Tennessee; 18 sites containing gordiids were identified. Watermolen and Haen (1994) compile 69 years of data for the state of Wisconsin; 15 sites were identified as containing worms. However, four of these sites were unsubstantiated verbal reports of sightings. These datasets give the impression that gordiids are geographically widespread within defined areas, although their distribution is spotty. Hanelt et al. (2001) found that basing distribution data on the presence of adults is problematic. First, the detection and collection of adults are extremely difficult. Gordiid adults are easily missed due to their seasonality, short life span (4–6 weeks), cryptic nature, hiding behaviours during mating, and the lack of effective sampling technique. A single collection locality, such as a small stream, can contain worms entwined in vegetation at the periphery, between rocks on the bottom, buried in the substrate, free swimming, wound around submerged sticks, or entangled in a knot near the substrate. Thus, thorough gordiid collection entails an extensive search of possible localities. Second, the finding of an adult gordiid does not necessarily indicate that the life cycle is occurring at that site. Thus, as mentioned above, finding a gordiid in a toilet bowl only serves to indicate that the definitive host carried the parasite into an unnatural environment. Thus, the use of gordiid cysts was suggested as an indicator for the presence of gordiids (Hanelt et al., 2001). This technique is

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preferable since many of the problems with using adults as indicators are eliminated. One female gordiid can produce several million cysts. Cysts are also long-lived, and are able to persist in snail paratenic hosts for more than a year (Hanelt, unpublished). Furthermore, the sampling of gordiids in this manner can be standardized by quantifying number of cysts per specific paratenic host. Using gordiid cysts within Physa sp. (Mollusca: Pulmonata), this technique was capable of identifying 35 out of 50 sites positive for gordiid cysts within Lancaster County, Nebraska (2193 km2) (Hanelt et al., 2001). Three years of intensive sampling had resulted in the detection of gordiid adults at only a single site within the same area. Of 1000 snails tested (20 from each of the 50 sites), 8073 cysts were found in 395 snails. Similarly, this study was repeated in Keith County, Nebraska (2874 km2), separated by 300 miles from Lancaster County. (Grother and Hanelt, unpublished data). In this area, 4 years of intensive sampling yielded adult gordiids from 3 sites. Testing for cysts in snails, of 15 sites tested, 10 proved positive for cysts. Of 251 snails examined, 2674 cysts were found in 75 snails. The results of these studies clearly indicate that the perception of how gordiids are distributed is greatly influenced by sampling methodology. Based on adult collections, gordiids appear to be rare and unevenly distributed. However, based on cysts, gordiids appear to be common and widespread (Hanelt et al., 2001). At the time these studies were made, the main drawback of this method was that species could not be determined from cyst morphology (Hanelt et al., 2001). Therefore, it was impossible to determine the biodiversity of gordiids by studying cysts. Subsequent morphometric techniques were developed allowing for the identification of species-specific characters and sets of characters making species-level identification of gordiid cysts possible (Hanelt and Janovy, 2002). In addition, the technique of sampling for cysts will allow investigators to identify sites that are worthwhile for intensive sampling of adults. This will make the study of gordiids more tractable, since lack of study material has been cited as a reason for making its study difficult (de Villalobos and Voglino, 2000; Inoue, 1958; Reutter, 1972).


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3.5.3. Temporal distribution Bolek and Coggins (2002) is the only study which seasonally tracks a population of gordiids. This study included 3 sample years (1996–1999) and involved the bimonthly collection and enumeration of specimens of G. difficilis. The combined data show that worms appear in June or July; their numbers peak in late July and early August, and disappear by September (Figure 7). When the data are considered by year, variation in number and timing is apparent. Population fluctuations were also noticed by year. In 1996, very few worms were collected, compared with the other years. Population peaks were in July in 1998, but not until August in 1997. Much more work, similar to this, on many more species is needed to understand fully the seasonality of gordiids. 3.5.4. Conclusion and future study The last decade has provided several important studies producing a framework for future work in the temporal and geographic distribution of gordiids. Large-scale collection efforts of adult worms in understudied areas will undoubtedly lead to the description of countless

Figure 7 Number of Gordius difficilis adults recorded and removed from man-made ponds. Based on data from Bolek and Coggins (2002).

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new species. In addition, since most gordiids have only been described from single localities, it is possible that many species have a very limited distribution. Large-scale sampling efforts would possibly lead to the discovery of new species in even the most well-sampled areas of the world. The use of cysts in paratenic hosts as indicators of gordiid distribution appears as a viable alternative to adults. Although morphological characters of cysts can be used to determine species-level identification, molecular markers could be established allowing individual or pooled paratenic hosts to be tested for the presence of cysts.

3.6. Humans and Gordiids 3.6.1. Gordiids in sources of potable water Numerous reports exist of nematomorphs ‘‘contaminating’’ city water systems. Cappucci and Schultz (1982) provides a detailed review of reported cases since 1870. These cases occurred in countries within a wide spectrum of economic development, from Zimbabwe and Malaysia to England and United States. More recently, such cases have been reported from areas such as Bosnia-Herzegovina (Pikula et al., 1996), Turkey (Aydemir et al., 1996) and Australia. Often these reports associate the presence of gordiids in water systems with water quality. For example, in a leading Australian farming magazine, an author wrote that gordiid adults (which do not feed nor have a working mouth) wreak havoc on water systems by eating out water filters (Walker et al., 1989). Gordiid worms within water supplies do not pose danger to humans, but are simply an indication that insectdefinitive hosts are capable of getting into the water source. 3.6.2. Gordiids around the home Gordiids are also frequently noted in and around the home. Worms often appear in the bathroom, where standing water is typical; examples are toilets, showers (see e.g. collection locality of P. varius in


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Hanelt and Janovy, 2002), bathtubs (Spiridonov et al., 1992) and hot tubs. Often, the worms are carried by their insect-definitive hosts. In other cases, crickets are disposed of in the toilet after being killed, only to have the worms within the cricket wiggle back up the pipes. After subsequent use of the facility, by either adults or especially children, people can become unduly alarmed at the sight of undulating worms (Herter and Neese, 1989). In another case brought to the attention of one of the authors (BH), a man recently returned home to Washington State from travel in Australia. Three days after his return, he was aghasted to find a gordiid in his bathtub. The worm was brought into the local health clinic where it was processed. The serial section revealed a male gordiid worm (Figure 8 is Plate 4.8 in the Separate Color Plate section). The worm was likely carried into the tub by the insect host, either before or during use, or brought in through the pipes. Pet owners also have reported finding worms within their pet’s water dish, leading to unnecessary trips to the veterinarian. In addition, worms are often reported from temporary standing water in the yard or the driveways after heavy rains. 3.6.3. Pseudoparasitism Since many of the reports of worms associated with humans are simply due to the spurious presence of worms in or near places of human habitation, reports of gordiids ‘‘infecting’’ humans must thus be interpreted with great caution. In one unpublished case, a worm was recovered from the underwear of a woman in Lincoln, Nebraska (Figure 9). The assumption was that the worm came from inside the patient, but no evidence was given for this assumption. Although exactly how the worm came to be inside the woman’s clothing is unknown, it is most likely that the worm was carried in by the host. However, it is clear that in some cases gordiids are resident within the digestive systems of humans. However, these cases do not represent real parasitism, but rather pseudoparasitism. Pseudoparasitism is defined as a parasite present in a host due to accidental circumstances. This host organism is not a natural host and usually the parasite does

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Figure 9 Sample of Paragordius varius female adult worm recovered from the ‘‘underclothing’’ of an adult female. Although the specifics of this case are unknown, this worm did not likely infect the woman but rather entered the clothing by being carried in by the insect host. This sample was brought to the attention of the authors by John Janovy Jr., University of Nebraska-Lincoln, USA.

not thrive (and often does not survive for extended periods) in this foreign environment. Most, if not all, cases reporting ‘‘infection’’ of humans with gordiids are instances of pseudoparasitism. For example, in a recent case, a girl in Korea was found to have vomited two Gordius sp. worms (Lee et al., 2003). It is clear from this report that the girl ate an insect (which looked like a cricket) shortly before she vomited. The most likely scenario for this event is that the cricket eaten by the patient was itself hosting the gordiids, which were subsequently released in the gastric juices of her stomach. Similar reports exist for worms vomited by a domestic cat (Saito et al., 1987) and by a domestic dog (Horton, 1986). Numerous reports exist of patients passing worms per rectum and per urethra (Beaver et al., 1984). In several instances, the infection of the urinary tract was reported. In one case, an adult male was admitted to a hospital in Brazil after having passed worms per urethram. While in the hospital,


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the patient expelled an additional adult worm, which was found in his chamber pot (de Lucena and Campelo, 1975). These reports are plausible, but since the documentation of most of these cases was less than thorough, it is unknown whether these patients were even suffering from pseudoparasitism. Furthermore, no diagnosis of an active case of infection with gordiids has ever been reported. It is clear that the sole method of infection of humans by gordiids is through the ingestion of the adult form. This could occur through the ingestion of adults in untreated water, or through the ingestion of infected insect-definitive hosts. Herter and Neese (1989) urged doctors to distinguish between pseudoparasitism from true helminthic infection. Misdiagnosis and ignorance of the benign nature of these worms has caused patients to undergo undue stress and financial burden of unnecessary regiments of antihelminthics (see e.g. Smith et al., 1990).

4. HOST BEHAVIOURAL ALTERATIONS 4.1. Introduction As seen before, hairworms are parasitic in arthropods when juveniles but they are free-living in aquatic environments (shallow puddles, marshes, streams and ponds) as adults. Because most host species are terrestrial arthropods, returning to water is a challenging task for the large majority of hairworm species. How these parasitic worms overcome such a problem is undoubtedly one of the most fascinating aspects of their ecology. It has been first argued that hairworms would rely on chance: larva, when mature, just waits until the hosts are close to water on their own and take this opportunity to emerge into water. Also this is possible in some cases; it has also been hypothesized that hairworms would manipulate the behaviour of their host to enhance their probability of reaching an aquatic habitat. Placed in a general context, this hypothesis is not an odd idea as, indeed, parasite-induced alteration of host phenotype is now documented for a wide range of parasites (for a review, see Barnard and

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Behnke, 1990; Combes, 1991; Moore, 2002; Poulin, 1998b). Numerous host phenotypic traits can be altered by parasites (e.g. behaviour, morphology and physiology), and these alterations can vary greatly in their magnitude, from slight shifts in the percentage of time spent in performing a given activity to the production of complex and spectacular behaviours (Moore, 2002; Poulin and Thomas, 1999). In the case of hairworms, hosts harbouring a mature worm have been hypothesized to display a behaviour originally not present in their repertoire: they seek water and enter it (Begon et al., 1996; Blunk, 1922; Dawkins, 1990; Jolivet, 1946; Poinar, 1991b; SchmidtRhaesa, 1997a; Thorne, 1940). From the parasite’s perspective, such a behavioural change is likely to be adaptive as it ensures the adult worm to be released in an appropriate location for reproduction. However, until recently the water-seeking behaviour of insects parasitized by hairworms was only supported from anecdotal observations. Most popular ones included, for instance, insects flinging themselves into toilets and dog watering dishes.

4.2. Study System Recently, Thomas et al. (2002) provided additional information concerning the behavioural changes of insects harbouring hairworms. Observations were made in southern France in a particular place; it was a private swimming pool located near a forest largely crisscrossed by several streams in which adult nematomorphs are commonly found during the summer. Every summer night, several insects (at least among the nine following species: Nemobius sylvestris infected by Paragordius tricupidatus; Meconema thalassinum, Pholidoptera griseoptera, Uromenus rugosicollis, Ephippiger cunii, Barbitistes serricauda, Leptophyes punctatissima, Antaxius pedestris and Yersinella raymondi infected by Spinochordodes telinii) ‘‘commit a mistake’’, instead of releasing their worm(s) into the streams, they jump into the water of the swimming pool (Thomas et al., 2002). A behavioural sequence of this phenomenon is, for instance, illustrated on the most abundant cricket species, N. sylvestris (Figure 10 is Plate 4.10 in the


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Separate Color Plate section). The majority of host species entered water during the first part of the night (i.e. before 1–2 a.m.). Except for the species A. pedestris and M. thalassinum, for which uninfected individuals could sometimes be found on the concrete area of the swimming pool, all the insect species mentioned before, when found around the swimming pool, were infected by a hairworm and sooner or later entered the water of the swimming pool. Insects entered the water by jumping into it or by entering gradually. After the host had entered the water, the emergence of the worm could be immediate (e.g. S. telinii emerging from M. thalassinum) or could take several minutes, i.e. after the host had drowned (frequently the case for P. tricuspidatus emerging from N. sylvestris). However, in the latter case, Thomas et al. (2002) noted that just after the host had jumped into the water, and was thus in contact with a liquid medium, the worm emerged 1–2 cm and returned inside the host, presumably because the end of the cricket’s abdomen was not directly in contact with water (the worm was always seen to emerge fully within 2–5 min). A few seconds after complete emergence from the host, the worm actively swims away and leaves its host. In accordance with the idea that the water-seeking behaviour in infected arthropods is a parasitic adaptation aimed at reaching a reproductive habitat, it is interesting to mention that the same behaviour was observed in two spider species (Pistius truncatus and Olios argelasius) harbouring mermithid nematodes (see also Maeyama et al., 1994; Thomas et al., 2002). Mermithids are phylogenetically distantly related to hairworms but they have similar biology (i.e. they also develop inside a terrestrial arthropod and are free-living in aquatic habitats when adults). This suggests the existence of an evolutionary convergence between nematomorphs and mermithids in their effect on host behaviour.

