Cumitech 28: Laboratory Diagnosis of Zoonotic Infections

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IN CLINICAL

MICROBIOLOGY

FEBRUARY

1996

Laboratory Diagnosis of Zoonotic Infections: Chlamydial, Fungal, Viral, and Parasitic Infections Obtained from.Companion and Laboratory Animals ANDREA GUICHON, and ANNE ZAJAC COORDINATING

THOMAS

AMERICAN

THOMAS

J. INZANA,

EDITOR

J. INZANA

SOCIETY

FOR MICROBIOLOGY

JOSEPH

M. SCIMECA,

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32 in a patient with com- firmationwith serologictestsisneeded.For C. trachpatible symptoms supports the clinical impression. omatis, the EIAs tend to be more specificbut less Cross-reactions may occur with sera from patients sensitivethan DFA tests (11). The kits that will previously infected or exposed to other chlamyd- detect C. psittaci include Clearvue (Unipath Labiae. The CF test titers are likely to be higher in oratory, Mountain View, Calif.), IDEIA (Bootsavian or mammalian chlamydiosis than in C. pneuCelltech Diagnostic,Ltd., Berkshire,United Kingmoniae infection, but early treatment with tetra- dom), and Kodak SureCell (Eastman Kodak, cyclinesmay delay or suppressthe complement- Rochester, N.Y.). Although these kits are being fixing antibody rise and obscurethe serological usedto test veterinary samples,false-positiveand diagnosis.The CF test may be performedin either -negativeresultsmay be more commonwith these test tube or microtiter format. If commercial specimensthan those from humans. Aberrant preparationsdo not offer an antigenwith a high resultswill vary widely dependingon the specienoughtiter, antigenscould be prepared in the men: fecal specimensmay result in unacceptably laboratory from yolk sacsof embryonated hen high false-positiveresults(34), whereasplacentas eggs.The other reagentsare commerciallyacces- andvaginal swabsfrom sheepabortionshavebeen sible and can be usedwith appropriate controls reported to have a 95.3% sensitivity and a 94.0% and standardization(84). specificitywith the Kodak SureCellkit (82). When The lesswidely availablemicroimmunofluores- testing bird specimens,the IDEIA kit may result cencetest, which measuresantibodiesto EB wall in too many false-positive results, whereas the antigens,is a more sensitiveand specificmethod. Kodak SureCell is lesssensitivethan cell culture It detects antibodiesto specificchlamydial sero- (34). As the genus-specifictests target the LPS vars (104). The antigenic suspensionis obtained antigen, additional differential staining with imfrom organismsgrown in yolk sacor tissueculture munofluorescentmonoclonal antibodies should monolayers.The test may be directed to the sus- be carried out to determine the species. pected antigenicserotype,and the immunoglobuThe DFA test allowsthe microscopistto evalulin classof the antibody can alsobe determined. ate the adequacyof the specimen,and it is conPaired serafrom patientswith humanpsittacosis venient for handling a smallnumber of samples. usually showrising immunoglobulinG (IgG) an- Subjectiveinterpretation and false-positiveresults tibody levelsand the presenceof IgM antibodies. with LPS from other gram-negative organisms The detectionof a high titer of IgM antibodiescan are a few disadvantages.In addition, a technoloassays be usedto confirm a recent infection (34). The gist trained in readingimmunofluorescence major disadvantageof microimmunofluorescence and a fluorescencemicroscopeare required for is that the genus-reactiveantigen exposedon the the DFA test. Although EIAs offer lesssensitivity than DFA surfaceof the EB can cross-reactwith antibodies to other chlamydialspecies(84). tests(88), they rely on a standardizedreadingand are suitable for batch processing;both features Suitability of Rapid Commercial Methods are convenient for the clinical laboratory. In adCommercial immunofluorescencetests have dition, the commercialEIAs conveniently include been developed to detect antibodies against a positive and negative controls in the assays. more broadly reactive antigen. Chlamydialinclusionsin cell monolayerspreviouslyinfected with a ZOONOTIC FUNGAL INFECTIONS cross-reactingstrain(suchasLGV 2) are available as the test antigen.The test is sensitiveand proDescription, Natural Habitat, and videsboth genus-and species-specific reactivity on Mode of Transmission the basisof the prominenceof LPS in the inclusion.It can be usedto detect avian and mamma- Dermatophytes lian chlamydiosisand other chlamydialinfections, Most of the fungi that infect animalscause but the resultsmay reflect the prevalenceof chla- similar or identical diseasesin humans.The facmydial antibodiescommonlyfound in the general tors and modesof transmissionof fungi to animals population. are also similar to those in humans(e.g., immuThe commercially available enzyme immuno- nocompromisedstate, inhalation of a large numassays(EIAs) and direct fluorescent-antibody ber of sporesfrom the environment, etc.). Since (DFA) reagentshave beendevelopedprimarily to mostfungi are normally saprophyticand opportudetect C. trachomatis. The kits that use genus- nistic pathogensof humansand animals,very few specificantibody reagentscan be usedon sputum fungal infections are zoonotic. Certain speciesof samplesto detect Chlamydia-specific LPS (88) dermatophytes, and recently the canine yeast and can benefit the patient if they allow antimi- Malassezia pachydemzatis, are exceptions. Unlike most saprophvticfungi, somedermatocrobial therapv to be started earlv in the courseof

CUMITECH

28

phytes have developed a close, though not obligate, relationshipwith certain host species.The predominantreservoir of dermatophytesmay be classifiedas the soil or environment (geophilic), humans (anthrophilic), or animals (zoophilic). The mostcommondermatophytesof pet animals (dogs and cats) are Microsporum canis, which includesM canis var. canis and 1M. canis var. distorturn; Microsponcm gypseum; and Trichophyton mentagrophytes var. mentagrophytes. Not only are dogsandcatsinfected by M. canis, they are the primary reservoirs for this agent. T mentagrophytes var. mentagrophytes is carried by a wide variety of laboratory and pet rodents and may infect a wide variety of animal speciesand humans.T. mentagrophytes var. erinacei is carried by hedgehogsin New Zealand and parts of Europe, and T. mentagrophytes var. quinckeanum is carried by mice.Theselatter two varietiesof T mentagrophytes are rarely transmitted to humansand will not be discussed here. M gypseum infections are geophilic and are unlikely to be spread from animalto animalor from animalto human.Of the infections caused by zoophilic dermatophytes, thosedueto M. canis are by far the mostcommon. However,becauseof the closeadaptationthat has developed,pets (particularly cats) may be asymptomatic carriers of M. canis. The suddenoccurrence of M canis infections in pets or ownersis usually associatedwith the addition of a new pet (usuallya kitten) to the household.Closeexamination of the pet may reveal partial alopecia, scaling,or broken hairs (67). The infectious particle of the dermatophyte is the arthrospore,which can remain infectious for years. Following contact, the arthrospore enters the skin through an abrasionand germinatesand hyphaebeginto grow in the stratumcorneum.The hyphaeinvade the hair follicle and enter the cortex of the hair by dissolvingthe keratin. The hyphaeand conidia are carried to the surfaceby the growinghair, which often breaksoff. Hair invasion may be endothrix (arthroconidia develop within the hair shaft only and the cuticle remainsintact) or ectothrix (arthroconidia develop outside the hair shaftand hyphaearewithin the hair shaft;the cuticle is destroyed). M. pachydermatis

The obligatory lipophilic yeastsMalasezzia furfur and Malassezia sympodialis normally inhabit human skin. In contrast, the nonobligatory lipophilic yeast M. pachydermatis is a commensalof the oily areasof the skin and earsof dogs,but it may alsobe isolatedfrom cats. When isolatedin high numbers,M pachydermatis hasbeen considered a causeof otitis externa in dogs,but rarely other infections. There have been no adequate epidemiologicstudiesdocumentingtransmission of M pachydermatis from dogsto humans.A sur-