4.3. Host Behaviour In addition to these observations, field and laboratory experiments in an Y-maze clearly indicate that crickets (N. sylvestris) infected by the

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nematomorph P. triscupidatus are more likely to jump into water than uninfected ones (Figure 11). Despite the significance of this result, the idea that the manipulation involved water detection from long distances by infected insects was not fully supported. Indeed, the results obtained by Thomas et al. (2002) were also in accordance with another, and maybe more realistic, hypothesis given the ecological context. For instance, we must remember that the necessity of water detection in this manipulation becomes questionable when we consider the ecological conditions in which this host–parasite system evolved. A behavioural alteration induced by nematomorphs could be the induction of an erratic behaviour: infected crickets would leave their microhabitat but in no particular direction. Given the abundance of streams in these native forests, this would undoubtedly bring the cricket close to a stream. Alternatively, if insects routinely encounter water during a time scale appropriate to worm development, there may be no need to induce erratic or water-seeking behaviour. In accordance with the former idea, all the crickets that were found in atypical habitats (car park, inside a hotel) harboured a worm. Thomas and colleagues even noticed that about 80% of the infected crickets collected in a summer season come from these atypical habitats, not from the edges of the swimming pool (F. Thomas, unpublished data). Along the same idea, it can be mentioned that even when the swimming pool was protected with a plastic cover at night (i.e. strong reduction of the humidity gradient), the mean number of infected crickets captured around the swimming pool remained unchanged (F. Thomas, unpublished data). Thus, all these results and observations support the idea that water detection from a long distance following a humidity gradient is not involved, at least in N. sylvestris. Once infected crickets encounter water, there is however another important behavioural difference with uninfected individuals. While crickets harbouring a worm often jumped into the water, uninfected ones most of the time were reluctant to enter it. This behavioural difference is undoubtedly a key step in the manipulative process as it allows the hairworm to emerge immediately after its host enters water. Whether infected crickets are attracted by the liquid, or they


Results of the choice experiment in an Y-maze (from Thomas et al., 2002).

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Figure 11

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simply do not perceive the danger linked to the presence of water is unclear. We cannot exclude that infected crickets do not react to a number of outside cues, including water, and therefore end up falling into it rather than avoiding it.

4.4. Host Changes Caused by Manipulation 4.4.1. Physiology and neurology Recent attempts to explore the physiological and neuronal basis of these behavioural changes revealed substantial changes in the brain of insects infected by hairworms (Thomas et al., 2003). Interestingly, the sampling procedure used by Thomas et al. (2003) allowed to disentangle nonspecific disorders by induced parasite from changes directly correlated with the manipulative process. The first category of crickets (N. sylvestris) was called ‘‘night-parasitized’’ crickets (NP) and corresponded to manipulated crickets, i.e. infected individuals captured between 10 p.m. and 1 a.m. near the edge of the swimming pool just before they jumped into water. As a control for this category, uninfected crickets at night were also collected in the nearby forest (‘‘night-uninfected’’ crickets, NU). Third, in order to obtain crickets harbouring a mature worm without being manipulated, manipulated crickets (i.e. NP category) were dissected only the day after their capture, between 1 and 3 p.m. (‘‘day-parasitized’’ crickets, DP), that is to say at a period of the day for which no behavioural change is observed under natural conditions (at least for N. sylvestris; F. Thomas, unpublished data). As a control for this category, uninfected crickets were also captured at night and kept until the day before being dissected between 1 and 3 p.m. (‘‘day-uninfected’’ crickets, DU). Finally, a last category of crickets corresponded to individuals that have released their worm into water (‘‘suicided’’ crickets, S). To obtain this category, infected insects arriving at the swimming pool were visually followed on the concrete area without disturbing them until they entered the swimming pool. Just after the emergence of the worm, the cricket was placed in a dry opaque plastic


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tumbler for 1 h. After this delay, most crickets were vigorous and were then dissected. This sampling design is interesting as it permits several sources of variation to be considered (e.g. infected versus uninfected, manipulated versus non-manipulated, etc.) when interpreting differences in the amount of neuroactive compounds among categories of crickets. Results obtained for polyamines, monoamines and amino acids revealed that the presence or absence of the parasite per se explained the largest part of the variation in compound concentrations: infected individuals display on average lower concentrations than uninfected ones (Figure 12a and b). The existence of several non-specific disorders may appear not really surprising given the relatively large size of the parasite relative to the host. This could

Figure 12 (A) Mean quantities of spermidine, spermine and dopamine among the five categories of crickets. Sample sizes are indicated above each bar (from Thomas et al., 2003). (B) Mean quantities (pmol/mg of proteins7SE) of amino acids among the five categories of crickets. Sample sizes: DU (day uninfected, n ¼ 15), DP (day parasitized, n ¼ 10), NU (night uninfected, n ¼ 24), NP (night parasitized, n ¼ 8) and S (suicided, n ¼ 13) (from Thomas et al., 2003).

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Figure 12 continued.

first result from a competition between the host and the nutrients sensu lato. Thomas et al. (2003) also did not hypothesis according to which this global depletion in dividuals results from infected individuals spending

parasite for exclude the infected inmore time


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searching for water than searching for food. In addition to these nonspecific effects, a significant part of the variation for several amino acids however correlated with the manipulative process. This concerns especially taurine, valine and tyrosine. Infected crickets, during the day, displayed the highest concentration of taurine (Figure 12b). Interestingly, taurine is considered as an important neurotransmitter in insects (one of the most abundant free amino acids in insect brains) and it participates in neurotransmission of mushroom bodies (Sinakevitch et al., 2001). Furthermore, taurine also regulates many biological phenomena including brain osmoprotection (Schaffer et al., 2000). In this context, we might be tempted to speculate that, as suggested long ago (see e.g. Blunk, 1922), hairworms cause thirst in its host during the day in order to motivate it to seek water at night. However, further studies are needed to confirm all these results as, for instance, we cannot determine from this study whether these changes are causes or consequences of the manipulative process. Thomas et al. (2003) also performed a histological study on the mushroom bodies of the brain in order to compare neurogenesis between infected and uninfected crickets. Interestingly, the mitotic index measured by counting all the cells in the M phase in the crickets mushroom bodies exhibited a two-fold increase in infected crickets as compared with uninfected ones (Figure 13 is Plate 4.13 in the Separate Color Plate section). Thus, neurogenesis was doubled in the brain of infected crickets. This strange result, as well as its precise role in the manipulative process, remains enigmatic at the moment. Knowing that taurine (at least in mammals) has been shown to stimulate neurogenesis (Chen et al., 1998), we might be tempted to establish a link between this enhanced neurogenesis and the fact that infected crickets displayed a higher concentration of taurine. Knowing that the mushroom bodies (where neurogenesis takes place) are the main sensory integrative centres of the insect brain, we can also speculate that this abnormal neurogenesis interferes with neural circuitry, perturbing the analysis of the environmental cues by the cricket. It is, for instance, known in rodents that uncontrolled addition of new neurons into existing circuits may potentially disrupt the function of the central nervous system (Feng et al., 2001).

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Alternatively, we could argue that the increased neurogenesis in infected crickets reflects a normal host adaptation when exposed to adverse conditions: a higher neurogenesis could be host-induced to improve environmental perception when this is urgently necessary, for instance in case of severe dehydration. Under this scenario, we could also speculate that the parasite dehydrates its host, thus simulating a dry environment so as to induce in return a cascade of adaptive host processes aimed at finding water rapidly. Preliminary results do not however support this hypothesis as, indeed, we even observe the reverse result: when crickets (A. domesticus) are placed 2 days without water or food, the mitotic index did not increase; instead it was significantly reduced compared to the one observed in normally fed controls (Cayre and Thomas, unpublished data). 4.4.2. Proteomics In a recent study, Biron and Thomas (unpublished data) used proteomic tools to identify the biochemical alterations that occur in the brain of the cricket N. sylvestris when it is driven to water by the hairworm P. tricuspidatus (Nematomorpha). In this study, simultaneously the host and the parasite proteomes were characterized at three strategic stages of the manipulative process, i.e. before, during, and after the expression of the water-seeking behaviour by the host. It was found that the parasitic worm produces effective molecules from the family WNT acting directly on the development of the central nervous system of the host. Interestingly, these WNT proteins display important similarities with those known in insects, suggesting a molecular mimicry. In the brain of manipulated crickets, there were also differential expressions of proteins specifically linked to the neurogenesis, the visual cycle, (circadian rhythm) and neurotransmitter activities. Finally, four proteins were detected in the brain of manipulated crickets for which the function(s) are still unknown (Biron and Thomas, unpublished data). These results support the hypothesis according to which the behavioural changes induced by hairworms in arthropod hosts rely on chemicals, some of them directly produced by the parasite.


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4.5. Manipulation or Collaboration? Until now, the water-seeking behaviour of insects harbouring hairworms has been interpreted as an adaptive parasite-induced behavioural change (i.e. a true manipulation) aimed at reaching a suitable place for reproduction. Although this is possible, other outcomes are theoretically possible. For instance, Biron et al. (2005) recently explored the idea that infected arthropods that would be collaborative, bringing mature hairworms into water, could achieve a higher fitness than those that do not collaborate (or are unable to find water). Conditions for host collaboration rather than host manipulation could be met if the host (i) does not always die after the emergence of the worm, (ii) is able to reproduce, at least partially, when the worm has been released into water (i.e. the host has not been totally castrated by the parasite and it can recover the damage caused by the parasite) and (iii) has no reproductive potential (e.g. complete castration) if it fails to bring the worm into water. To explore this hypothesis, Biron et al. (2005) quantified the life expectancy and the gonad development of infected crickets when allowed to release their parasite into water (i.e. collaborative behaviour) versus infected individuals prevented from bringing the parasite into a suitable aquatic environment (i.e. non-collaborative behaviour). Biron et al. (2005) first showed that hairworm emergence is not lethal per se for the host, instead the cricket can even live several months after having released the parasite. Interestingly, this work also revealed that a substantial proportion of the females (23%) that liberated the worm into water produced eggs or had developed ovaries several weeks post emergence, while such a phenomenon is absent when females were prevented from releasing the parasite. Thus, it seems that for infected females, bringing the parasite into a suitable aquatic environment is indeed a necessary condition before any gonad development becomes possible. The inability of non-collaborative females to produce eggs is observed even after the death of the parasite inside the host, indicating that failing to bring the parasite into water, or waiting for its death, is clearly not a profitable option for infected females. What remains to be elucidated beyond this study, is the quality of the eggs

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produced by collaborative females, as well as, the fitness of their offspring if these eggs are fertilized. Indeed, at this moment, preliminary tests suggest that collaborative females, even when they are recognized as sexual partners by males and are able to take the spermatophore like uninfected females, have serious problems ovipositing. Thus, until this aspect is clarified, we cannot exclude the hypothesis that egg production by certain collaborative females has no evolutionary role (e.g. eggs are not fertile, females are unable to oviposit, etc.), being just a non-adaptive consequence (a ‘‘by-product’’) of the worm emergence. In addition, all these results would also deserve to be confirmed under natural conditions. Indeed, in the field, the collaborative behaviour is likely to come with costs for infected crickets as jumping into water is a risky behaviour, increasing exposure to predators (fish, frogs) and drowning. Results obtained with male crickets were substantially different. From an evolutionary point of view the most important difference is the complete castration of infected individuals regardless of their behaviour (collaborative or not). Hence, it is difficult to argue that males bringing the parasite into water obtain fitness compensation for such collaborative acts.

4.6. Conclusion and Future Studies In conclusion, it is clear that hairworms do not only rely on chance to reach water, instead they alter the behaviour of their hosts in a way that increases their probability to reach an aquatic environment. How? Despite considerable progress, underlying reasons for why infected insects ‘‘capitulate’’ and act in ways that benefit the parasite remain clearly enigmatic. Hairworm–insect interactions remains more than ever a fascinating model of research, full of challenging questions for anyone interested in parasitic strategies based on host manipulation. Further research needs to be integrative with specific efforts made to develop collaborations between parasitologists and researchers from other subdisciplines, especially physiology, neurobiology, and biochemistry. The answer to many questions on the


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evolution of insect manipulation by hairworms might come from this starting convergence between these disciplines.

5. GENERAL CONCLUSION A few years ago, Poulin wondered why there was such a paucity of biologists investigating the phylum Nematomorpha (Poulin, 1998a). Reasons that have been cited as cause for this lack of study include the difficulty in finding reliable and consistent sources of study material, the unknown phylogenetic relationships within this group, the lack of a domesticated model system, and the lack of specific information on life cycles. Missing this most basic information, seasoned biologists as well as students have often overlooked this group in favour of more well-known and reliable systems. However, over the last 10 years, a small but dedicated group of investigators has made a sustained effort in bringing the knowledge of nematomorphs out of the dark ages and into the third millennium. The most immediate need is to understand the basic gordiid biology better. Using a mixture of very traditional and modern techniques, we now have a greater understanding of their taxonomy, structure, ecology, and evolution. Detailed descriptions and redescriptions now exist for dozens of nematomorphs species. The structural features of several nematomorph species and developmental stages have been described, allowing us a glimpse into how these parasites grow from free-living larvae (50 mm long) to adults (some as long as 2 m) within relatively small insect hosts. The distribution of gordiids has finally been documented at several unique levels. Broad scale collections of adults, combined with extensive literature and voucher specimen reviews, has provided us with new information on the extent of individual gordiid species distribution. Focused collections of nematomorphs at much more local scales appear to indicate that gordiids are much more common than previously suggested. The tracking of a gordiid population over several years has revealed fluctuations of numbers and in seasonal timing. Our understanding of their life cycle has improved. Much of the speculation of how gordiids

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transition between their free-living and parasitic life cycle stages has been tested, supporting the involvement of a paratenic host. This work on the life cycle has culminated in the domestication of a gordiid species, which has been used to test field observations and further understand parasite development. Our knowledge of nematomorphs has also benefited from recent studies employing molecular and proteomic techniques. Taxonomy based on ribosomal sequences supports the broad relationships suggested by character-based methods. Finally, studies of the host– parasite interaction at the end of the parasitic phase have begun to provide a framework for understanding how the parasite ensures transmission to aquatic environments. Experiments using proteomic tools suggest that rather than relying on chance, the parasite manipulates the chemical composition of the host’s brain to ensure favourable transmission. Although we do not suggest that the studies undertaken within the last 10 years have allowed us to get a complete understanding of this phylum, we do think that a solid foundation now exists for fruitful continued study. As usual, this research has led to more questions than answers. So, throughout this review, we have provided various avenues of future research that can now be taken, by building on existing data. In addition, many facets of gordiid biology are still completely unknown. One of the main areas within this group in desperate need of further research is the general biology of the nectonematids (marine hairworms). Life cycle, hosts, distribution, and reproduction of nectonematids are completely unknown. Although getting at this information sounds like a daunting task, basing these kinds of studies on what is already known about gordiids (freshwater hairworms) and combining this with modern techniques, the risk in undertaking this kind of study is greatly reduced. For example, the documentation of the nectonematid life cycle has proven extremely difficult due to their rarity and habitat. It is unknown whether there are one or two hosts in the life cycle. Knowing that gordiids use a paratenic hosts, a fair assumption is that the same could be true for nectonematids. Using molecular techniques and nectonematid-specific primers, possible


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paratenic hosts could be screened in large batches to determine infection status. These primers can now be made since a nectonematid ribosomal DNA sequence has recently become available. This kind of method and insight will enable a relatively fast determination of who is involved in the life cycle, and will allow for a targeted study of the hosts. Interestingly, Poulin’s comments on the lack of researchers working on nematomorphs appear to have come at a time when the study of nematomorphs is enjoying a Renaissance. Ultimately, we hope that the information now available on this unique phylum will not only make its study more tractable but also more attractive to others.