DIAGNOSIS

OF ZOONOTIC

INFECTIONS

5

vey of yeastsrecovered from a reference laboratory indicated that M pachydemzatis is capableof colonizing multiple human anatomic sites (36). Mickelsen et al. (69) isolatedM. pachydermatis from 30 hospitalizedpatients without any clinical symptoms,most of whom were infants in a neonatal intensivecare unit. Most of the isolateswere recovered from clean-catchor baggedurine samples; the remainingisolateswere recoveredfrom endotracheal aspiratesand swabsof the nares, rectum, or vagina. The most serious infection causedby M. pachydermatis is catheter-associated sepsis,in which the organismsare thought to gain accessto the catheter after colonization of the skin and then to invade the bloodstream(61). Although not documented,it is possiblethat hospital personnelbecomecolonizedwith M pachydermatis by their petsandthen serveasa reservoir of the organismfor hospitalizedpatients,particularly newborninfantswho havenot yet established a normal flora. Human

Infections

Dermatophytes

Dermatophytosisdue to a zoophilic fungus is clinically similarto that causedby an anthrophilic fungus. In general,zoophilic fungi causea more intense inflammatory responsein humans than anthrophilic fungi, and the former may cause pustular lesions(kerions). The typical lesionis an annular scalingpatch with a raised margin; the degree of inflammation will vary. The lesion results from a hyperkeratosiswith septatehyphae and arthroconidia in the stratum corneum. The inflammatory responseis mostintenseat the area of recent invasion. The infection spreadsin all directionswith a central area of healing,resulting in a ring-like appearance.The clinical appearance will vary dependingon the site of infection, the fungal speciesinvolved, and the host’s immune response(39). Tinea capitis(infection of the scalp, eyebrows,and eyelashes)is commonlycausedby M. canis, while the zoophilic T mentagrophytes varieties may causetinea barbae(infection of the beard and moustache)and tinea pedis (infection of the feet) (107). M. pachydermatis While M. furfur is usually associatedwith the skin infection pityriasisversicolor, both IM. furjfur and M. pachydermatis may causecatheter-associ-

ated sepsis.Almost all confirmed clinical casesof infections in humanshave occurred in infants in neonatal intensive care units (36, 57, 69). Most infections are associatedwith placementof a central venouscatheter (CVC) for administration of fluids containing lipid emulsions.Isolateshave beenrecoveredfrom cerebrospinalfluid, urine, eyeand ear discharge,catheter

M pachydemzatis

6

GUICHON

ET AL.

blood, peripheral blood, central line catheter tips, and tracheal aspirates. All infants were premature and had multiple complications. Most of the infants were receiving broad-spectrum antibiotics and a parenteral lipid emulsion through a CVC. Sepsis due to M. pachydermatis has been associated with fever, lethargy, respiratory distress,and bouts of bradycardia. Many infected infants recover upon removal of the CVC. In general, a Malassezia infection shouldbe consideredin any caseof sepsisinvolving a patient receiving lipid emulsionsthrough a CVC. There has also been one case of M. pachydermatis canaliculitis in a 61-year-oldman (80). Treatment Dermatophytes

When a zoonotic dermatophyte infection is diagnosed,the suspectingcontact animal should be examinedfor the samefungus.By far the most commonagent is M. canis. Dermatophyte lesions in animalswill usuallyclear within 8 to16 weeks becauseof the immuneresponseof the hostto the infecting agent. Bedding and other materialsthe pet hashad closecontact with need to be decontaminated becausethe arthrosporescan remain infectiousfor years.The most effective treatment is oral administration of griseofulvin; ketoconazole,itraconazole,andterbinafinehave alsobeen used,dependingon the siteof infection. However, topical treatmentsshouldbe usedfirst. The keratolytic agent Whitfield’s ointment (salicylic and benzoic acids) is more effective on heavily keratinized areas.Agents with specificantifungal activity include chlorphenesin,undecylenate,tolnaftate, and a wide variety of azole antimycotic agents:miconazole,clotrimazole,oxiconazole,and others(39). If the infection is in the nail or hair, oral therapy is usually the only successfultreatment, but it requires long-term administration, as the nail grows slowly and treatment must be continued until a completely clean nail has formed. M. pachydermatis

Treatment of catheter-associated sepsisdue to should involve removal of the CVC and a short courseof amphotericinB. Sepsis due to M. fi&r is usuallymore seriousthan that due to M. pachydermatis and may require more aggressiveantifungal therapy (61).

M. pachydermatis

Isolation

and Identification

Dermatophytes

Samplesof skin scrapingsor hair or both may be submittedin a clean,nonstaticcontainer, such asa paper envelope.Hairs shouldbe epilatedwith sterileforceps.The baseof the hair, includingthe

CUMITECH

28

follicle, is preferable. In young, noncompliant patients, a strip of clear tape may be placed over the hair and then removed. The border of the active region of skin infections should be disinfected with alcohol and scrapedwith a scalpelto collect epidermalscales.If nailsare infected, the waxy, subungualdebris,which containsthe viable fungi, shouldbe collected (107). Scrapingsshould be collectedfrom the proximal to the distal end of the nail, with the first four to five scrapings discarded.Large piecesof nail shouldbe pulverized in a sterile container prior to culture and microscopicexam (66). Hairs infected with M. canis will usuallygive off a yellow-greenfluorescencein a darkenedroom under a UV light of 3660 A, such as a Wood’s lamp. Fluorescencecanbe usedto selecthairsfor culture but should not be used for diagnosis becauseshampoosandother treatmentscan cause a false-positive fluorescenceor inhibit fluorescence. However, not all strains of M. canis will causefluorescenceof hairs,andM. gypseum and T. mentagrophytes will never fluoresce.M. canis, M. gypseum, and T. mentagrophytes all cause ectothrix-type hair invasion,which is characterizedby the presenceof arthroconidia on the outside of the hair shaft and the destruction of the cuticle (66). The arthroconidia of M. canis are small(1 to 3 pm) and form a mosaicsheatharound the hair. The arthroconidiaof M. gypseum and T. mentagrophytes are larger (3 to 4 km), and they are few in number or form chainson the hair surface(108). A rapid diagnosiscan presumptivelybe madeby microscopicexaminationof hair or skin scrapings placedin 20% KOH and gently warmed.Narrow, hyaline, septatehyphaeand chainsof arthrospores may be seen (107). Adequate time must be allowed for the epithelial cells to dissolve,or cell membranesmay be confusedwith hyphae. Falsenegative resultsare commonwith this technique, asare false-positiveresultsdue to artifacts suchas threads, fibers, etc. (67). For this reason,swabs should never be used to collect dermatological specimens. The final diagnosismust be basedon culture and identification of the dermatophytein order to differentiate a zoonotic dermatophyte infection from an anthrophilic or geophilic dermatophyte infection. Samplesshould be inoculated onto a selectivemedium, suchas Sabouraud’sagar with cycloheximideand chloramphenicol.An excellent selectiveand differential mediumis dermatophyte test medium. Although the cycloheximidein the medium inhibits the growth of most saprophytic fungi, somecontaminants(suchas.Aspergillus spp., Penicillium spp.,etc.) may grow in the presenceof cycloheximide.The pH indicator in dermatophyte test mediumturns red during the growth of dermatophytesbecauseof the utilization of proteins; saprophytes preferentially utilize the carbohy-

CUMITECH

28

drates(turning the mediumyellow) but will utilize protein when the carbohydratesare exhausted. Therefore, the red color changedue to dermatophyte growth will occur during growth, rather than after growth hasoccurred.However, a teasepreparation of the fungus should still be made to confirm the identity of the dermatophyte.Because of the growth of dermatophyteson selectivemedia and their distinctive conidia and hyphae, slide culturesusuallydo not needto be madefor these organisms. All zoonotic dermatophytesfrom pet and laboratory animalsare hyaline.M. canis will usuallybe fluffy and present with a colorless or yelloworangereverse.M. gypseum will be more powdery to granular and pale to rosy buff with a white border; the reversewill be buff to reddishbrown. Zoophilic T mentagrophytes will be powdery to flu@, with a cream-tanor pink color. The reverse is light tan, yellow, red, or reddish brown. A diffusible pigment may be produced by some strains.Microscopically, the macroconidiaof M. canis var. canis are 8 to 125by 5 to 25 pm, are ellipsoidto fusiform, and have rough, thick walls with up to 15 septations.An asymmetricknob on the apex maybe visible. Microconidia may or may not be present.The macroconidiaof M. canis var. dislortum are bizarre in shapeand are 20 to 60 by 7 to 27 pm. Microconidia are abundant. Macroscopicallyand microscopicallyM. gypseum looks similar to M. canis. Macroconidia are abundant and are 25 to 60 by 7.5 to 15 pm and ellipsoidto fusiform. A key differential feature of M. gypseum isthat there are usuallyonly up to six septationsin the macroconidia,and the cell wall is thin and smooth(107). Coloniesof T. mentagrophytes are cream, tan, or pink and powdery to granular or fluffy. The reverse is light tan, yellow, red, or reddishbrown. Someisolatesproduce a diffusible melanoid pigment. The microconidia are abundant and are round to pyriform in clustersor are singlealongthe hyphae.Clavatemacroconidiaare not commonbut are produced by somestrains. Coiled or spiral hyphae are often seen,particularly in older cultures.The ureasetest is positive within 7 to 8 days, and this specieswill grow at 37’C. T. mentagrophytes is not nutritionally deficient and will grow on caseinmedium without additionalvitamins (Trichophyton test agar #l). T. mentagrophytes can be distinguishedfrom the morphologically similar T. rubrum, which is a rapid ureaseproducer and is able to causeperforations in the hair perforation test (66). M. pachydermatis Although M. pachydermatis is not asfastidious for long-chainfatty acidsasM. fir&r is, fatty acids will stimulate the growth of M. pachydermatis. Convenient culture media for M. pachydemzatis

DIAGNOSIS

OF ZOONOTIC

INFECTIONS

7

agar, or brain heart infusion agar with 5% sheep blood. Optimum growth occursat 35 to 37°Cfor 2 to 3 days.Growth is poor on Sabourauddextrose agar, malt extract agar, and other media at 25°C (61). A medium composedof glucose-yeastextract-peptone agar supplementedwith olive oil, Tween80, and glycerol monosterate(23) growsall Malassezia spp.well and is commerciallyavailable as MAL agar from Becton Dickinson Microbiology Systems,Cockeysville,Md. Upon microscopicexamination, M. pachydermatis cells will appear subgloboseto ellipsoidal and 2.5 to 5.5 by 3.0 to 6.5 pm in size.Hyphae are not produced in vivo or in vitro. A broad-base, monopolarbud isoften seenat oneend of the cell, separated from the mother cell by a septum. Separationof the daughtercell by fissionresultsin a scar from which later buds emerge.Successive scarsform a collarette, which is a visibly distinct feature of Malassezia spp. Thus, microscopicexaminationisusuallyadequateto identify the yeast in the genusMalassezia. While Malassezia spp. may be identified in blood drawn through the catheter by any of several stains (e.g., Giemsa, Wright, or Gram), most organismsare identified following culture of blood drawn through the catheter. Unlessa sourceof lipids, suchasTween 80, is added to the bottles, M. fur/k can be identified only on blind subculture.However, M. pachydewnatis has been recovered from blood cultures (61). Use of the DuPont Isolator tubes (Wampole Laboratories, Cranbury, N.J.) with subsequentculture to lipid-supplementedmedia may be the best single method for isolation of Malassezia, aswell asother fungi. M. pachydermatis is mosteasilydifferentiated from M. furfur by its ability to grow on mediawithout lipid supplementation. Fermentation tests for M. pachydemtatis are negative, but assimilationtests for glucose, mannitol, andglucitol are positive (92). All Malassezia spp. are positive for ureaseproduction but are relatively inactive in the commercial yeast identification systems.In addition, M. pachydermatis may be positive for glucose,glycerol, and sorbitol in the API20C system(Analytab Products, Plainview,N.Y.) and may therefore be misidentified as Candida Zipolytica without microscopic confirmation (61). Additional details of culture and identification techniquesand reagents and stainsare describedin reference 66 and in a previousCumitechon identification of fungi in the clinical laboratory (38). LYMPHOCYTJC CHORIOMENINGITIS

VIRUS

Description, Natural Habitat, Mode of Transmission

and

Lymphocytic choriomeningitisvirus (LCMV) is include chocolate agar, tryptic soybean digest classifiedin the family of Arenaviridae and the

8

GUICHON

ET AL.

genus Arenavirus. Serologically the speciesare subdivided into “Old World” (LCMV, Lassa, and Mozambique) and “New World” or Tacaribe (Junin, Machupo, Tacaribe, Pichinde,and other) viruses (87). The viral envelope, derived during budding from the host cell cytoplasmic membrane, is covered with club-shapedprojections. LCMV virions are round to pleomorphicand 60 to 280 nm in diameter. LCMV has three major structural proteins: the envelope glycoproteins GP-1 and GP-2 and the nucleocapsidprotein NP (46). Within the laboratory, LCMV growsin variousmammalian,avian, and insectcell lines.Vero (monkey), L-929 (mouse),and BHK-21 (hamster) lines are used for preparation of antigen, virus isolation,and virus titration (58, 71). LCMV is indigenousin wild rodent populations, the primary hosts being both speciesof common house mouse:Mus musculus and i&s domesticus (29). Infection of colonized hamsters (Mesocricetus auratus) has been reported (33). Natural infectionscan occur in the roden.tAoodemus sylvaticus, monkeys,guineapigs,and humans. Only mice and hamstersreadily transmit LCMV to other speciesthrough shedding of virus in urine, saliva,and milk. In mice,horizontal spread occurs initially, but once the infection becomes enzootic, intrauterine transmissionoccurs with high efficiency (87). Common sources of human infection from LCMV are pet or laboratory rodents.Within the laboratory, an important source of infection is LCMV-contaminated tumor cell lines and tissues transplanted in mice and/or hamsters.Humans may acquirethe virus by inhaling dust from contaminated bedding, by direct skin or mucous membranecontact with infected urine, or by parenteral exposure. In laboratory animals,LCMV infectionscanbe persistentor short lived and can be subclinicalor cause morbidity and mortality. The course depends on virus strain, inoculation route, animal species,and age of the animal. Natural LCMV infections in laboratory and wild mice are usually subclinical(90, 91). Mice infected at severaldays of ageor older may becomeill following horizontal infection, but they usually recover. Mice infected in utero or as neonatesbecomecarriers, usually without clinical signs, but occasional deathsmay occur. Human Infections

In humans,LCMV infection mayproducemeningoencephalitis,but manycasesare subclinicalor manifestas a mild febrile illnesswithout central nervoussystem(CNS) manifestations.The tyPical diseasebeginswith fever, malaise,weakness,anorexia, nausea,myalgia, and severe headache, which often occursin the retrorbital region with photophobia.The patient may presentwith a sore

CUMITECH

28

throat, vomiting, and arthralgia. Chest pain and pneumonitisoccur lessfrequently. Physicalexamination revealspharyngealinflammation, usually without exudate. Severely affected patients have meningeal signs,including nuchal rigidity, and about one-third develop encephalopathy,while the remainderexhibit an asepticmeningitis(65). A mild to moderate infiltration of lymphocytes may be present within the leptomeninges,and evidence of perivascular infiltrations of macrophagesin multiple areas of the brain has been reported (105). Diagnosis

Sincemany of the natural rodent infectionsare generallyasymptomaticand humaninfectionsresult in nonspecificclinical signs,LCMV infection is best diagnosedby virus isolation or detection and serology (16, 46, 87). Attempts should be madeto isolateor detect virus from rodent coloniesthat are seldommonitored and may be enzootically infected and from suspecthuman infections with nonspecific clinical signs. Tissue or serumsamplessubmittedto a laboratory for virus isolation should be frozen at -70°C usingcryovials, packedin dry ice, and shippedby overnight mail. When submittingspecimensfor virus isolation, blood,body fluids,andtissuescanbe utilized. When submittinghamstertissues,urine and kidneys should be included. Cerebrospinalfluid in humansis testedif the patient is showingsignsof CNSdisease(7,46,71,87). The presenceof latent LCMV or infection of tissueswith LCMV can be determinedby a diagnostictest called the mouse antibodyproduction test, or in the caseof rats, the rat antibody production test (74). When attempting to isolate LCMV in tissue culture cells, a phenomenonknown as autointerferencemay be observed with specimenscontaining very high titers of LCMV, aswell asof other arenaviruses. Autointerference resultsin the absenceof plaque formation. This interference can be avoided by inoculatingcell lineswith serialdilutions of specimen (71). LCMV can be more reliably detected in cells by using immunocytochemistryor serologic assaysto demonstrateviral antigens(94). Serology is of questionablevalue for mice becausethey are infected congenitally and mount only weak antibody responses.Serodiagnosisof LCMV in humansrequiresa fourfold rise in titer betweenthe acute- andconvalescent-phase serum samplesor an indirect immunofluorescence antibody (IFA) titer of 1:128in a singlespecimenin whichIgM anti-LCMV ispresent(6). In summary, serologyis most effective for routine monitoring of rodents and for diagnosisof infection in humans(87); the methodsinclude CF, the neutralization test, IFA, EIA, and radioimmunoassay.