ACKNOWLEDGEMENTS F.T. was supported by an ACI jeune chercheur from the Centre National de la Recherche Scientifique. A.S.-R. was supported by the Deutsche Forschungsgemeinschaft, the Deutscher Akademischer Austauschdienst and the European Union’s Training and Mobility of Researchers program. B.H. was supported by a grant from the Initiative for Ecological and Evolutionary Analysis, University of Nebraska-Lincoln (UN-L) and an award from the Special Funds Committee, School of Biological Sciences, UN-L. In addition, we thank Cristina de Villalobos and Fernande Zanca (La Plata, Argentina), Sergej Spiridonov (Moscow), John Janovy, Jr. and Matt G. Bolek (UN-L) for providing useful insights and discussions on the biology of gordiids. Finally, the authors thank Elizabeth Dalbec for proofreading this manuscript.

REFERENCES Adrianov, A.V., Malakhov, V.V. and Spiridonov, S.E. (1998). Fine morphology of the larvae of hairworm Gordius sp. (Nematomorpha). Doklady Akademii Nauk 361, 558–561.

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Aydemir, M., Yorgancigil, B., Demirci, M. and Akkaya, H. (1996). A report of Gordius aquaticus Linnaeus, 1758 isolated from Isparta city drinking water. Turkiye Parazitoloji Dergisi 20, 3–4. Baek, K.M. (1993). Two species of genus Chordodes (Gordioidea, Nematomorpha) from Korea. Korean Journal of Systematic Zoology 9, 221–228. Baek, K.M. and Noh, Y.T. (1992). Two species of genus Gordius (Gordioidea, Nematomorpha) from Korea. Korean Journal of Systematic Zoology 8, 223–230. Baer, J.G. (1951). Ecology of Animal Parasites. Urbana, IL: Illinois Press. Barnard, C.J. and Behnke, J.M. (1990). Parasitism and Host Behaviour. London: Taylor & Francis. Barus, V. (1964). Freshwater snails as reservoir hosts of invasive larvae of the nematode Syngamus trachea (Montagu 1811). In: Parasitic Worms and Aquatic Conditions (R. Ergens and B. Rysavy´, eds), pp. 83–90. Prague: House of the Czechoslovak Academy of Sciences. Beaver, P.C. (1969). The nature of visceral larva migrans. Journal of Parasitology 55, 3–12. Beaver, P.C., Jung, R.C., Cupp, E.W. and Craig, C.F. (1984). Clinical Parasitology. Philadelphia, PA: Lea & Febiger. Begon, M., Harper, J.L. and Townsend, C.R. (1996). Ecology: Individuals, Populations, and Communities. Oxford: Blackwell Science. Biron, D.G., Ponton, F., Menigoz, A., Hanelt, B. and Thomas, F. (2005). Water seeking behavior in insects harboring hairworms: should the host collaborate? Behavioral Ecology 16, 656–660. Bleidorn, C., Schmidt-Rhaesa, A. and Garey, J.R. (2002). Systematic relationships of Nematomorpha based on molecular and morphological data. Invertebrate Biology 121, 357–364. Blunk, H. (1922). Die Lebensgeschichte der im Gelbrand schmarotzenden Saitenwu¨rmer I. Zoologischer Anzeiger 54, 110–132. Bohall, P.J., Wells, M.R. and Chandler, C.M. (1997). External morphology of larvae of Chordodes morgani (Nematomorpha). Invertebrate Biology 116, 26–29. Bolek, M.G. and Coggins, J.R. (2002). Seasonal occurrence, morphology, and observation on the life history of Gordius difficilis (Nematomorpha: Gordioidea) from southeastern Wisconsin, United States. Journal of Parasitology 88, 287–294. Bresciani, J. (1991). Nematomorpha. In: Microscopic Anatomy of Invertebrates (F.W. Harrison, ed.), pp. 197–218. New York: Wiley-Liss, Inc. Brittain, J.E. and Eikeland, T.J. (1988). Invertebrate drift—a review. Hydrobiologia 166, 77–93. Brivio, M.F., de Eguileor, M., Grimaldi, A., Vigetti, D., Valvassori, R. and Lanzavecchia, G. (2000). Structural and biochemical analysis of the


295

parasite Gordius villoti (Nematomorpha, Gordiacea) cuticle. Tissue and Cell 32, 366–376. Brown, S.P., Renaud, F., Guegan, J.F. and Thomas, F. (2001). Evolution of trophic transmission in parasites: the need to reach a mating place? Journal of Evolutionary Biology 14, 815–820. Cappucci, D.T., Jr. and Schultz, M.G. (1982). Gordian worms (hairworms): biological and public health aspects. CRC Handbook Series in Zoonoses. Section C: Parasitic Zoonoses. Boca Raton, FL: CRC Press. Capuse, I. (1966). Sur le Dacochordodes bacescui Capuse (ord. Chordodida, fam. Chordodidae). Travers du Museum da Histoire Naturale ‘‘Grigore Antipa’’ 6, 17–23. Chandler, C.M. (1985). Horsehair worms (Nematomorpha, Gordioidea) from Tennessee, with a review of taxonomy and distribution in the United States. Journal of the Tennessee Academy of Sciences 60, 59–62. Chen, X.C., Pan, Z.L., Liu, D.S. and Han, X. (1998). Effect of taurine on human fetal neuron cells: proliferation and differentiation. Advances in Experimental Medicine and Biology 442, 397–403. Choisy, M., Brown, S.P., Lafferty, K.D. and Thomas, F. (2003). Evolution of trophic transmission in parasites: why add intermediate hosts? American Naturalist 162, 172–181. Cochran, P.A., Newton, A.K. and Korte, C. (2004). Great Gordian knots: sex ratio and sexual size dimorphism in aggregations of horsehair worms (Gordius difficilis). Invertebrate Biology 123, 78–82. Combes, C. (1991). Ethological aspect of parasite transmission. American Naturalist 138, 866–880. Combes, C. (2001). Parasitism: the Ecology and Evolution of Intimate Interactions. Chicago: University of Chicago Press. Daengsvang, S. (1968). Further observations on the experimental transmission of Gnathostoma spinigerum. Annals of Tropical Medicine and Parasitology 62, 88–94. Dawkins, R. (1990). Parasites, desiderata lists and the paradox of the organism. Parasitology 100, S63–S73. de Lucena, D.T. and Campelo, M. (1975). Accidental parasitism of man with two gordiids. Revista do Instituto de Medicina Tropical de Saõ Paulo 17, 390–393. de Miralles, D.A.B. and de Villalobos, L.C. (1993a). Distribucion geografica de los Gordiaceos en la region neotropical. In: Fauna de agua dulce de la Repulica Argentina (Z.A. de Castellanos, ed.), pp. 17–32. La Plata: Profadu (Conicet). de Miralles, D.A.B. and de Villalobos, L.C. (1993b). Distribucion geografica de los Gordiaceos en la republica Argentina. In: Fauna de agua dulce de la Repulica Argentina (Z.A. de Castellanos, ed.), pp. 1–16. La Plata: Profadu (Conicet).

296

B. HANELT ET AL.

de Miralles, D.A.B. and de Villalobos, L.C. (1995). A review of the Gordiacea (Nematomorpha) in the collection of the California Academy of Sciences with the description of a new species. Graellsia 51, 17–22. de Miralles, D.A.B. and de Villalobos, L.C. (1996a). Dos nuevas especies de Gordiaćeos para Ventania, Argentina (Nematomorpha). Comunicaciones do Museo de Ciencias y Tecnologia, PUCRS, Se´rie Zoologia 9, 3–9. de Miralles, D.A.B. and de Villalobos, L.C. (1996b). New Gordius for Argentina (Gordiacea Nematomorpha). Acta Zoologica Mexicana Nueva Serie 68, 67–73. de Miralles, D.A.B. and de Villalobos, L.C. (1996c). Nueva especie de Neochordodes (Gordiacea Nematomorpha) parasita de Scapteriscus sp. (Grillotalpidae). Revista de la Academia de Ciencias de Zaragoza 51, 237–246. de Miralles, D.A.B. and de Villalobos, L.C. (1996d). Species of Neochordodes in Argentina. Iheringia Serie Zoologia 81, 145–150. de Miralles, D.A.B. and de Villalobos, L.C. (1996e). Una nueva especie de Gordius (Linne, 1758) (Gordiaceae, Nematomorpha) de la Patagonia. Naturalia Patagońica, Ciencias Biolo´gicas 4, 41–45. de Miralles, D.A.B. and de Villalobos, L.C. (1997). Ultraestructura cuticular de una nueva especie de Chordodes Creplin, 1874, para la Argentina (Nematomorpha—Gordioidea—Chordodidae). Estudos de Biologia 42, 41–48. de Miralles, D.A.B. and de Villalobos, L.C. (1998). Dos nuevos especies y una nueva combinacion para el genero Acutogordius (Nematomorpha: Gordiidae). Neotropica 44, 75–79. de Miralles, D.A.B. and de Villalobos, L.C. (2000). Noteochordodes, un nuevo geńero para la republica Argentina (Nematomorpha, Gordiaceo). Revista del Museo de La Plata (NS) Zoologia 14, 279–283. de Villalobos, L.C. (1995). A new species of the genus Chordodes Creplin, 1874, in Argentina. Neotropica (La Plata) 41, 63–65. de Villalobos, L.C. and Camino, N. (1999). Two new species of Gordiacea (Nematomorpha) parasites of Stagmatoptera hyaloptera (Mantidae) from Argentina. Iheringia, Serie Zoologia 86, 71–76. de Villalobos, L.C. and de Miralles, D.A.B. (1997). Una nueva especie de Chordodes (Gordiaceo Nematomorpha) parasita de Blaptica sp. (Blaberidae, Blataria). Comunicaciones do Museo de Ciencias y Tecnologia, PUCRS, Se´rie Zoologia 10, 45–51. de Villalobos, L.C. and Restelli, M.A. (2001). Ultrastructural study of the cuticle and epidermis in Pseudochordodes bedriagae. Cell and Tissue Research 305, 129–134. de Villalobos, L.C., Ribera, I. and Downie, I.S. (1999). Hairworms found in Scottish agricultural land, with descriptions of two new species of


297

Gordionus Mu¨ller (Nematomorpha: Gordiidae). Journal of Natural History 33, 1767–1780. de Villalobos, L.C., Schmidt-Rhaesa, A. and Zanca, F. (2003). Revision of the genus Beatogordius (Gordiida, Nematomorpha) II. South American species with description of two new species. Memo´rias do Instituto Oswaldo Cruz 98, 115–128. de Villalobos, L.C. and Voglino, D. (2000). Description of Gordionus porosus n. sp. and redescription of Gordionus ondulatus (Nematomorpha, Parachordodinae) with scanning electron microscopy. Journal of Parasitology 86, 358–365. de Villalobos, L.C. and Zanca, F. (2001). Scanning electron microscopy and intraspecific variation of Chordodes festae Camerano, 1897 and C. peraccae (Camerano, 1894) (Nematomorpha: Gordioidea). Systematic Parasitology 50, 117–125. Degrange, C. and Martinot, J.P. (1996). Gordiacea, parasites of Orthoptera in Vanoise National Park. Travaux Scientifiques du Parc National de la Vanoise 19, 161–182. Dogiel, V.A. (1966). General Parasitology. New York: Academic Press. Donin, C.L.L. and Cotelli, F. (1977). The rod-shaped sperm of Gordioidea (Aschelminthes, Nematomorpha). Journal of Ultrastructural Research 61, 193–200. Dorier, A. (1930). Recherches biologiques et syste´matiques sur les Gordiace´s. Annales Universite´ de Grenoble 7, 1–183. Dorier, A. (1935a). Etude biologique et morphologique de la larva de Parachordodes gemmatus (Villot). Travaux du Laboratoire d’Hydrobiologie et de Pisciculture de Grenoble 25, 147–161. Dorier, A. (1935b). Sur le passage a la vie latente des larves des Gordiace´s. Comptes Rendus de l’Acade´mie des Sciences, Paris 200, 491–494. Feng, R., Rampon, C., Tang, Y.P., Shrom, D., Jin, J., Kyin, M., Sopher, B., Miller, M.W., Ware, C.B., Martin, G.M., Kim, S.H., Langdon, R.B., Sisodia, S.S. and Tsien, J.Z. (2001). Deficient neurogenesis in forebrainspecific presenilin-1 knockout mice is associated with reduced clearance of hippocampal memory traces. Neuron 32, 911–926. Feyel, T. (1936). Researches histologiques sur Nectonema agile Verr. e´tude de la forme parasitaire. Archives d0 Anatomie Microscopique 32, 197–234. Garey, J.R. and Schmidt-Rhaesa, A. (1998). The essential role of ‘‘minor’’ phyla in molecular studies of animal evolution. American Zoologist 38, 907–917. Giribet, G., Distel, D.L., Polz, M., Sterrer, W. and Wheeler, W.C. (2000). Triploblastic relationships with emphasis on the acoelomates and the position of Gnathostomulida, Cycliophora Plathelminthes and