CUMITECH

28

DIAGNOSIS TABLE

LCMV”

Herpesvirus

Species Means of spread

Rodents, primarily mice Urine and bodily secretions, aerosol

Human clinical signs

Asymptomatic infections occur; febrile illness without CNS manifestations to weakness, myalgia, anorexia, nausea, often severe retrobulbar headache with photophobia; fatality rate low

Control procedures

Monitoring rodent control program for adventitious infections Education and training Personal protective equipment Appropriate biosafety work practices and procedures Serology for man and rodents; IFA test and EIA are replacing less-sensitive CF test and NT. RIA is most sensitive, but not routinely used yet. Virus isolation and detection for rodents not monitored routinely or for patients with nonspecific signs

a Abbreviations: NT, neutralization Infection

Control

INFECTIONS

9

2. Virus reference chart of LCMV and herpesvirus B

Characteristic

Diagnostic procedures

OF ZOONOTIC

B

Nonhuman primates, primarily macaques Bites, scratches, and contact with infected tissues, caging, or research aparatus Vesicular eruptions or ulcers near or at exposure site, severe pain or itching at the site, regional lympadenopathy, fever, numbness paresthesia, muscle weakness, conjunctivitis, CNS impairment, ascending encephalomyelitis; fatility rate high Minimize risk of exposure; chemical restraint of nonhuman primates for exams and procedures Education and training Personal protective equipment; especially face shields and waterproof gowns Utilizing equipment having “squeezeback cages” Serology and direct virus isolation for humans and nonhuman primates; utilizing a nonhuman primate exposure kit containing equipment for serology and transportation of all specimens

test; RIA, radioimmunoassay.

Procedures

Recently (1992), an outbreak of LCMV in sevenworkers at a cancer researchinstitute was reported (20). It was the first time this virus was associatedwith infectionsin nude mice. The outbreak demonstratedthat laboratory transmission of LCMV remainsinsidiousand a persistentproblem. Institutions shouldenactproceduresto safeguard personnelfrom an LCMV outbreak. These proceduresshouldfollow a five-stepapproachthat includes education and training (especially in work practices and procedures), physical plant and appropriate engineeringcontrols, personal protective devices, a rodent control program to prevent adventitiousinfectionsof laboratory mice andhamsters,and diagnosticmethodsto evaluate tissuesand reagentspassagedthrough rodents (78). Microisolator cagingsystemswith appropriate techniquesand trained personnelhave been useful in controlling the spreadof somerodent viral pathogensin the event of an outbreak or during testing (17, 58). Investigatorsand researchpersonnelshouldbe familiar with or have training in the potential of zoonotic spreadof LCMV. Animal resourcesstaff, as well as investigatorsand technicians,should know the health statusof their animalsprior to any experimentalmanipulations.The animal resourcesstaff shouldbe informed of what type of tissue(s)is transplanted,the sourceof tissue,and whetherthe tissuehashadpreviousscreeningtests

for LCMV or other pathogenscapable of producing intercurrent disease.Surveillance guidelines for rodent pathogens through a sentinel program havebeenreported (17,56). In addition, tissuesused for transplantation, including blood and tumor cells,shouldbe testedfor LCMV prior to use in rodents. Thus, the zoonotic diseasepotential within institutions can be minimized with the adoption of modern laboratory animal management practices, routine diseasesurveillance, appropriate sanitary procedures, good personal hygiene, and a good personnelhealth and educational program. The epidemiology, symptoms,control procedures, and diagnosisof LCMV infections are summarizedin Table 2. HERPESVIRUS

B OR HERPESVIRUS

Description, Natural Habitat, Mode of Transmission

SIMIAE and

Herpesvirus B, or herpesvirus simiae, is an alpha herpesvirusclosely related antigenically to human herpes simplex virus types one and two (73). Herpesvirussimiaewasfirst isolatedin 1933 by Gay and Holden from the spinal cord of a researchinvestigator who died of a rapidly progressivemeningoencephalitis after beingbitten by a macaque(28). Herpesvirussimiaeis frequently carried asymptomaticallyby Old World primates such as rhesusMacaca mulatta and cynomolgus

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Macaca fasicularis monkeysandhasbeenreported in other membersof the genusMacaca, including M. radiata, M. fuscata, and M. arctoides (52, 54).

Macaques are native to Asia and northern Africa, but many animalsare housedin research facilities and zoos and are kept aspets in private homesthroughout the world. HerpesvirusB infection is prevalent in both captive and feral populations of macaques.Serologicalsurveyshave indicated that the infection rate in these animals approaches80%. The prevalenceof infection in young rhesusmacaquesrisesby the time of sexual maturity and during the peak breeding season, indicating new infections occur at that time. Investigatorshave speculatedthat genital contact is an important route of transmission,although the specificroute of transmissionhasnot been determined becauseof the natural stressesassociated with breeding(e.g., fighting, dominance,and agonisticinteractions)(52). However, the surveysmay underestimatethe numberof infected animalsfor two reasons:(i) persistentlyinfected animalscan lose detectable antibody titers and (ii) animals may take up to 16 months to seroconvert after initial infection (106). In addition, latently infected monkeysactively shedvirus only intermittently and are more likely to shedherpesvirusB whenthey are ill, under stress,or immunocompromised,in addition to during the breedingseason (52, 53, 102,116). HerpesvirusB can be cultured with equalfrequencyfrom the conjunctiva,buccal mucosa,and genital areasof sheddingmonkeys (53, 102,106, 116). Human Infections

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28

to have declined, which may be due to early diagnosisand treatment with antiviral therapy, better supportive care, and/or the diagnosisof milder or early infections. Diagnosis

Specimensfor herpesvirusB culture shouldbe collected from the deepestmarginsof the wound with a sterilecotton or Dacron swabwith wooden or paper handlesand immediately placed into a screw-capvial containing 1 to 2 ml of virus transport- medium. In someinstances,a full thickness skin biopsy sampleor a smallpiece of skin from the margin of the wound for culture may be more appropriate dependingon the site and nature of the injury. Adequate diagnostictestingis available from three licensedlaboratories(seeAppendix 1). If refrigeration is available,Eagle’sminimumessentialmediumwith Earle’s saltsplusgentamicin (50 kg/ml) and bovine serum (2%) is a stable medium that can be stored for 6 months at 4OC. Hanks’buffered salt solutionwith gentamicinmay be used when refrigeration is not available. A licensedtestinglaboratory (Appendix 1) shouldbe consultedregardingwhat the recommendedtransport medium is for use in the health clinic or which “primate exposurekit” should be used at primate facilities (Appendix 2). Samplesof herpesvirusB cultures, as well as clinical specimensknown or reasonablybelieved to contain herpesvirusB, must be packaged,labeled, and shippedaccordingto Code 42 of Federal Regulations,part 72 (“Interstate Shipmentof Etiologic Agents”). Samplesshouldbe securedin watertight primary containersand placed in secondary durable watertight containerswith sufficient absorbent material to handle the entire contentsof the primary container in the event of leakage.A copyof this regulationmay be obtained from the Biosafety Branch, Office of Health and Safety, Centersfor DiseaseControl and Prevention, Atlanta, GA 30333. Specimensare usually shippedby overnight delivery on dry ice; if dry ice is not available, freezer packs may be placed around samples. Specimensfor serologyare usefulfor determining seroconversionof an individual suspectedof exposure to herpesvirus B. If serum obtained within the last 6 monthsis not available, a single serumspecimencanbe collectedfrom the injured personwithin 24h after exposure,labeledwith the person’snameand date, andstoredat -2OOC.The specimenis then paired with a follow-up serum sampleobtained approximately 3 weeksafter exposureto test for herpesvirusB seroconversion.

Most workers are concernedwith exposureto herpesvirusB becauseinfectionsin humanscommonly present as a rapidly ascendingencephalomyelitis after a prodrome characterizedby nonspecificfebrile malaisewith or without herpetic blisters and/or peripheral neuresthesias.Clinical symptomsof active infection with herpesvirusB can be divided into early, intermediate, and late manifestations.The presenceof both early and intermediate clinical manifestationsis inconsistent. Early clinical signsinclude vesicular eruptions or ulcerationsat or near the exposuresite, severepain or pruritis at the exposuresite, and regionallymphadenopathy.Intermediate signsinclude fever, numbness,paresthesiaor other neuresthesiaat or near the exposure site, muscle weaknessor paralysisin the exposedextremity, conjunctivitis, and persistenthiccups.Late signs includesinusitis,neckstiffness,headachefor more than 24 h, nauseaand vomiting, altered mental status,and other signscompatiblewith CNS imInfection Control Procedures pairment or viral encephalitis(44). Historically, the case-fatalityrate associated with herpesvirusB One of the most important factors that can infection hasbeen approximately 70% (73). Re- reduce the risk of infection after exposure to cently the herpesvirusB case-fatalityrate appears herpesvirusB is the adequacyand timelinessof

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wound decontamination procedures. For this reason, primate exposure kits should be available at all institutions with primate holding facilities (Appendix 2). The most critical period for the prevention of herpesvirus B infection is the first few minutes after exposure. Thorough cleansing within minutes of injury or exposure is the only means of preventing herpesvirus B contamination of a wound from progressing to infection. First aid management is started by cleaning the wound. The mechanical action of scrubbing or irrigating the wound for at least 15 min to wash away virus is probably the most important aspect of the cleansing procedure, even though detergents can destroy the viral envelope and inactivate the virus (44). Following cleaning, a specimen should be collected by swabbing the wound for viral culture after rinsing with water, and blood should be obtained as acute-phase serum for herpesvirus B-specific serology. Collection of specimens for herpesvirus B culture before cleansing is not recommended because it will delay cleansing and force viral particles deeper into tissues. Postcleaning culture specimens from any human wound potentially contaminated by herpesvirus B are still valuable for detection of infection. Isolation of virus from these specimens indicates residual contamination and increased potential for infection. False-negative results are possible if cleansing residue on the specimens inactivates any remaining viral particles during the transport process. Reporting the incident and recording events associated with the injury in a bite-and-scratch log preserve information that will help the examining physician to evaluate the risk of exposure. Medical evaluation of patients having wounds acquired from simian species or from objects contaminated with their body fluids requires immediate reporting to appropriate supervisors by a previously identified physician consultant. An emergency primate exposure kit is employed in facilities where primates are housed. The kit includes instructions, sampling materials for both humans and nonhuman primates, and instructions on how to proceed in a step-by-step method in case of an exposure. If diagnostic specimens have been collected by the primate facility, they may be packed in dry ice and sent with the employee to the physician’s office. The specimen can also be sent directly to a diagnostic service, and orders specifying that the results should be sent directly to the consulting physician in order to protect the confidentiality of the patient’s medical records and to ensure proper medical communication and follow-up should be issued. Evaluation of serum samples and swabs, including the results from the primate, is discussed in detail by Holmes et al. in reference 44. This reference includes an algorithm to aid in making decisions for the disposition of the patient, for treatment, and for management strategies.

DIAGNOSIS

OF ZOONOTIC

INFECTIONS

11

Procedures for primate facilities are centered around prevention and advanced preparation. Most monkey-inflicted injuries and needle-stick exposures are preventable by training, education, and following standard operating procedures that have been previously published for the prevention of exposure to herpesvirus B (8, 9, 108). Preventative measures include wearing protective clothing, such as masks, gloves, disposable outer clothing, and especially safety glasses and/or face shields. Laboratory practices should minimize the risks of bites or scratches; for example, routine chemical restraint on nonhuman primates should be used for physical examinations and equipment should be designed so animals can be manipulated with minimal risk to the handler, such as escapeproof squeeze-back cages or enclosed runs with built in squeeze-back cages (8). Rapid evaluation and management of persons with macaque-related injuries will be facilitated by the proper use of organized first aid, specimen collection or “exposure kits,” and written procedures for the assessment and referral of these employees to a knowledgeable physician. The facility is responsible for having these kits and protocols readily available and for educating employees about their use. Heating, ventilation, and air handling systems should be designed so personnel are not exposed to infectious aerosol. In-house service programs should be performed on a routine basis, especially in larger facilities in which personnel and investigators change routinely. Serum samples from employees who have significant exposure to macaques or their tissues or body fluids should be banked to establish a baseline level of antibody titer for evaluation after exposure to herpesvirus B. In addition, an exposure (bite-and-scratch) log to assist with evaluations can be used to improve work practices. The record should not be used in a punitive way and should contain the injured person’s name, date, location, severity of injury, identification numbers of the animals involved or specimens from these animals, details of procedures, and/or equipment associated with the injury. In addition, veterinary and medical personnel should carefully document and report information on the circumstances of exposure, first aid, clinical presentation, initial evaluation, clinical course, laboratory studies, medical management, and follow-up reports. The clinical and virological status of any macaque associated with a human injury should be assessed by a veterinarian as soon as possible and be conducted, if possible, with chemical restraint. An injury associated with an ill animal or an animal that is immunocompromised or has observable lesions may constitute a high-risk category for exposure. Viremia has rarely been documented in immunocompromised monkeys or animals with severe clinical illness (89). Alterna-

12

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ET AL.

tively, shedding of herpesvirus B by clinically healthy animals has been reported and is most likely to occur during the breeding season or under stress conditions (106). For any monkey associated with a bite, scratch, needlestick, or other exposure, blood can be withdrawn for serological evaluation and swab samples can be collected for viral culture from the conjunctiva (bilaterally), buccal mucosa, and/or urogenital sites. A fourfold or greater rise in antibody to herpesvirus B between acute- and convalescent-phase serum samples suggests a primary infection and a higher probability the macaque was shedding virus at the time of the employee’s exposure. Results from the veterinary examination and laboratory testing will help the consulting physician decide which specimens should be sent for culture and whether the patient should receive prophylactic therapy while waiting for results and follow-up care. The epidemiology, symptoms, control procedures, and diagnosis of herpesvirus B infections are summarized in Table 2. ZOONOTIC PARASITES OF COMPANION ANIMALS Introduction

and Sample Preparation

Several protozoan and helminth (worm) parasitesof smallanimalscan be transmitted to humans.Some of these organismsare highly prevalent in dogs and/or cats, while others are uncommonor rare. Many of theseparasiteshave complexlife cycles,and specifictermsare usedto describedifferent stagesin thesecycles.The “definitive” or “final” host is the animalin which the parasite reachesmaturity and often undergoes sexualreproduction. “Intermediate” and “transport” hostsare usedby the parasiteon the way to the definitive host. Asexual reproduction may occur in thesehosts.The “trophozoite” stageof a protozoan parasiteis the active, feeding form, in contrastto the “cyst” and “oocyst,” which are the stagesof theseprotozoan parasitesspecializedfor transmission. Diagnosis of zoonotic parasitic infections is usually accomplishedeither by microscopicfecal examination or by use of immunologictests to identify parasite antigen or specific antibody to the parasite. In caseswhere either traditional microscopicanalysisor immunologictestscan be used, factors such as expense,expertise of staff, and conveniencemay determinethe type of test chosen.The type of diagnostictest selectedwill determinehow samplesshouldbe handledon the way to the laboratory. Whenever a commercial diagnostic kit is used, the label directions for samplepreparation must be followed to ensure accurateresults. If feces are to be evaluated microscopically,