298

B. HANELT ET AL.

Chaetognatha: a combined approach of 18S rDNA sequences and morphology. Systematic Biology 49, 539–562. Halvorsen, O. and Wissler, K. (1973). Studies on the helminth fauna of Norway XXVIII: an experimental study of the ability of Dypyllobothrium latum (L.), D. dendriticum (Nitzsch), and D. ditremum (Creplin) (Cestodes Pseudophyllidea) to infect paratenic hosts. Norwegian Journal of Zoology 21, 201–210. Hanelt, B. and Janovy, J., Jr. (1999). The life cycle of a horsehair worm, Gordius robustus (Gordioidea: Nematomorpha). Journal of Parasitology 85, 139–141. Hanelt, B. and Janovy, J., Jr. (2002). Morphometric analysis of non-adult characters of common species of American gordiids (Nematomorpha: Gordioidea). Journal of Parasitology 88, 557–562. Hanelt, B. and Janovy, J., Jr. (2003). Spanning the gap: identification of natural paratenic hosts of horsehair worms (Nematomorpha: Gordioidea) by experimental determination of paratenic host specificity. Invertebrate Biology 122, 12–18. Hanelt, B. and Janovy, J., Jr. (2004a). Life cycle and paratenesis of American gordiids (Nematomorpha: Gordiida). Journal of Parasitology 90, 240–244. Hanelt, B. and Janovy, J., Jr. (2004b). Untying a Gordian knot: the domestication and laboratory maintenance of a Gordian worm, Paragordius varius (Nematomorpha: Gordiida). Journal of Natural History 39, 939–950. Hanelt, B., Grother, L.E. and Janovy, J., Jr. (2001). Physid snails as sentinels of freshwater nematomorphs. Journal of Parasitology 87, 1049–1053. Heinze, K. (1952). U¨ber Gordioidea. Eine systematische Studie u¨ber Insektenparasiten aus der Gruppe der Nematomorpha. Zeitschrift fu¨r Parasitenkunde 15, 183–202. Hemsworth, R.J. and Brooker, M.P. (1979). The rate of downstream displacement of macro invertebrates in Upper Wye Wales UK. Holarctic Ecology 2, 130–136. Hershey, A.E., Pastor, J., Peterson, B.J. and Kling, G.W. (1993). Stable isotopes resolve the drift paradox for Baetis mayflies in an Arctic River. Ecology 74, 2315–2325. Herter, C.D. and Neese, R.E. (1989). Pseudoparasitism with Gordius robustus. American Family Physician 39, 139–142. Horton, M.L. (1986). Nonparasitic worm vomited by a dog. Modern Veterinary Practice 67, 905. Inoue, I. (1958). Studies on the life history of Chordodes japonensis, a species of Gordiacea. I. The development and structure of the larva. Japanese Journal of Zoology 12, 203–218.


299

Inoue, I. (1960a). Studies on the ecology of the larvae of the hairworm, Chordodes japonensis. Bulletin of the Tokyo Gakugei University 11, 21–25. Inoue, I. (1960b). Studies on the life history of Chordodes japonensis, a species of Gordiacia. II. On the manner of entry into the aquatic insect-larvae of Chordodes larvae. Annotationes Zoologicae Japonenses 33, 132–141. Inoue, I. (1962). Studies on the life history of Chordodes japonensis, a species of Gordiacea. III. The modes of infection. Annotationes Zoologicae Japonenses 35, 12–19. Inoue, I. (1994). Gordiod specimens deposited at the Toyama Science Museum. Bulletin of the Toyama Science Museum 17, 101–103. Jolivet, P. (1946). Nouvelle station franc- aise et nouvel hoˆte du ver Gordius aquaticus. Miscellanea Entomologica 42, 139–140. Lanzavecchia, G., De Eguileor, M., Valvassori, R. and Scari, G. (1995). Body cavities of Nematomorpha. In: Body Cavities: function and phylogeny (G. Lanzavecchia, R. Valvassori and M.D. Candia Carnevali, eds), pp. 45–60. Modena: Mucchi. Lee, K.J., Bae, Y.T., Kin, D.H., Deung, Y.K., Ryang, Y.S., Im, I.K. and Young, T.S. (2003). A Gordius worm found in a three year old girl’s vomitus. Yonsei Medical Journal 44, 557–560. Maeyama, T., Terayama, M. and Matsumoto, T. (1994). The abnormal behaviour of Colobopsis sp. (Hymenoptera, Formicidae) parasitized by Mermis (Nematoda) in Papua New Guinea. Sociobiology 24, 115–119. McManus, M.L. (1988). Weather, behaviour and insect dispersal. Memoirs of the Entomological Society of Canada 146, 71–94. Meissner, G. (1856). Beitra¨ge zur Antonomie und Physiologie der Gordiaceen. Zeitschrift fu¨r wissenschaftliche Zoologie 7, 1–140. Montgomery, T.H., Jr. (1898). The Gordiacea of certain American collections with particular reference to the North American fauna II. Proceedings of the California Academy of Sciences. 3rd ser: Zoology 1, 333–344. Moore, J. (2002). Parasites and the Behaviour of Animals. Oxford: Oxford University Press. Morand, S., Robert, F. and Connors, V.A. (1996). Complexity in parasite life cycles: population biology of cestodes in fish. Journal of Animal Ecology 64, 256–264. Mottram, J.C. (1932). The living drift of rivers. Transactions of the Newbury District Field Club 6, 195–198. Mu¨hldorf, A. (1914). Beitra¨ge zur Erwicklungsgeschichte und zu den phylogenetischen Beziehungen der Gordius Larve. Zeitschrift fu¨r wissenschaftliche Zoologie 111, 1–75. Mu¨ller, G.W. (1927). U¨ber Gordiaceen. Zeitschrift fu¨r die Morphologie und O¨kologie der Tiere 7, 134–270.

300

B. HANELT ET AL.

Mu¨ller, K. (1982). The colonization cycle of fresh water insects. Oecologia 52, 202–207. Mu¨ller, M.C.M., Jochmann, R. and Schmidt-Rhaesa, A. (2004). The musculature of horsehair worm larvae (Gordius aquaticus, Paragordius varius, Nematomorpha): F-actin staining and reconstruction by cLSM and TEM. Zoomorphology 123, 45–54. Myers, M.J., Sperling, F.A.H. and Resh, V.H. (2001). Dispersal of two species of Trichoptera from desert springs: conservation implications for isolated vs connected populations. Journal of Insect Conservation 5, 207–215. Olsen, O.W. (1986). Animal Parasites: Their Life Cycles and Ecology. New York: Dover. Parker, G.A., Chubb, J.C., Ball, M.A. and Roberts, G.N. (2003). Evolution of complex life cycles in helminth parasites. Nature 425, 480–484. Peterson, K.J. and Eernisse, D.J. (2001). Animal phylogeny and the ancestry of bilaterians: inferences from morphology and 18S rDNA gene sequences. Evolutionary Development 3, 170–205. Pikula, Z.P., Spiridonov, S.E. and Drljevic, E.T. (1996). Dacochordodes bacescui Capuse, 1966: report of the first two isolations from humans. Canadian Journal of Infectious Diseases 7, 144–146. Poinar, G.O., Jr. (1991a). Hairworm (Nematomorpha: Gordioidea) parasites of New Zealand wetas (Orthoptera: Stenopelmatidae). Canadian Journal of Zoology 69, 1592–1599. Poinar, G.O., Jr. (1991b). Nematomorpha. In: Ecology and Classification of North American Freshwater Invertebrates (J.H. Thorp and A.P. Covich, eds), pp. 273–282. New York: Academic Press, Inc. Poinar, G.O., Jr. (2001). Nematoda and Nematomorpha. In: Ecology and Classification of North American Freshwater Invertebrates (J.H. Thorp and A.P. Covich, eds), pp. 255–295. San Diego: Academic Press. Poinar, G.O., Jr. and Brockerhoff, A.M. (2001). Nectonema zealandica n. sp. (Nematomorpha: Nectonematoidea) parasitizing the purple rock crab Hemigrapsus edwardsi (Brachyura: Decapoda) in New Zealand, with notes on the prevalence of infection and host defense reactions. Systematic Parasitology 50, 149–157. Poinar, G.O., Jr. and Doelman, J.J. (1974). A reexamination of Neochordodes occidentalis (Montg.) comb. n. (Chordodidae: Gordioidea): larval penetration and defense reaction in Culex pipens L. Journal of Parasitology 60, 327–335. Poinar, G.P. (1999). Paleochordodes protus n.g., n.sp. (Nematomorpha, Chordodidae) parasites of a fossil cockroach, with a critical examination of other fossil hairworms and helminths of extant cockroaches (Insecta: Blattaria). Invertebrate Biology 118, 109–115. Poulin, R. (1996). Observations on the free-living adult stage of Gordius dimorphus (Nematomorpha: Gordioidea). Journal of Parasitology 82, 845–846.


301

Poulin, R. (1998a). Book review: Nematomorpha. Journal of Parasitology 84, 35. Poulin, R. (1998b). Evolutionary Ecology of Parasites: From Individuals to Communities. London: Chapman and Hall. Poulin, R. and Thomas, F. (1999). Phenotypic variability induced by parasites. Parasitology Today 15, 28–32. Protasoni, M., de Eguileor, M., Congiu, T., Grimaldi, A. and Reguzzoni, M. (2003). The extracellular matrix of the cuticle of Gordius panigettensis (Gordioiidae, Nematomorpha): observations by TEM, SEM and AFM. Tissue Cell 35, 306–311. Restelli, M.A., de Villalobos, L.C. and Zanca, F. (2002). Ultrastructural description of the musculature, the intraepidermal nervous system, and their interrelation in Pseudochordodes bedriagae (Nematomorpha). Cell and Tissue Research 308, 299–306. Reutter, K. (1972). Gordius das Wasserkalb. Mikroskosmos 61, 198–204. Robert, F., Renaud, F., Mathieu, E. and Gabrion, C. (1988). Importance of the paratenic host in the biology of Bothriocephalus gregarius (Cestoda, Pseudophyllidea), a parasite of the turbot. International Journal for Parasitology 18, 611–621. Saito, Y., Inoue, I., Hayashi, F. and Itagaki, H. (1987). A hairworm, Gordius sp., vomited by a domestic cat. Nippon Juigaku Zasshi 49, 1035–1037. Schaffer, S., Takahashi, K. and Azuma, J. (2000). Role of osmoregulation in the actions of taurine. Amino Acids 19, 527–546. Schmidt-Rhaesa, A. (1996a). The nervous system of Nectonema munidae and Gordius aquaticus, with implications for the ground pattern of the Nematomorpha. Zoomorphology 116, 133–142. Schmidt-Rhaesa, A. (1996b). Ultrastructure of the anterior end in three ontogenetic stages of Nectonema mundiae (Nematomorpha). Acta Zoologica 77, 267–278. Schmidt-Rhaesa, A. (1997a). Nematomorpha. In: Su¨X wasserfauna Mitteleuropas (J. Scwoerbel and P. Zwick, eds), pp. 1–124. Stuttgart: Gustav Fischer. Schmidt-Rhaesa, A. (1997b). Ultrastructural features of the female reproductive system and female gametes of Nectonema munidae Brinkmann 1930 (Nematomorpha). Parasitology Research 83, 77–81. Schmidt-Rhaesa, A. (1997c). Ultrastructural observations of the male reproductive system and spermatozoa of Gordius aquaticus L., 1758. Invertebrate Reproduction and Development 32, 31–40. Schmidt-Rhaesa, A. (1998a). Muscular ultrastructure in Nectonema munidae and Gordius aquaticus (Nematomorpha). Invertebrate Biology 117, 37–44. Schmidt-Rhaesa, A. (1998b). Phylogenetic relationships of the Nematomorpha—a discussion of current hypothesis. Zoologischer Anzeiger 236, 203–216.