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28

they will usualyneedto be concentrated.A partial list of concentrationtechniquesisgiven in Appendix 3. If it cannot be examined shortly after collection, the samplemust be preserved.This is particularly necessaryif the feces need to be tested for the presenceof fragile protozoan trophozoitesthat will deteriorate rapidly in the environment. Fixing human feces will also reduce exposureto infectious agentsduring transport. In general, fixation solutions should be added to samplesat the ratio of 3 parts fixative to 1 part sample(2, 25, 76). One of the most commonly usedcommercially available fixatives for fecal samplesis polyvinyl alcohol(PVA). Adding fecesto PVA will preserve trophozoites for examination in stained slides. When usingthis compoundasa fixative, an equal amountof fecesisusuallycombinedwith 5 to 10% formalin becausePVA-preserved material is less satisfactoryfor use in concentration procedures. Formalin-fixed fecesare not appropriatefor making permanentstainedfecal smears(2, 25). MIF (merthiolate-iodine-formalin) is another compoundoften used for fixation of fecal material. MIF-treated samplescan be examined by direct wet mount, stainedfecal smear,or sedimentation concentration techniques.Iodine is added at the sametime as the sampleand provides a stain for parasites as well as a fixative. SAF (sodiumacetate-aceticacid-formalin) is a similar fixative that can be usedwith the acid-faststainfor Cryptosporidiumspp.,aswell asfor usein concentration procedures(21, 25, 76). Concernover the useof hazardouschemicalsin laboratory solutionshasincreasedin recentyears. PVA containsa substantialproportion of mercuric chloride, and safedisposalof this materialmay be difficult. Someworkers have substitutedcupric or zinc sulfate to replace mercuric chloride, but resultsobtainedwith thesefixatives do not appear to be as satisfactory (26). MIF also contains mercury. In an effort to replacecompoundscontaining heavy metals,a solution containingglycerine, alcohol, and formaldehyde (PIF) has been introduced as an alternative fixative and is commercially available (76). However, it should be rememberedthat formaldehyde is a known carcinogen and may alsopresent disposalproblems (26) Veterinary fecal samplesare usually examined on the premiseswhere they were collected, so there is lessneed to preservethe material. Public health risks are also reduced with animal feces, and freshmaterial can often be senton cold packs by overnight mail for examination.If preservation is desired, formalin fixation is appropriate for helminth eggs,but protozoan trophozoiteswill be destroyed; an alternative fixative, such as PVA, shouldbe used.Fecesfixed with PVA, however,

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DIAGNOSIS

28

cannot be analyzed by routine fecal flotation techniques (2) . PROTOZOAN

ZOONOSES

Toxoplasma gondii Description, transmission

natural

habitat,

and mode of

T. gondii is one of the mostimportant zoonotic parasitic infections. The definitive hosts of this coccidian parasite are domestic cats and other felids, but the parasite is capable of utilizing virtually all warm-bloodedanimals,including humans,as intermediate hosts.In cats, T. gondii is found principally in the intestinal tract, where it reproducesin epithelial cells (77). Following a period of both asexualand sexual development, oocysts are produced and are excreted in the feces.Someorganismsin catsmay alsoenter other organsof the body, where they multiply asexually and form cysts containing variable numbers of parasites (18). Cats rarely show clinical signs associatedwith Toxoplasma infection, although occasionallysevereillnessmay develop following systemicinfection (19). After Toxoplasma oocysts reach the environment in the feces,they require a period of at least 1 to 2 daysto becomeinfective for the next host. If infective oocystsare ingestedby a cat they can completetheir life cycle. If the oocystsare eaten by another animalspecies,including humans,organismswill invade the tissuesand form cysts.If the cysts are later ingestedby a cat, the parasite will completeits life cycle in the cat’sintestines.If thesecystsshouldbe eatenby anotheranimalor a humanthe parasiteswill simply emergefrom the cysts,penetrate the gut wall and repeat the processof encystment(18). Although cat contact is often describedas the source of human Toxoplasma infection, direct contactwith catsusuallywill not result in exposure to the parasite.Epidemiologicalevidencesuggests that the most frequent causeof infection in the United States is the ingestionof raw or undercooked meat containing tissuecysts (15, 18, 77, 103). Consequently, persons at risk of severe infection should be advised not to eat raw or undercookedmeat and to washhandsthoroughly after handlinguncookedmeat. To avoid infection from the parasitic oocysts,it is also advisedthat at-risk individualsshouldnot clean out cat litter boxes,let their cats hunt, or feed their cats raw meat. They shouldalsowear gloveswhen gardening (18). Human infections

and treatment

Most often, human Toxoplasma infections are never diagnosedand usually present with an asymptomaticcervical lymphadenopathy.In im-

OF ZOONOTIC

INFECTIONS

13

munocompetentpersons,the parasiteusuallyproduces no clinical signs; 10 to 20% of patients developa flu-like condition or infectiousmononucleosis syndrome. Chorioretinitis may also develop in acute cases.Rarely, fatal diseasewith myocarditis, pneumonitis, or encephalitisoccurs (5). Once immunity develops,it is very unlikely that successfulreinfection will occur. Chronic (latent) infection occursin all infected people after resolution of the acute phasebecauseof asymptomatic persistenceof T. gondii in cyst form. Reactivation of chronic infection resulting in toxoplasmosisoccurs almost exclusively in patients who are severelyimmunocompromised (5). Toxoplasmicencephalitisis the mostcommonsequela of toxoplasmosis in AIDS patientsand individuals on immunosuppressive drugs.Pneumonitis,chorioretinitis, and multiorgan involvement with acute respiratory failure and hemodynamicabnormalities may alsooccur (5). It is estimatedthat 25 to 50% of AIDS patientsseropositiveto Toxoplasma spp.will eventually develop toxoplasmicencephalitis if prophylaxis is not undertaken (63). In addition, seriousdiseasemay result from congenital toxoplasmosisdue to infection during pregnancy. If a woman is first infected with T. gondii during pregnancy, the organism can becomedistributed to the fetus, where it may cause seriousdevelopmentalabnormalities.If infection occursprior to pregnancy,immunity developsand subsequentexposureto the parasitedoesnot lead to fetal infection (63, 64). The incidenceof fetal infection increaseswith later stagesof pregnancy. There may be no sequelae,but sometimes,afterbirth clinical manifestationsthat mimic herpes simplex virus, cytomegalovirus,or rubella virus infections may occur (5). Toxoplasma infection is frequently treated with pyrimethamineand sulfonamides,althoughspiramycin and other agentshave been usedin pregnant women. In immunosuppressedpatients, treatment is very difficult and many compounds have been used experimentally. Pyrimethamine, trimethoprim-sulfamethoxazole,and roxithromytin have been used as treatment of toxoplasmic encephalitisin AIDS patients (63). Diagnosis

A numberof techniqueshave beenusedin the laboratory for diagnosisof human Toxoplasma infections. Blood and cerebrospinalfluid may be examinedfor the presenceof the parasite.Following centrifugation of the sample,Giemsa-stained slides should be prepared from the sediment and examinedfor evidenceof free or intracellular organisms.Tissue imprints can be similarly stained,but thesecytological techniquesmay not be sufficiently sensitive to provide an adequate diagnosis.Inoculation of mice with tissue suspected of containing the parasite, followed by

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examination of peritoneal exudate and tissue, is a much more sensitive diagnostic technique. Mouse inoculation with placental tissue is helpful in diagnosing cases of congenital infection (64, 111). Examination of the feces is not helpful, because in humans Toxoplasma only formscystsin the tissues and doesnot produce oocysts. At this time, serologictechniquesfor antibody are the primary tests usedfor diagnosisof toxoplasmosisin humans.Serum,plasma,cerebrospinal fluid, and eyefluid specimens may all be tested for antibody. The Sabin-Feldmandye test is consideredthe referencetest for the parasiteagainst which other tests are measured.The Sabin-Feldmandye test principally detectsIgG antibody. Organismsare lysedin the presenceof specificantibody andcomplementand fail to stainwith a blue dye (81). The resultingtiter canbe converted into international units by using a World Health Organization standard.This test, although it is very specificand sensitive,is not performed in many laboratoriesbecauseit requireslive organisms. IFA testsare more widely usedthan the SabinFeldmandye testbecausethey yield similarresults without requiring the useof live parasites.Falsepositive reactions may occur in samplesfrom patientswith antinuclearantibodies.Several IFA kits are available,and the test hasbecomewidely used in commercialand public health laboratories. This test is very useful for laboratoriesthat screenfewer than 25 samplesa weekfor Toxoplasma-specificantibody. Although IFA testsare also availablefor measuringIgM titers, EIAs are preferred (111). EIAs are available for determining levels of both IgG and IgM antibody to Toxoplasma spp. Thesetestsare now the most commonly usedin the United States.The EIAs are more easilyquantified than the IFA test and can be more readily automated.In a survey of someof the EIA kits available,it wasshownthat resultscomparableto those obtained with the IFA test were obtained when IgG wasmeasured.However, Toxoplasmaspecific IgM antibodiesmay reduce measurable IgG titers, and heat ‘inactivation of serum may causefalse-positiveresults. IgM titers are most accuratelydeterminedby the capture IgM EIA. In this assay,antibodiesto IgM capture IgM in the sampleas a first step, allowing IgG to be washed away to prevent interference with IgM detection (110, 112). Selection of the appropriate serologictest for diagnosisof suspectedcasesof toxoplasmosis will vary dependingon the type of infection (congenital, ocular, cerebral toxoplasmosis,etc.) and immunologicstatusof the host. Individuals are usually tested initially for the presenceof IgG. If a positivetiter is detectedin a pregnantwomanand it isimportant to establishwhetherthe infection is acute,IgM levelscanbe determined.Although an

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28

IgM titer may persist for as much as 18 months after infection, a high IgM titer suggestsa recent infection. In infants with suspectedcongenital Toxoplasma infection, a positive IgM titer after birth in the absenceof any placental leak is diagnostic.The capture IgM EIA is preferred in these cases.In immunosuppressed patients with Toxoplasma encephalitis,antibody tests may not provide reliable resultsand additional diagnostic information can be obtainedfrom tissuebiopsies, therapeutic trials, and other procedures(64, 111). In cats,Toxoplasma infection is frequently diagnosed serologically.Oocysts are usually present for only 1 to 2 weeksand are seenon fecal flotation exams,but they are not unique in appearanceand can only be interpreted assuggestiveof infection. Antibody titers can be usedto diagnose systemiccat infections, but they provide little information on when infection wasacquired.A latex agglutinationassayand indirect hemagglutination assay,which potentially detect all classesof Toxoplasma-specific antibodies,are available for cats and other host species.However, they do not accurately reflect levelsof parasite-specific IgM (55). Cryptosporidium

spp.

Description, natural habitat, mode of transmission, and animal infections Cryptospotidium pawum is a protozoan belong-

ing to the large coccidiangroup of parasites.This parasitecan be found primarily in the gastrointestinal (GI) tract of a wide variety of mammalsand humans.Birds and reptiles are infected with different speciesof Cryptosporidium. The parasite infects the epithelial cells of the GI tract of the host and undergoesboth asexualand sexual reproduction. Following sexual reproduction, oocystsare passedin the feces(101). Theseoocysts appearto be quite resistantto environmentalconditions and can survive in cool, moist conditions for long periods (24). Recently, Csyptosporidium spp. have received increasednational attention following a diseaseoutbreak in Milwaukee, Wis., in which approximately400,000peopledeveloped clinicalcryptosporidiosis. It remainsunclearwhether humansor animalsprovided the sourceof the parasitethat contaminatedthe city’s drinking water (21). Although humanscan acquire Cyptosporidium spp. from contact with an infected animal, the parasite can also be maintained in people without animal association.Personsat greatest risk of cryptosporidiosisare AIDS patients and other immunocompromised individuals (101). Human

infections

and treatment

In all mammalianspecies,the primary clinical sign associatedwith Cryptosporidium infection is diarrhea. Infection may also be associatedwith fever, vomiting, or other signs.Diarrhea usuallv

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28 TABLE

DIAGNOSIS

OF ZOONOTIC

3. Diagnostic kits available for the direct detection of Cryptosporidium Test (company)

Type

ProSpectT/Cryptosporidium (Alexon, Inc.) IDEIA Cryptosporidium (Dako Corp.) MeriFluor Cryptosporidium/Giardia (Meridian Diagnostics Inc.) Color Vue Cryptosporidium (Seradyn Inc.) Giardia ProSpectT/Giardia (Alexon Inc.) GiardEIA (Antibodies Inc.) ColorVue Giardia (Seradyn Inc.) Giardia lamblia Direct Detection System (Trend Scientific Inc.)

EIA EIA DFA EIA EIA EIA EIA EIA

INFECTIONS

and Giardia

15

species

Sensitivity

Specificity

100 100 93 96 91 97 95

98 100 100 93 100 96 94 98

persistsfor a few days to a few weeksand then tine for diagnosisof infection (13, 93; seeAppenresolvesasthe host developsimmunity. In domes- dix 3). The collectedmaterial can either be examtic animal species,young animalsare most likely ined directly or be usedto makea smearthat can to acquire the infection. In a recent national be stainedwith an acid-fasttechnique (13). survey, approximately60% of dairieswere found Commercial kits for diagnosisof Cryptospoto have Cvptosporidium-infectedcalves(27). The ridium infectionsare alsoavailable.Thesekits are parasitecan alsobe found in cats and dogs,but becomingincreasinglypopular becauseresultscan ratesof infection are much lower in thesespecies, usually be more easily interpreted than by scanrarely exceedinga few percent of animalssampled ning stainedsmearsand are lessdependenton the (22,70). In immunocompromised individualsnor- experienceof the technician performing the test mal immunity to the parasite does not develop (Table 3). The most sensitiveand specificof the and the organismcontinuesto parasitize the GI available tests is a fluorescent-antibodytest that tract, producingsevere,chronic diarrheathat may identifiesoocystsin either concentratedor unconbe life-threatening. In some immunosuppressed centrated samples.This test is alsoavailableas a patients,cholecystitis,hepatitis,pancreatitis,reac- combined kit for Giardia detection. Other kits tive arthritis, and respiratory diseasehave also available are EIAs which detect Cryptosporidium been detected(100, 101). antigen in fecal samples(27, 60, 110). Because Specific therapy for cryptosporidial diarrhea is Cryptosporidium oocystsare immediatelyinfective usually not undertaken in uncomplicatedcases, when passedin the feces,it is important to handle and treatment consistsof providing hydration samplesfrom patients with suspectedinfections and/or symptomaticrelief. In AIDS or other im- with care. munosuppressed patients, control of Cryptosporidium infection is difficult. Many antiprotozoal Giardia spp. and antibiotic compoundshave beenused,though unsuccessfully,in theseindividuals.Azithromycin Description, natural habitat, and mode of and paromomycinappear to have someefficacy transmission againstthisorganism,but they are not consistently Giardia spp. are some of the most common effective (113). intestinal parasitesencounteredin humans,dogs, and cats, Theseflagellateprotozoan parasiteslive in the smallintestine of the host. They occur in Diagnosis In the laboratory, Cryptosporidium infection can only two forms: as a motile trophozoite, which be diagnosedby histologic examinationof tissue reproducesby asexualbinary fission,and asa cyst, samples.Parasiteinfection canalsobe determined which ispassedin the feces.The latter form of the by identification of oocystsin fecal samples(spu- parasiteis surroundedby a cyst wall and is spetum samplesmay alsobe examinedif respiratory cialized for transmission.It can survive in the infection is suspected).Cryptosporidium oocysts environment for up to several months in cool are small (4 to 5 I.L in diameter) and can be water, although its survival is shorter under other confusedwith yeast on a wet mount. To allow less-favorableconditions (115). Human infection is readily transmittedbetween differentiation from yeast, severalstainingprocedures, including Giemsa, auramine-rhodamine, individuals, especiallyunder conditionswhere feand acid-fast stains, have been used on fecal cal-oral contact is common, such as day care smears(13). The modified Ziehl-Neelsen and centers and nursery schools(43, 114, 115). HowKinyoun’s acid-fast stains are most commonly ever, the extent to which humansacquire Giardia used,but oocystspreservedwith PVA do not stain spp.directly from animalsis controversial.Several well with acid-fasttechniques(27). A Sheather’s outbreaksof diseasehave beenlinked to infection sugarflotation procedurecan be usedto concen- in wild animals,and giardiasishas been called trate fecal oocystsin fresh specimens,and this “beaver fever” in Canada.In contrast, the high technique is frequently usedin veterinary medi- prevalenceof the parasitein the pet population