302

B. HANELT ET AL.

Schmidt-Rhaesa, A. (1999). Nematomorpha. In: Encyclopedia of Reproduction (E. Knobil and J.D. Neill, eds), pp. 333–341. San Diego: Academic Press. Schmidt-Rhaesa, A. (2000). Rasterelektronenmikroskopische Untersuchungen an Gordionus violaceus Baird 1853 und Gordius elongiporus spec. nova (Nematomorpha) aus Niederosterreich. Linzer Biologische Beitraege 32, 455–461. Schmidt-Rhaesa, A. (2001a). Are the genera of Nematomorpha monophyletic taxa? Zoologica Scripta 31, 185–200. Schmidt-Rhaesa, A. (2001b). The life cycle of horsehair worms (Nematomorpha). Acta Parasitologica 46, 151–158. Schmidt-Rhaesa, A. (2001c). Problems and perspectives in the systematics of Nematomorpha. Organisms, Diversity and Evolution 1, 161–163. Schmidt-Rhaesa, A. (2001d). Reinvestigation and reinterpretation of Chordodiolus echinatus Linstow, 1901, a peculiar nematomorph species. Mitteilungen des Museums fu¨r Naturkunde, Berlin, Zoologische Reihe 77, 91–94. Schmidt-Rhaesa, A. (2001e). Variation of cuticular characters in the Nematomorpha: studies on Gordionus violaceus (Baird, 1853) and G. wolterstorffii (Camerano, 1888) from Britain and Ireland. Systematic Parasitology 49, 41–57. Schmidt-Rhaesa, A. (2002). Australian species of Chordodes (Nematomorpha) with remarks on the genus and its life history. Journal of Natural History 36, 1569–1588. Schmidt-Rhaesa, A. (2004a). Gordius longitriatus a new species or Nematomorph from Hawaii. Records of the Hawaii Biological Survey for 2003 Bishop Museum Occasional Papers 78, 53–55. Schmidt-Rhaesa, A. (2004b). Japanese horsehair worms (Nematomorpha) from the Lake Biwa Museum. Species Diversity 9, 97–107. Schmidt-Rhaesa, A. (2004c). Ultrastructure of an integumental organ with probable sensory function in Paragordius varius (Nematomorpha). Acta Zoologica 85, 15–19. Schmidt-Rhaesa, A. (2005). Morphogenesis of Paragordius varius (Nematomorpha) during the parasitic phase. Zoomorphology 124, 33–46. Schmidt-Rhaesa, A. and Bryant, M.S. (2004). Filling the Gondwana gaps: new species and new reports of Beatogordius Heinze, 1934 (Nematomorpha) from Australia and Madagascar. Systematic Parasitology 57, 173–181. Schmidt-Rhaesa, A., Chung, P.R. and Sohn, W.M. (2003a). Parachordodes megareolatus, a new species of horsehair worm (Nematomorpha) from Korea. Korean Journal of Systematic Zoology 19, 161–166. Schmidt-Rhaesa, A. and de Villalobos, L.R. (2001). Revision of the genus Beatogordius (Gordiida, Nematomorpha). I. African species with


303

inclusion of Chordodiolus echinatus (Linstow, 1901). Annales du Museé royal de l’Afrique Centrale 206, 1–25. Schmidt-Rhaesa, A. and Ehrmann, R. (2001). Horsehair worms (Nematomorpha) as parasites of praying mantids with a discussion of their life cycle. Zoologischer Anzeiger 240, 167–179. Schmidt-Rhaesa, A., Hanelt, B. and Reeves, W. (2003b). Redescription and compilation of Nearctic freshwater Nematomorpha (Gordiida), with the description of two new species. Proceedings of the Academy of Natural Sciences of Philadelphia 153, 77–117. Schmidt-Rhaesa, A. and Menzel, L. (2005). Central American and Caribbean species of horsehair worms (Nematomorpha), with the description of three new species. Journal of Natural History 15, 515–529. Schmidt-Rhaesa, A., Thomas, F. and Poulin, R. (2000). Redescription of Gordius paranensis Camerano, 1892 (Nematomorpha), a species new for New Zealand. Journal of Natural History 34, 333–340. Schultz, R.S. and Davtian, E.A. (1954). The forms of host-parasite relationships in helminthology. Zoologicheskii Zhurnal 33, 1201–1205. Shoop, W.L. (1988). Trematode transmission patterns. Journal of Parasitology 74, 46–59. Sinakevitch, I., Farris, S.M. and Strausfeld, N.J. (2001). Taurine-, aspartate- and glutamate-like immunoreactivity identifies chemically distinct subdivisions of Kenyon cells in the cockroach mushroom body. Journal of Comparative Neurology 439, 352–367. Skaling, B. and MacKinnon, B.M. (1988). The absorptive surfaces of Nectonema sp. (Nematomorpha: Nectonematoidea) from Pandalus montagui: histology, ultrastructure, and absorptive capabilities of the body wall and intestine. Canadian Journal of Zoology 66, 289–295. Smith, D.R., Watson, D.D., Clevenger, R.R. and McBride, C. (1990). Paragordius varius, Archives of Pathology and Laboratory Medicine 114, 981–983. So¨derstro¨m, O. (1987). Upstream movements of invertebrates in running waters—a review. Archiv fu¨r Hydrobiologie 111, 197–208. Sørensen, M.V., Funch, P., Willerslev, E., Hansen, A.J. and Olesen, J. (2000). On the phylogeny of the Metazoa in the light of Cycliophora and Micrognathozoa. Zoologischer Anzeiger 239, 297–318. Spiridonov, S.E. (1993). Gordius annamensis sp. n. (Nematomorpha: Parachordodadidae) a new species of gordian worm from central Vietnam. Parazitologiya 27, 429–432. Spiridonov, S.E. (1998). Horse hairworms of the genus Gordius Linne, 1776 (Gordiacea, Nematomorpha) in the collection of Scientific Zoological Museum M.V. Lomonosov Moscow State University. Byulleten’ Moskovskogo Obshchestva Ispytatelei Prirody Otdel Biologicheskii 103, 19–26.

304

B. HANELT ET AL.

Spiridonov, S.E. (2000). A new hairworm in the genus Chordodes (Nematomorpha: Chordodidae) from the Minusinsk depression. Parazitologiia 34, 437–441. Spiridonov, S.E. (2001). Chordodes guineensis sp.n., a new species of horsehair worms from West Africa (Nematomorpha: Chordodidae). Zoosystematica Rossica 10, 275–279. Spiridonov, S.E., Pikula, Z.P. and Drljevic, E.T. (1992). Redecription of Dacochordodes bacescui Capuse, 1966 (Nematomorpha: Chordodidae). Helminthologica 29, 193–196. Sva´benı´ k, J. (1925). Parasitismus a metamorfosa druhu Gordius tolosanus Dujardin. Publications de la Faculte´ des Sciences de l’Universite´ Masaryk 58, 1–48. Thomas, F., Schmidt-Rhaesa, A., Martin, G., Manu, C., Durand, P. and Renaud, F. (2002). Do hairworms (Nematomorpha) manipulate the water seeking behaviour of their terrestrial hosts? Journal of Evolutionary Biology 15, 356–361. Thomas, F., Schmidt-Rhaesa, A. and Poulin, R. (1999). Microhabitat characteristics and reproductive status of male Euchordodes nigromaculatus (Nematomorpha). Journal of Helminthology 73, 91–93. Thomas, F., Ulitsky, P., Augier, R., Dusticier, N., Samuel, D., Strambi, C., Biron, D.G. and Cayre, M. (2003). Biochemical and histological changes in the brain of the cricket Nemobius sylvestris infected by the manipulative parasite Paragordius tricuspidatus (Nematomorpha). International Journal of Parasitology 33, 435–443. Thorne, G. (1940). The hairworm, Gordius robustus Leidy, as a parasite of the Mormon cricket, Anabrus simplex Haldeman. Journal of the Washington Academy of Sciences 30, 219–231. Valvassori, R., Scarı` , G., De-Eguileor, M., Lernia, L.D., Magnetti, P. and Melone, G. (1988). Gordius villoti (Nematomorpha) life cycle in relation with caddis fly larvae. Bollettino di Zoologia 55, 269–278. Vejdovsky, F. (1888). Studien u¨ber Gordiiden. Zeitschrift fu¨r wissenschaftliche Zoologie 46, 188–216. Villot, A. (1874a). Monographie des dragonneaux (Genre Gordius Dujardin). Archives de Zoologie expe´rimentale 3, 39–72. Villot, A. (1874b). Monographie des dragonneaux (Genre Gordius Dujardin). Deuxie´me partie. Anatomie et Physiologie. Archives de Zoologie expe´rimentale 3, 181–238. Voigt, E. (1938). Ein fossiler Saitenwurm (Gordius tenuifibrosus n. sp.) aus der eozaën Braunkohle des Geiseltales. Nova acta Leopoldina new series 5, 351–360. Walker, T.K., Rollinson, D. and Simpson, A.J. (1989). A DNA probe from Schistosoma mansoni allows rapid determination of the sex of larval parasites. Molecular Biochemistry and Parasitology 33, 93–100.


305

Watermolen, D.J. and Haen, G.L. (1994). Horsehair worms (phylum Nematomorpha) in Wisconsin, with notes on their occurrence in the Great Lakes. Journal of Great Lakes Research 9, 7–11. White, D.A. (1966). A new host record for Paragordius varius (Nematomorpha). Transactions of the American Microscopical Society 85, 579. White, D.A. (1969). The infection of immature aquatic insects by larval Paragordius (Nematomorpha). Great Basin Naturalist 29, 44. Zapotosky, J.E. (1971). The cuticular ultrastructure of Paragordius varius (Leidy, 1851)(Gordioidea: Chordodidae). Proceedings of the Helminthological Society of Washington 38, 228–236. Zapotosky, J.E. (1974). Fine structure of the larval stage of Paragordius varius (Leidy, 1851) (Gordioidea: Paragordidae). I. The preseptum. Proceedings of the Helminthological Society of Washington 41, 209–221. Zapotosky, J.E. (1975). Fine structure of the larval stage of Paragordius varius (Leidy 1851) (Gordioidea: Paragordidae). II. The postseptum. Proceedings of the Helminthological Society of Washington 42, 103–111. Zrzavy´, J., Mihulka, S., Kepka, P., Bezdek, A. and Tietz, D. (1998). Phylogeny of the metazoa based on morphological and 18S ribosomal DNA evidence. Cladistics 14, 249–285.

Plate 2.10 Novel, multinucleated structures formed upon the fusion of sporozoites of C. parvum.

Plate 4.3 Histological section of developing Paragordius varius males and females within the definitive host Gryllus firmus, 25 days post-exposure.

Plate 4.8 Longitudinal section of male worm recovered from the bath tub of a man presumed to be infected. The worm was recovered from Oregon State, USA; the man was presumed to have become infected during travel to Australia. After looking at this section, it was determined to be a gordiid species commonly found in North America, and likely entered the bathtub by being carried in by the insect-definitive host. Top: complete section, bottom: close up of cuticle layer. This sample was brought to the attention of the authors by Hector C. Aldape.

Plate 4.10 Illustration of the water-seeking behaviour of Nemobius sylvestris in an artificial water area followed by the emergence of the hairworm Paragordius tricupidatus.

Plate 4.13 Histological sections of Nemobius sylvestris mushroom bodies treated by the nuclear coloration of Feulgen-Rossenbeck. (a) frontal section of the whole brain showing the position and structure of the mushroom bodies. (b) The mushroom body cortex is shown enlarged, allowing the visualization of the group of neuroblasts at the apex of the structure. A figure of mitosis is indicated by the arrow. (c–e) Pictures of anaphase and metaphases among the neuroblasts. Nb, neuroblasts; Ca, calyx; Kc, Kenyon cells; M, mitosis (from Thomas et al., 2003).

Index

Accessory molecules, murine leishmaniasis, mechanisms of 27–28 Acetyl CoA, in Cryptosporidium parvum 116–118 Acheta domestica, Paragordius varius infection 270–271 Actin polymerisation-dependent motility, Cryptosporidium host–parasite relationship 100–101 Active immunity, ichthyophthiriosis 202–204 Adaptation, Nematomorpha life cycle, paratenic host and 266–269 Adaptive immune response, murine cutaneous leishmaniasis 17–21 dendritic cells 13–14 regulatory T cells 21–22 T cell mechanisms 17–18 response to Leishmania major 19–20 T helper cell polarisation 18–19 Admixture studies, human cutaneous leishmaniasis, single nucleotide polymorphism (SNP) analysis 42 Advanced intercross lines (AILs), murine cutaneous leishmaniasis, quantitative trait loci (QTL) mapping 35 Affected family member studies, human cutaneous leishmaniasis, genetic analysis 41 Alternative oxidase (AOX), in Cryptosporidium parvum 117–118 Amastigote parasites Leishmania species 4–7


macrophages, innate immune response, murine leishmaniasis 10–12 murine cutaneous leishmaniasis, susceptibility studies 44–49 Amino acid metabolism in Cryptosporidium parvum 121–122 manipulation of host changes and 285–288 Amplification techniques, Cryptosporidium maintenance 96–99 Amprolium, in situ ichthyophthiriosis control using 216–217 Animal models, in vivo Cryptosporidium 96–97 Antibody responses, cryptosporidiosis immunobiology 103–104 Antibody transfer, murine leishmaniasis, adaptive immune response 17–18 Antimonial drug therapy, cutaneous leishmaniasis 6–7 Apocope oscula carringtoni, Ichthyophthirius multifiliis trophont growth and development in 185–186 Ascaris lumbricoides, zoonotic potential 127 Ascorbic acid, ichthyophthiriosis control and treatment 209–210 Asexual development, Cryptosporidium life cycle 90–91 Association studies, human cutaneous leishmaniasis, genetic analysis 39–40


308 Azithromycin, Cryptosporidium chemotherapy 133–136 Backcrosses, murine cutaneous leishmaniasis quantitative trait loci (QTL) mapping 34–35 susceptibility studies 43–49 Bacterial artificial chromosome (BAC) leishmaniasis animal models 8 murine cutaneous leishmaniasis, gene discovery and validation 38 Bacterial infection, cryptosporoidiosis in cattle 106–107 B cells, ichthyophthiriosis immunity 201–204 Beatogordius biogeographical distribution of 256–257 taxonomy of 252–255 Binary fission, Ichthyophthirius multifiliis reproduction, in fish host 188–190 Biochemical regulation, in Cryptosporidium parvum 111–123 amino acid metabolism 121–122 Cryptosporidium parvum genome 112–115 core energy metabolism 115–118 fatty acid polyketide synthesis 118–120 nucleic acid metabolism 120–121 Biodiversity, Nematomorpha 251–255 Biogeographical patterns, Nematomorpha 255–257 Bronopol, ichthyophthiriosis control using 214–215 Carassius auratus, Ichthyophthirius multifiliis epidemiology 161–163 Carassius carassius, ichthyophthiriosis pathogenesis in 208 Cattle, cryptosporidiosis in 105–107 zoonotic potential 124–127

INDEX CCL2/MCP1 chemokine, murine leishmaniasis, cellular and noncellular innate response mechanisms 16–17 CD4+ cells cryptosporidiosis 102–104 human infection 105 murine leishmaniasis adaptive immune response 17–18 helper T cells 18–19 regulatory T cells 20–21 interferon-g and IL-12 mechanisms 24–27 Leishmania major infection, early T cell response 19–20 CD8+ cells, cryptosporidiosis immunobiology 102–104 CD25+ marker, murine leishmaniasis, adaptive immune response 20–21 CD40 signaling, murine leishmaniasis, dendritic cell response 13–14 CD40-CD40L interaction, murine leishmaniasis 28 Cell-free media, Cryptosporidium chemotherapy 132–136 Cell-mediated immune response cryptosporidiosis 102–104 human cutaneous leishmaniasis 30–31 Cellular infiltration, ichthyophthiriosis immunity 202–204 Chemokines, murine leishmaniasis, cellular and noncellular innate response mechanisms 16–17 Chemotherapy Cryptosporidium control 132–136 ichthyophthiriosis control and 212–217 free-living parasite stage, administration during 213–214 in situ trophont control 215–217 Chilodonella sp., evasive mechanisms of 205–207 Chlarias batrachus, ichthyophthiriosis control in 214

INDEX Chloramine-T, ichthyophthiriosis control using 214 Chordodes, biogeographical distribution of 256–257 Chordodes japonensis cysts infectivity, paratenic host increase of 266–267 paratenic hosts 263–264 Chordodiolus echinatus, taxonomy of 252–255 Chorioallantoic membrane (CAM), in vitro Cryptosporidium cultivation 97–99 Clopidol, in situ ichthyophthiriosis control using 216–217 Coccidiostat compounds, in situ ichthyophthiriosis control using 216–217 Co-infection, leishmaniasis and role of 53 Collaboration, manipulation of host changes vs. 289–290 Common haplotype mapping, murine cutaneous leishmaniasis, quantitative trait loci 36 Companion animals, human cryptosporidiosis and 126–127 Complement opsonisation, murine leishmaniasis, macrophages, innate immune response 10–12 Complement receptors 1 and 3 (CR1, CR3), murine leishmaniasis, macrophages, innate immune response 10–12 Complex proteins, host–parasite relationship, Cryptosporidium 100–101 Confinement stress, ichthyophthiriosis control and treatment 209–210 Congenic strains (CS), murine cutaneous leishmaniasis genetic susceptibility studies 44–49 quantitative trait loci 37 Contractile vacuoles, Ichthyophthirius multifiliis excretory system 183–184 exit from fish host 190–193

309 Copper sulphate, ichthyophthiriosis control using 214 Core energy metabolism, in Cryptosporidium parvum 115–118 CpLDH1/CpMDH1 genes, in Cryptosporidium parvum 117–118 Cricket hosts, Nematomorpha invasion, water-seeking behaviour and 282–284 Crowned aeroles, Chordodes taxonomy and 254–255 Cryopreservation, Ichthyophthirius multifiliis 193–194 Cryptosporidiosis in cattle 105 zoonotic potential 127–127 human infection epidemiology 127–130 immunobiology 104–105 zoonotic potential 124–127 immunobiology 101–104 immunoprophylaxis 136–137 in pigs 108–109 in poultry 109 vaccine development 136–137 Cryptosporidium biochemical regulation 111–123 amino acid metabolism 121–122 Cryptosporidium parvum genome 112–115 core energy metabolism 115–118 fatty acid polyketide synthesis 118–120 nucleic acid metabolism 120–121 control 130–137 chemotherapy 132–136 detection 130–132 immunotherapy and immunoprophylaxis 136–137 epidemiology and transmission 123–130 cycles, zoonotic potential and 123–127 human infection 127–130

310 Cryptosporidium (contd.) future research issues 138 host–parasite relationship 99–111 cattle infection 105–107 dog/cat infection 110 fish infections 111 horse infection 110 human infection 104–105 immunobiology 101–104 pathogenesis 99–101 pig infection 108–109 poultry infection 109 reptile infection 110–111 sheep/goat infection 107–108 life cycle and development 88–99 establishment 88–89 macrogamonts 94 maintenance and amplification 95–99 meront I 92 meront II 92–93 merozoites 93 microgamonts 93–94 oocysts 94–95 sporozoites 95 trophozoite stage 91–92 in vitro cultivation 97–99 in vivo maintenance, animal models 96–97 pathology 78–79 phylogenetic relationships and taxonomy 79–87 biological and molecular characteristics 79–85 current taxonomy 85–87 Cryptosporidium andersoni, cattle infection 107 Cryptosporidium baileyi, poultry infection and 109 Cryptosporidium hominis, human infection epidemiology 127–130 Cryptosporidium meleagridis human infection epidemiology 129–130 poultry infection and 109

INDEX Cryptosporidium molnari, fish infection 111 Cryptosporidium nasorum, fish infection 111 Cryptosporidium parvum biochemical regulation 111–123 genome structure 112–115 biological and molecular characteristics, 81-85 cattle infections 105–110 host–parasite relationship, pathogenesis 100–101 human infection 105 epidemiology 127–130 life cycle and development 88–99 taxonomy and pathology 79 in vitro maintenance 98–99 Cutaneous leishmaniasis human infection genetic susceptibility studies 49–50 host resistance to disease 29–31 genetic analysis 38–42 affected family member studies 41 association studies 39–40 future research issues 41–42 linkage analysis 40–41 mouse models 8–29 adaptive immune response 17–21 regulatory T cells 21–22 T cell mechanisms 17–18 T cell response to Leishmania major 19–20 T helper cell polarisation 18–19 cytokines and immunomodulatory molecules 21–28 interferon-g and IL-2 24–27 interleukin-4 and -10 21–24 TNF-a, TGF-b and accessory molecules 27–28 innate immune response mechanisms 9–17 dendritic cells 12–14

INDEX immune system characteristics 14–17 macrophages 9–12 transcription factors, Th1/Th2 response 28–29 Cutaneous ulcers, Leishmania species as cause of 6–7 Cuticle formation, Gordiida morphology 246–249 b-Cyclodextrin Cryptosporidium chemotherapy 135–136 in situ ichthyophthiriosis control using 216–217 Cyprinus carpio, Ichthyophthirius multifiliis epidemiology 162–163 Cytokines human cutaneous leishmaniasis host resistance to 30–31 linkage analysis 40–41 susceptibility genetics 49–50 ichthyophthiriosis immunity 200–204 murine cutaneous leishmaniasis 21–28 adaptive immune response, helper T cells 18–19 interferon-g and IL-2 24–27 interleukin-4 and -10 21–24 macrophages, innate immune response 10–12 TNF-a, TGF-b and accessory molecules 27–28 Dacochordodes, biodiversity of 251–255 Damage–response framework, leishmaniasis 3–4 Decoquinate, Cryptosporidium chemotherapy 136 Definitive host, gordiid transition to 259–260 Dendritic cells (DC), murine leishmaniasis, host resistance to disease basic principles 8 cytokine mechanisms 24 innate immune response 12–14

311 Detection techniques, Cryptosporidium control 130–132 Diclazuril, Cryptosporidium chemotherapy 135–136 Diethylcarbamazine, in situ ichthyophthiriosis control using 216–217 Dispersal mechanisms, of paratenic hosts 267–269 Distribution mechanisms, Nematomorpha 272–276 fine scale distribution 273–274 temporal distribution 275 DNA microarray analysis, murine cutaneous leishmaniasis, congenic strains 37 Dogs and cats, cryptosporidoisis in 110 Drift events, nematomorpha paratenic hosts 268–269 Dugesia tigrina, paratenesis 264–265 Eagle’s minimum essential medium (EMEM), Ichthyophthirius multifiliis trophont reproduction 189–190 in vitro culture and cryopreservation 193–194 Endoplasm structure, Ichthyophthirius multifiliis 184–185 Environmental factors ichthyophthiriosis control and treatment 210–212 leishmaniasis epidemiology 51–53 Epidermal invasion, fish hosts, Ichthyophthirius multifiliis 173–175 Epithelioma papulosum cyprini, Tetrahymena corlissi culture 193–194 Epizootics, ichthyophthiriosis epidemiology 162–163 Euchordodes nigromaculatus, sex ratio 271–272

312 Evasive mechamisms Ichthyophthirius multifiliis, host immune response, evasion of 205–207 leishmaniasis and host immune system 2 Excretory system, Ichthyophthirius multifiliis trophonts 183–184 Extracellular mechanisms, Cryptosporidium life cycle 89–91 Fas/FasL system, murine leishmaniasis, macrophages, innate immune response 12 Fatty acid synthase (FAS), in Cryptosporidium parvum 118–120 ‘‘Feeder organelle,’’ Cryptospiridium species 82–85 Feeding and digestion, Ichthyophthirius multifiliis trophonts 186–188 Fertilisation, Cryptosporidium macrogamonts 94 Fibronectin receptor, murine leishmaniasis, macrophages, innate immune response 10–12 Fine scale distribution, Nematomorpha 273–274 Fish farming, ichthyophthiriosis epidemiology 162–163 Fish host infection cryptosporidiosis 111 Ichthyophthirius multifiliis 173–177 epidermal invasion 173–175 infection site 176–177 trophont reproduction in 188–190 Formalin, ichthyophthiriosis control with 213–214 Free-living stages, Ichthyophthirius multifiliis 166–172 chemotherapeutic control during 213–214 theront 169–172 tomont 166–169

INDEX Garlic, ichthyophthiriosis control using 214 Genetic analysis. See also Proteomics host response in leishmaniasis 42–50 human susceptibility genetics 49–50 mouse susceptibility genetics 42–49 human cutaneous leishmaniasis 38–42 affected family member studies 41 association studies 39–40 future research issues 41–42 linkage analysis 40–41 murine cutaneous leishmaniasis 31–38 common haplotype mapping 36 congenic strains 37 gene discovery and validation 37–38 intercrosses or backcrosses 34–35 loci mapping 33–34 multiple strain comparisons 33 recombinant congenic strains 36 recombinant inbred strains 35 Genome sequencing, Cryptosporidium parvum 112–115 G1 isolate, Ichthyophthirius multifiliis immunity, i-antigens 194–198 Glycoinositolphospholipids (GIPLs), murine leishmaniasis, macrophages, innate immune response 12 Glycophosphatidyl inositol (GPI) anchor, Ichthyophthirius multifiliis immunity, i-antigen isolation 195–198 Glycoproteins, murine leishmaniasis, macrophages, innate immune response 10–12 Glyphosate toxicity, ichthyophthiriosis control and treatment 210–212 Goblet cells, Ichthyophthirius multifiliis, epidermal invasion 174–175 Golgi complex, Ichthyophthirius multifiliis somatic cortex and 181–183

INDEX Gordiida biodiversity of 251–255 biogeographical distribution of 256–257 biology of, future research issues 291–292 development in host 250–251 distribution mechanisms 272–276 fine scale distribution 273–274 temporal distribution 275 human–gordiid interaction 276–279 household distribution 276–277 potable water sources 276 pseudoparasitism 277–279 larval development 250 life cycle and ecology 258–279 general principles 258–260 laboratory conditions 269–271 longevity increases in 267 morphology of 246–249 paratenic hosts 260–269 dispersal and maintenance functions 268–269 evolutionary role of 269 identity of 261–263 life cycle role of 265–269 lost infective stage recovery 267 natural hosts 263–264 parasite infectivity and 266–267 parasite longevity and 267 paratenesis 264–265 site dispersal/maintenance 267–269 research background on 244–245 Gordius difficilis paratenic hosts 263–264 sex ratio 271–272 temporal distribution 275 Gordius robustus development in host 250–251 laboratory life cycles 270–271 paratenic hosts 263–264 increase in infectivity 266–267 sex ratio 271–272

313 Gordius aquaticus larval development 250 morphology of 246–249 Gordonius genus intraspecific variation 255 taxonomy of 253–255 Gregarina tribolorum, pairing behaviour 82–85 Gryllus firmus, laboratory life cycles 270–271 Gut-associated lymphoid tissue (GALT), ichthyophthiriosis immunity 200–204 Halfuginone lactate, Cryptosporidium chemotherapy 135–136 HCT-8 cells, Cryptosporidium life cycle 91–92 Heterogeneity, in Leishmania parasites 7–8 H-11 gene, murine cutaneous leishmaniasis, susceptibility studies 44–49 H-2 loci, murine cutaneous leishmaniasis, genetic susceptibility studies 44–49 Home conditions, human–gordiid interaction 276–277 Horses, cryptosporidoisis in 110 Host finding and contact, Ichthyophthirius multifiliis 172–173 Host–parasite relationship Cryptosporidium 99–111 cattle infection 105–107 dog/cat infection 110 fish infection 111 horse infection 110 human infection 104–105 immunobiology 101–104 pathogenesis 99–101 pig infection 108–109 poultry infection 109 reptile infections 110–111 sheep/goat infections 107–108

314 Host–parasite relationship (contd.) ichthyophthiriosis immunity 204 Ichthyophthirius multifiliis exit from fish host 190–193 parasite evasion of host immune response 205–207 protective immunity mechanisms 204 reproduction in fish host 188–190 Nematomorpha 250–251 host behavioural alterations 279–291 field and laboratory experiments 281–284 manipulation vs. collaboration mechanisms 289–291 physiology and neurology manipulations 284–288 proteomics 288 study system for 280–281 Host resistance to disease human cutaneous leishmaniasis 29–31 genetic analysis 38–42 leishmaniasis, genetics of 42–50 murine cutaneous leishmaniasis 8–29 adaptive immune response 17–21 regulatory T cells 21–22 T cell mechanisms 17–18 response to Leishmania major 19–20 T helper cell polarisation 18–19 cytokines and immunomodulatory molecules 21–28 interferon-g and IL-2 24–27 interleukin-4 and -10 21–24 TNF-a, TGF-b and accessory molecules 27–28 genetic analysis 31–38 common haplotype mapping 36 congenic strains 37 gene discovery and validation 37–38 intercrosses or backcrosses 34–35 loci mapping 33–34 multiple strain comparisons 33

INDEX recombinant congenic strains 36 recombinant inbred strains 35 innate immune response mechanisms 9–17 dendritic cells 12–14 immune system characteristics 14–17 macrophages 9–12 transcription factors, Th1/Th2 response 28–29 Host response loci mapping, murine cutaneous leishmaniasis 33–34 Human cryptosporidiosis epidemiology 127–130 immunobiology 104–105 zoonotic potential 124–127 Human cutaneous leishmaniasis genetic susceptibility studies 49–50 host resistance to disease 29–31 genetic analysis 38–42 affected family member studies 41 association studies 39–40 future research issues 41–42 linkage analysis 40–41 Human–gordiid interaction 276–279 household distribution 276–277 potable water sources 276 pseudoparasitism 277–279 Human leukocyte antigens (HLAs), human susceptibility to leishmaniasis 49–50 Humoral immune response, cryptosporidiosis immunobiology 103–104 Hyaluronidase, fish hosts, Ichthyophthirius multifiliis, epidermal invasion 174–175 Hymenostomatida, ichthyophthiriosis immunity 203–204 Hyperimmune bovine colostrum, cryptosporidiosis immunotherapy 136–137

INDEX IAG48[G1] gene, Ichthyophthirius multifiliis immunity, i-antigen isolation 196–198 i-Antigens ichthyophthiriosis vaccine development and 218–219 Ichthyophthirius multifiliis immunity 194–198 Ichthyophthirioides browni 162 trophont morphology 178 Ichthyophthiriosis control and treatment 209–219 chemical use 212–217 free-living parasitic stages 213–215 in situ trophont destruction 215–217 vaccine development 217–219 water management 211–212 epidemiology 162–163 i-antigen and immunity to 197–198 pathogenesis 207–209 protective immunity 198–204 Ichthyophthirius multifiliis fish host infection 173–177 epidermal invasion 173–175 infection site 176–177 free-living stages 166–172 theront 169–172 tomont 166–169 historical background and taxonomy 163–166 host finding and content 172–173 immunity 194–207 i-antigens 194–198 parasite invasion, host immune response 205–207 protective immunity 198–204 importance and global distribution 160–163 life cycle 163 research overview 160 trophont transformation 177–194 cell structure and function 179–183 endoplasm 184–185 excretory system 183–184

315 exit from host 190–193 feeding and digestion 186–188 growth and development 185–186 host reproduction 188–190 morphological features 177–178 in vitro culture and cryopreservation 193–194 Immune response Ichthyophthirius multifiliis, parasite evasion of 205–207 murine cutaneous leishmaniasis 17–21 dendritic cells 13–14 regulatory T cells 21–22 T cell mechanisms 17–18 response to Leishmania major 19–20 T helper cell polarisation 18–19 parasite evasion mechanisms and, leishmaniasis 2 Immunization routes, ichthyophthiriosis inoculation 199–204 Immunobiology Cryptosporidium species 101–104 Ichthyophthirius multifiliis 194–207 i-antigens 194–198 parasite invasion, host immune response 205–207 protective immunity 198–204 Immunoglobulins host finding and contact mechanisms, Ichthyophthirius multifiliis 172–173 immunoglobulin G (IgG), Ichthyophthirius multifiliis, protective immunity against 204 immunoglobulin M (IgM), ichthyophthiriosis immunity 201–204 Immunotherapy, cryptosporidiosis 136–137 Inducible nitric oxide synthase (iNOS) human cutaneous leishmaniasis 31 murine leishmaniasis, macrophages, innate immune response 11–12

316 Infection site cryptosporidiosis in cattle 106–107 Ichthyophthirius multifiliis trophonts 176–177 murine leishmaniasis and role of 52–53 paratenic gordiid host identity 262–263 Infectivity of parasites, paratenic host and increase in 266–267 INF-g-inducible protein 10 (IP-10), murine leishmaniasis, cellular and noncellular innate response mechanisms 16–17 Innate immune response, murine cutaneous leishmaniasis 9–17 dendritic cells 12–14 immune system characteristics 14–17 macrophages 9–12 Inoculation, cutaneous leishmaniasis, Leishmania major parasites 6–7 In silico reconstitution, Cryptosporidium parvum genome 112–115 In situ trophont control, ichthyophthiriosis infection 215–217 Intercrosses, murine cutaneous leishmaniasis quantitative trait loci (QTL) mapping 34–35 susceptibility studies 43–49 Interferon-g (IGF-g) cryptosporidiosis immunobiology 102–104 murine leishmaniasis basic mechanisms of 24–27 cellular and noncellular innate response mechanisms 15–17 macrophages, innate immune response 10–12 transcription factors, Th1/Th2 response 28–29

INDEX Interferon-g receptor 1 (IFNGR1), human leishmaniasis, genetic susceptibility studies 50 Interferons-a and -b (IFNa/b), murine leishmaniasis, macrophages, innate immune response 11–12 Interleukin-4 (IL-4), murine leishmaniasis basic mechanisms of 21–24 cellular and noncellular mechanisms 15–17 dendritic cells 14 interferon-g and IL-12 mechanisms 24–27 Leishmania major infection, early T cell response 19–20 Interleukin-8 (IL-8), murine leishmaniasis, transcription factors, Th1/Th2 response 28–29 Interleukin-10 (IL-10), murine leishmaniasis basic mechanisms of 21–24 macrophages, innate immune response 10–12 Interleukin-12 (IL-12), murine leishmaniasis basic mechanisms of 24–27 cellular and noncellular innate response mechanisms 16–17 dendritic cell response 13–14 disease outcome and 22 genetic susceptibility studies 47–49 innate immune respone 10–12 Leishmania major infection, early T cell response 19–20 Interleukin-12p35 and -12p40, murine leishmaniasis, mechanisms of 26–27 Intraepithelial lymphpocytes (IEL), cryptosporidiosis immunobiology 102–104 Intraspecific variation, in nematomorphs 255

INDEX In vitro techniques Cryptosporidium pathology 79 Ichthyophthirius multifiliis 193–194 In vivo techniques, Cryptosporidium maintenance 97–99 pathology 96–97 Ir-2 gene, murine cutaneous leishmaniasis, susceptibility studies 44–49 JAK-STAT pathway, murine leishmaniasis, transcription factors, Th1/Th2 response 28–29 Kaolin, Cryptosporidium chemotherapy 133–136 Knockout mouse models, murine leishmaniasis, interleukin-4 and -10 mechanisms 23–24 Laboratory parasites, environmental factors in leishmaniasis and 52–53 LACK (Leishmania homologue of mammalian RACK1) antigen, murine leishmaniasis, Leishmania major infection, early T cell response 19–20 Larval morphology gordiid life cycle and 259–260 Nematomorpha 250 Leishmania amazonensis, murine leishmaniasis interleukin-4 and -10 mechanisms 22–24 macrophages, innate immune response 10–12 Leishmania braziliensis cutaneous leishmaniasis and 6–7 murine leishmaniasis, macrophages, innate immune response 10–12 Leishmania donovani 42 Leishmania guyanesis, human cutaneous leishmaniasis, host resistance to 30–31

317 Leishmania major animal models 7–8 cutaneous leishmaniasis and 6–7 environmental factors influencing 51–53 human cutaneous leishmaniasis, host resistance to 30–31 murine leishmaniasis adaptive immune response early T cell response 19–20 T cell mechanisms 17–18 innate immune response cellular and noncellular mechanisms 14–17 dendritic cells 12–14 macrophages 9–12 interferon-g and IL-12 mechanisms 24–27 interleukin-4 and -10 mechanisms 21–24 susceptibility studies 43–49 Leishmania mexicana, murine leishmaniasis dendritic cell response 13–14 interleukin-4 and -10 mechanisms 22–24 Leishmaniasis. See also Cutaneous leishmaniasis animal models 7–8 damage–response framework 3–4 environmental factors in 51–53 etiology of 2 host response to, genetics of 42–50 human susceptibility 49–50 mouse susceptibility genetics 42–49 Leishmania life cycle and 4–7 Leishmania sp. digenetic structure 2 leishmaniasis and 4– 7 Leishmania tropica, cutaneous leishmaniasis and 6–7 Letrazuril, Cryptosporidium chemotherapy 135–136 Leucocytes, Ichthyophthirius multifiliis feeding and digestion 187–188

318 Life cycle and development mechanisms Cryptosporidium 8 8–99 establishment 88–89 macrogamonts 94 maintenance and amplification 95–99 meront I 92 meront II 92–93 merozoites 93 microgamonts 93–94 oocysts 94–95 sporozoites 95 trophozoite stage 91–92 in vitro cultivation 97–99 in vivo maintenance, animal models 96–97 Ichthyophthirius multifiliis 163 Nematomorpha 258–279 experimental conditions 269–271 general conditions 258–260 laboratory conditions 269–271 paratenic host, role of 265–269 Linkage analysis human cutaneous leishmaniasis 40–41 murine cutaneous leishmaniasis 45–49 Lipophosphoglycan (LPG), murine leishmaniasis, macrophages, innate immune response 10–12 lmr loci, murine cutaneous leishmaniasis, genetic susceptibility studies 45–49 Localized cellular response, ichthyophthiriosis pathogenesis and 208–209 Loci mapping, murine cutaneous leishmaniasis genetic susceptibility studies 47–49 host response to disease 33–34 Longevity of parasites, paratenic increase in 267 Loperamide, Cryptosporidium chemotherapy 133–136 Lost infective stages, paratenic host recovery of 267 Lsh gene. See Slc11a1 gene

INDEX Macrogamonts, Cryptosporidium life cycle 94 Macrophages, murine leishmaniasis, innate immune response 9–12 Maintenance mechanisms, of paratenic hosts 267–269 Major histocompatibility complex (MHC) human leishmaniasis susceptibility genetics 49–50 ichthyophthiriosis immunity 200–204 murine cutaneous leishmaniasis, genetic susceptibility studies 44–49 Malachite green, in situ ichthyophthiriosis control with 215 Manipulative behaviour, nematophora–host interactions 284–288 collaboration vs. 289–290 physiology and neurology 284–285 proteomics 288 Mannose receptor, murine leishmaniasis, macrophages, innate immune response 10–12 Mattesia dispora, life cycle 82–85 Mattesia geminate, life cycle 83–85 Melanotaenia eachamensis, ichthyophthiriosis control and treatment in 210 Membrane proteins, Ichthyophthirius multifiliis somatic cortex and 179–183 Merogony, Cryptosporidium life cycle 90–91 Meront I development, Cryptosporidium life cycle 92 Meront II development, Cryptosporidium life cycle 92–93 Merozoites, Cryptosporidium life cycle 90–91, 93 Methylene blue, ichthyophthiriosis control using 214

INDEX Methylene-tetrahydrofolate (MTHF), |in Cryptosporidium parvum 122 Metronidazole, in situ ichthyophthiriosis control using 216–217 Miamiensis avidus, ichthyophthiriosis pathogenesis and 209 Microgamonts, Cryptosporidium life cycle 93–94 Microtubular structures, Ichthyophthirius multifiliis excretory system 183–184 somatic cortex 181–183 MIP-1a and -1b, murine leishmaniasis, cellular and noncellular innate response mechanisms 16–17 MIP-1a/CCL3 chemokine, murine leishmaniasis, cellular and noncellular innate response mechanisms 16–17 ‘‘Missing genes,’’ in Cryptosporidium parvum 112–115 Monoclonal antibodies (mAb), Ichthyophthirius multifiliis, protective immunity 204 Monocyte chemotactic protein 1 (MCP-1), murine leishmaniasis, cellular and noncellular innate response mechanisms 16–17 Morphometric analysis, paratenic gordiid host identity 261–263 Mouse models leishmaniasis 7–8 susceptibility studies 42–49 in vivo Cryptosporidium 96–97 Mucocutaneous leishmaniasis (MCL), human susceptibility genetics 49–50 Mucocysts, Ichthyophthirius multifiliis epidermal invasion 174–175 somatic cortex structure and 181–183 Multiple strain comparisons, murine cutaneous leishmaniasis, genetic analysis 33 Murine cutaneous leishmaniasis environmental factors influencing 51–53

319 host resistance to disease 8–29 adaptive immune response 17–21 regulatory T cells 21–22 T cell mechanisms 17–18 response to Leishmania major 19–20 T helper cell polarisation 18–19 cytokines and immunomodulatory molecules 21–28 interferon-g and IL-2 24–27 interleukin-4 and -10 21–24 TNF-a, TGF-b and accessory molecules 27–28 genetic analysis 31–38 common haplotype mapping 36 congenic strains 37 gene discovery and validation 37–38 intercrosses or backcrosses 34–35 loci mapping 33–34 multiple strain comparisons 33 recombinant congenic strains 36 recombinant inbred strains 35 genetic susceptibility studies 42–49 innate immune response mechanisms 9–17 dendritic cells 12–14 immune system characteristics 14–17 macrophages 9–12 transcription factors, Th1/Th2 response 28–29 MyD88 protein, murine leishmaniasis, dendritic cell response 13–14 Natural killer (NK) cells host resistance to disease, cutaneous leishmaniasis, mouse models 8 murine leishmaniasis cellular and noncellular innate response mechanisms 15–17 interferon-g and IL-12 mechanisms 25–27 Natural paratenic hosts, nematomorpha 263–264

320 Nectonema agile, morphology of 249–250 Nectonema munidae, morphology of 249–250 Nectonematids biodiversity of 252–255 biogeographical distribution of 256–257 biology of 292–293 life cycle and ecology 258 morphology of 249–250 paratenic gordiid host identity 261–263 research background on 244–245 Nematomorpha distribution 272–276 fine scale distribution 273–274 future research issues 275–276 temporal distribution 275 gordiid–human interaction 276–279 household distribution 276–277 potable water sources 276 pseudoparasitism 277–279 host behavioural alterations 279–291 field and laboratory experiments 281–284 manipulation vs. collaboration mechanisms 289–291 physiology and neurology manipulations 284–288 proteomics 288 study system for 280–281 life cycle 258–260 experimental life cycles 269–271 morphology 245–251 Gordiida species 246–249 in host 250–251 larval characteristics 250 Nectonema species 249–250 paratenic hosts 260–269 evolutionary role of 269 identity of 261–263 life cycle role of 265–269 lost infective stage recovery 267 natural hosts 263–264

INDEX parasite infectivity and 266–267 parasite longevity and 267 paratenesis 264–265 site dispersal/maintenance 267–269 research background 244–245 sex ratio 271–272 taxonomy and systematics 251–258 biodiversity 251–255 biogeography 255–257 intraspecific variation 255 phylogeny 257–258 Nematomorpha occidentalis, paratenic hosts 263–264 Neochordodes biogeographical distribution of 256–257 taxonomy of 253–255 Neoichthyophthirius scholtfeldti 162 Neurological behaviour, manipulation of host changes 284–285 Neutrophils host resistance to disease, cutaneous leishmaniasis, mouse models 8 murine leishmaniasis, cellular and noncellular innate response mechanisms 15–17 New York isolate of Ichthyophthirius multifiliis (NY1), ichthyophthiriosis immunity 198 Night-parasitized crickets, physiology and neurology 284–285 Night-uninfected crickets, physiology and neurology 284–285 Niridazole, in situ ichthyophthiriosis control using 216–217 Nitazoxanide, Cryptosporidium chemotherapy 135–136 Nitric oxide (NO) human cutaneous leishmaniasis, host resistance to 31 murine leishmaniasis interferon-g and IL-12 mechanisms 24–27 macrophages, innate immune response 11–12

INDEX Non-transmitted chromosome controls, human cutaneous leishmaniasis, genetic analysis 39–40 Noteochordodes, biodiversity of 252–255 Nucleic acid metabolism, in Cryptosporidium parvum 120–121 Oocysts Cryptosporidium life cycle 94–95 detection and control 130–132 zoonotic potential in Cryptosporidium 127 Ophryoglena sp., Ichthyophthirius multifiliis taxonomy 164–166 theront development 170–172 Oral cortex, Ichthyophthirius multifiliis cells 183 Oreochromis mossambicus, ichthyophthiriosis immunity 202–204 Organelle of Lieberku¨hn Ichthyophthirius multifiliis theronts 170–172 epidermal invasion by 175 Ichthyophthirius multifiliis trophonts 186–188 Palitomy, Ichthyophthirius multifiliis reproduction 167–168 Pantacordodes, taxonomy of 252–255 Parachordodes, taxonomy of 253–255 Paragordius varius biogeographical distribution of 256–257 development in host 250–251 laboratory domestication of 245 laboratory life cycles 270–271 larval development 250 morphology of 246–249 natural life cycle of 258–260 paratenesis 264–265 paratenic hosts 263–264 increase in infectivity 266–267 longevity increases and 267

321 Paramecium sp. evasive mechanisms of 205–207 Ichthyophthirius multifiliis comparisons with 181–184 Parasitic evolution, paratenic host functions in 269 Paratenesis, Nematomorpha 264–265 Paratenic hosts, Nematomorpha 260–269 evolutionary role of 269 identity of 261–263 life cycle role of 265–269 lost infective stage recovery 267 natural hosts 263–264 parasite infectivity and 266–267 parasite longevity and 267 paratenesis 264–265 site dispersal/maintenance 267–269 Paromomycin, Cryptosporidium chemotherapy 133–136 Passive immunity, ichthyophthiriosis 201–204 Pathogenesis framework Cryptosporidium, host–parasite relationship 99–101 ichthyophthiriosis 207–209 leishmaniasis, damage–response framework 3–4 Pectin, Cryptosporidium chemotherapy 133–136 Perforatorium characteristics, fish hosts, Ichthyophthirius multifiliis, epidermal invasion 173–175 Peripheral blood mononuclear cells (PBMC), human cutaneous leishmaniasis, host resistance to disease 30–31 Phlebotomine sandfly, Leishmania parasite life cycle and 4–7 pH levels, ichthyophthiriosis control and treatment and changes in 211–212 Phosphoenolpyruvate (PEP), in Cryptosporidium parvum 116–118 Phototaxis, Ichthyophthirius multifiliis theront contact 172–173

322 Phylogenetic relationships Cryptosporidium 79–87 biological and molecular characteristics 79–85 current taxonomy 85–87 taxonomic techniques 87 of Nematomorpha 257–258 Physiological conditions, manipulation of host changes 284–285 Pigs, cryptosporidiosis in 108–109 Plasma cells, ichthyophthiriosis immunity 201–204 Plasmodium, motility behaviour 83–85 Polyclonal antibodies, cryptosporidiosis immunobiology 104 Polyketide synthesis, in Cryptosporidium parvum 118–120 Polymerase chain reaction (PCR) Cryptosporidium detection and control 131–132 taxonomy 85–87 Tetrahymena corlissi culture 193–194 Polymorphisms human cutaneous leishmaniasis, genetic analysis 39–40 murine cutaneous leishmaniasis, genetic analysis 32 Post-kala-azar dermal leishmaniasis syndrome (PKDL), human susceptibility studies 50 Potable water, human–gordiid interaction 276 Potassium permanganate, ichthyophthiriosis control using 214 Poultry, cryptosporidiosis in 109 Probiotics, cryptosporidiosis immunotherapy 137 Promastigote parasites Leishmania species 4–7 macrophages, innate immune response, murine leishmaniasis 10–12 murine cutaneous leishmaniasis, susceptibility studies 44–49

INDEX natural vs. laboratory environmental conditions and 52–53 Protective immunity, ichthyophthiriosis 198–204 Proteomics of Nematomorpha. See also Genetic analysis future research issues 292–293 manipulation of host changes and 288 Proteophosphoglycans (PPG), murine leishmaniasis, macrophages, innate immune response 10–12 Pseudochordodes bedriagae morphology of 246–249 taxonomy 252–255 Pseudochordodes sp. biogeographical distribution of 256–257 taxonomy of 253–255 Pseudoparasitism, human–gordiid interaction 277–279 Puntius gonionotus, ichthyophthiriosis control in 214 Pyloric region, Cryptosporidium pathogenesis 101 Pyrophosphate-dependent phosphofructokinase (PPi-PFK) homologues, in Cryptosporidium parvum 116–118 Quantitative trait loci (QTL), murine cutaneous leishmaniasis 34 common haplotype mapping 36 congenic strains 37 gene discovery and validation 37–38 intercrosses and backcrosses 34–35 recombinant congenic strains 36 recombinant inbred strains 35 Rainbow trout protective immunity against ichthyophthiriosis 198–204 in situ ichthyophthiriosis control in 216–217 Rana esculenta, Ichthyophthirius multifiliis host finding and contact 173

INDEX RANTES (regulated on activation normal T cell-expressed andsecreted) protein, murine leishmaniasis, cellular and noncellular innate response mechanisms 16–17 Recombinant congenic strains (RCS), murine cutaneous leishmaniasis genetic susceptibility studies 47–49 quantitative trait loci mapping 36 Recombinant inbred strains (RIS), murine cutaneous leishmaniasis, quantitative trait loci mapping 35 Recombinant vaccines, for ichthyophthiriosis 218–219 Reptiles, cryptosporidoisis in 110–111 Residum cells, Cryptosporidium microgramonts 93–94 Resistance alleles, murine cutaneous leishmaniasis, genetic susceptibility studies 45–49 Resistant phenocopies, murine cutaneous leishmaniasis, genetic susceptibility studies 46–49 Restriction fragment length polymorphism (RFLP), in Cryptosporidium 124–127 detection and control 131–132 Rhamdia quelin, ichthyophthiriosis control and treatment in 211–212 Ronidizole, ichthyophthiriosis control using 214 Roxithromycin, Cryptosporidium chemotherapy 133–136 Salinomycin sodium, in situ ichthyophthiriosis control using 216–217 Salivary gland preparations, leishmaniasis management and 51–53 Salmo trutta, ichthyophthiriosis vaccine in 217–218 Schistosoma mansoni, epidermal invasion mechanisms of 175

323 Secnidazole, in situ ichthyophthiriosis control using 216–217 Sex ratio, Nematomorpha 271–272 Sexual development Cryptosporidium life cycle 90–91 Gordiida morphology and 247–249 Sheep and goats, cryptosporidiosis in 107–108 Sib-pairing, human cutaneous leishmaniasis, genetic analysis 41 Silver nitrate, ichthyophthiriosis control using 214 Single nucleotide polymorphism (SNP) analysis human cutaneous leishmaniasis 41–42 murine cutaneous leishmaniasis, common haplotype mapping 36 Slc11a1 gene human leishmaniasis, genetic susceptibility studies 50 identification of 42 murine cutaneous leishmaniasis, loci mapping 33–34 Sodium chloride (NaCl), ichthyophthiriosis control and treatment 211–212, 214 Sodium percarbonate, ichthyophthiriosis control using 214 Somatic cortex, Ichthyophthirius multifiliis cell structure 179–183 Sonicate vaccine compounds, for ichthyophthiriosis 217–218 Spinochordodes, taxonomy of 252–255 Spongiome structures, Ichthyophthirius multifiliis excretory system 183–184 Sporozoites, Cryptosporidium life cycle 90–91, 95 in vitro maintenance 97–99 ‘‘Stream drift paradox,’’ Nematomorpha paratenic hosts 268–269 STS alleles, murine cutaneous leishmaniasis, genetic susceptibility studies 47–49 Survival conditions, Ichthyophthirius multifiliis tomonts 168–169

324 Susceptibility genetics human leishmaniasis 49–50 murine leishmaniasis 42–49 Taurine, manipulation of host changes and 287–288 Taxonomy Cryptosporidium 85–87 Ichthyophthirius multifiliis 163–166 Nematomorpha 251–258 biodiversity 251–255 biogeography 255–257 intraspecific variation 255 phylogeny 257–258 T cells ichthyophthiriosis immunity 200–204 murine cutaneous leishmaniasis adaptive immune response 17–18 regulatory T cells 20–21 Leishmania major infection, early response mechanism 19–20 Teleosts, ichthyophthiriosis immunity 199–204 Temperature fluctuations, ichthyophthiriosis control and treatment 209–210, 212 Temporal distribution, Nematomorpha 275 Tetrahymena species evasive mechanisms of 205–207 ichthyophthiriosis immunity 203–204 vaccine developed from 217–219 Ichthyophthirius multifiliis and feeding and digestion systems 186–188 i-antigen isolation 196–198 parasomal sac structure and function 179–183 trophont morphology 178 trophont reproduction in fish host 189–190 in vitro culture and cryopreservation 193–194

INDEX Tetrahymena thermophila, ichthyophthiriosis vaccine from 218–219 T helper cells (Th1/Th2) environmental factors in leishmaniasis and 52–53 host resistance to disease, cutaneous leishmaniasis, mouse models 8 murine leishmaniasis adaptive immune response 18–19 interferon-g and IL-12 mechanisms 24–27 interleukin-4 and -10, mechanisms of 21–24 Leishmania major infection, early T cell response 19–20 TNF-a, TGF-b, and accessory molecules 27–28 transcription factors 28–29 Theronts ichthyophthiriosis transmission control and treatment and exposure to 210–211 immunity 202–204 pathogenesis and 207–209 Ichthyophthirius multifiliis 167, 169–172 epidermal invasion 174–175 host finding and contact 172–173 protective immunity against 204 in vitro culture and cryopreservation 193–194 Thioesterase (TE) domain, in Cryptosporidium parvum 119–120 TLR4, murine leishmaniasis, dendritic cell response 13–14 TNFRp55 and p57, murine leishmaniasis, basic mechanisms 27–28 Toltrazuril, in situ ichthyophthiriosis control with 216 Tomont development, Ichthyophthirius multifiliis 166–169 reproduction phase 167–168 survival conditions 168–169

INDEX Toxoplasma, motility behaviour 83–85 Transcription factors, murine leishmaniasis, Th1/Th2 response 28–29 Transforming growth factor-b (TGF-b), murine leishmaniasis basic mechanisms 27–28 macrophages, innate immune response 10–12 Transmission mechanisms, in Cryptosporidium 123–127 Treatment regimens, ichthyophthiriosis 209–219 chemical use 212–217 free-living parasitic stages 213–215 in situ trophont destruction 215–217 vaccine development 217–219 water management 211–212 Trichodina, evasive mechanisms of 205–207 Trophonts, Ichthyophthirius multifiliis evasion of host immune response by 205–207 exit from fish host 190–193 feeding and digestion 186–188 growth and development 185–186 in situ control of infection from 215–217 infection sites 176–177 reproduction in fish host 188–190 structure and function 177–185 cell structure and function 179–183 endoplasm 184–185 excretory system 183–184 morphology 177–178 in vitro culture and cryopreservation 193–194 Trophozoite, Cryptosporidium life cycle 91–92 Tumor necrosis factor-a (TNF-a), murine leishmaniasis, mechanisms of 27–28

325 Vaccine development cryptosporidiosis 136–137 for ichthyophthiriosis 203–204, 217–219 recombinant vaccines 218–219 whole cell/sonicate compounds 217–218 Vacuoles, in Ichthyophthirius multifiliis endoplasm 184–185 feeding and digestion system 187–188 Variant-specific surface glycoproteins (VSGs), ichthyophthiriosis immunity 197–198 Viral infections, cryptosporidiosis in cattle 106–107 Virulence factors, leishmaniasis, damage–response framework 3–4 Visceral leishmaniasis (VL) environmental factors influencing 51–53 human susceptibility genetics 49–50 Water gordiid transition to 259–260 human–gordiid interaction, potable water and 276 management, ichthyophthiriosis control and treatment 211–212 Nematomorpha, water-seeking behaviour 280–281 Whole cell vaccine compounds, for ichthyophthiriosis 217–218 WNT protein family, manipulation of host changes and 288 Xenosomes, Ichthyophthirius multifiliis endoplasm 185 Xiphophorus maculates, ichthyophthiriosis control and treatment in 210 Yeast artificial chromosomes (YAC), leishmaniasis animal models 8 Zoonotic potential, in Cryptosporidium 123–127

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