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does not seem to be associated with a similar rate of infection in their owners (40). Repeated efforts to show biochemical or genetic identity between Giardia spp. isolated from animals and those isolated from people have not been persuasive (99). It appearslikely that there may be strain variation in the abilitiesof Giardia spp.to infect a range of host species.At this time, it is probably prudent to assumethat all animal infections are potentially zoonotic. Human infections

and treatment

Many casesof Giardia infection are asymptomatic. In clinical casesinvolving both animalsand humans,the primary symptomis diarrhea. In humans,diarrheamay alsobe accompaniedby bloating, flatulence, cramps,and anorexia. Vomiting, fever, and tenesmusis lesscommon.The courseof giardiasisis either an acute, self-limited diarrhea or a chronicsyndromeof diarrhea,malabsorption, and weight loss.Stoolsmay be profuseandwatery but later may become’greasy and foul-smelling and may float. Gross blood, pus, mucus, and polymorphonuclearcellsare usually absent(43). Quinacrine, metronidazole, furazolidone, and paromomycinhave all beenusedin the treatment of giardiasis.Outside the United States, other drugs,including tinidazole, ornidazole, and some of the benzimidazoleanthelmintics(albendazole and mebendazole),are also being used in the treatment of this infection (42).

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frequently in human hospitals,but it does not concentratethe parasitecystsaseffectively asthe 33% zinc sulfate flotation procedure. The test sampleis mixed with the flotation solution and centrifuged, and the material at the surface is collectedfor examination(Appendix 3). Zinc sulfate, or any other flotation solution, will rapidly destroy Giardia trophozoites, so they are unlikely to be seenin slidespreparedby this technique(2, 114). Becausecyst production is intermittent, a single negative samplecannot rule out Giardia infection. In general,it is recommendedthat three fecal samplescollected over the courseof a week shouldbe examined(43, 115). Several immunodiagnostickits have become availablefor Giardia identification (Table 3) and asin the casewith Cyptosporidium spp., the ease of performing these tests comparedwith that of traditional techniqueshas led to their increased use in diagnostic laboratories. These tests are becomingvery popular and appearto offer sensitivity equal to or greater than that of traditional methodsof fecal examination, particularly when only a singlefecal sampleis available.Among the EIAs, some detect both trophozoite and cyst antigenswhile others detect only cyst antigens. These tests also vary in their abilities to use preserved(frozen or fixed) samples(110). HELMINTHS

Diagnosis

Diagnosisof Giardia infection is performed by visual recognition of the organismin the stool or by immunologic identification of fecal Giardia antigen. The trophozoite form may be seen in diarrheic feces,but it is unlikely to be presentin stool of normal consistency.These delicate trophozoitesusuallydie quickly outsidethe intestine, and thin salinewet mountsshouldbe madefrom fresh fecal samples.Giardia trophozoites are approximately 20 k in length and move with a falling-leaf-like motion. On high power (x450), several flagella can often be visualized and the organismin profile canbe seento have a concave ventral surface (115). Fecal smearscan also be madefrom PVA-fixed specimens,but trophozoites will be lesseasily recognizedwhen they are not motile. Trophozoitesmay alsobe observedin duodenal endoscopicaspiratesor samplescollected by the Enterotest (HEDECO, Mountain View, Calif.) (2, 115). The cyst form of Giardia spp. is most often presentin the fecesandcan be detectedby several techniques.Staining of cystsand trophozoites in preserved fecal smearsis usually performed by trichrome or iron hematoxylin staining,but concentration procedurescan alsobe performed. The formalin-ether sedimentationtest has been used

28

Visceral Natural

habitat

and Ocular

Larval Migrans

and mode of transmission

Visceral and ocular larval migrans(VLM and OLM, respectively)are conditionsassociatedwith migration of parasitelarvae through the tissuesof the body or eye,respectively.In the United States, mostcasesof VLM and OLM are associatedwith the migrating larvae of Toxocara canis, the common roundworm of dogs(31). Recently, larvae of the raccoon roundworm, Baylisascaris procyonis, have beendescribedin VLM-associatedmeningoencephalitis;they are alsoableto causeOLM (51). Adults of these roundworm speciesare parasites of the GI tract of the host, where they produceeggsthat are passedin the feces.Following a developmentperiod in the environment,the eggsbecomeinfective. If ingestedby the definitive host, the larvae are releasedin the GI tract and then undergoa migration through the body. The larvae may then return to the intestine and mature, or they may becomearrestedin the tissue. Most T. canis larvae will becomearrested in the tissuesof dogs older than a few months of age. Adult Toxocara infections in the small intestine are uncommon in adult dogs. In female dogs, these dormant larvae are stimulated by changes associatedwith pregnancy and lactation and are

CUMITECH

28

DIAGNOSIS

transmittedto young animalsthrough the milk or placenta(30). If infective eggsare ingestedby animalsother than the definitive host, larvae releasedfrom the eggswill enter the tissuesand eventually become dormant.If they are later eatenby the appropriate host of the parasite, these larvae will resume development and become adult worms in the small intestine. Small rodents and birds act as transport hostsfor theseparasites(30). If humans ingest the infective eggs, they too can act as transport hostsfor the parasites. Human infections

and treatment

OF ZOONOTIC

INFECTIONS

17

by fecal flotation examfor parasiteeggs.Diagnostic techniquesfor identification of arrestedtissue larvae are not available. Prevention of human infection with these parasitesrequires frequent removal of pet feces from the environment and limiting contact with raccoonsin both urban and rural areas(30). Hookworms Description, transmission

natural

habitat,

and mode of

The canine hookworm, Ancylostoma caninum, is a commonparasiteof the smallintestineof dogs in much of the United States. Another canine hookworm,AncyZostoma braziliense, is found principally in the southernUnited States.Adult hookworms are blood-suckingparasitesof the small intestine (30). Adult femalesproduce eggsthat are passedin dog feces.Developmentof the egg occursin the environment,resultingin the hatching of a first-stagelarva. This processcan occur within a day under hot, humid conditions. The hookwormlarva undergoesa further developmental period in the environment until it reachesthe infective third larval stage.This larva is able to infect a dog by severaldifferent routes. The dog may eat the parasitelarva directly. Alternatively, a transport host (small rodent, etc.) may become infected and then, if later eatenby a dog, transmit the parasite. Larvae of A. caninum may become arrestedin the tissuesof femaledogsand produce transmammary infection of puppies. A final method of infection involves the direct penetration of host skin by the infective larva. In dogs, these parasitesoften reach the small intestine (30). If larvae penetrate human skin, however, they are typically unableto completethe life cycle.

The presenceof migrating larvae in the tissues will produce the clinical signsof VLM, although infections with very low numbersof larvae are often asymptomatic.Heavier infections are associated with eosinophilia, hepatomegaly, hyperglobulinemia, pulmonary symptoms, and fever (14,72). Fulminant diseasewith death may occur. The liver is the organ most frequently involved, and hepatosplenomegalyis common. Lung involvement and skin lesionsmay also occur. Neurologic lesions,though infrequent, may occur in severecasesand appearto be more likely to occur in casesof infection with Baylisascarisspp. VLM occursmost commonly in children fewer than 6 years of agewith a history of pica. OLM occurs when larvae of one of theseworm speciesreach the eye. It appearsto be more likely to occur in older children and adults than VLM. Endophthalmitismay be present,and infection may result in somedegreeof permanentvisual damage. Treatment with anti-inflammatory drugsis important in humancasesof VLM. Corticosteroids and antihistamineshave been usedextensivelyin these cases.Anthelmintics (benzimidazolesand diethylcarbamazine)are also used, but their effects are more controversial. In casesof OLM, Human infections and treatment treatment typically consistsof corticosteroids.AlThe larvae of Ancylostoma spp. are capableof though anthelminticsare used, their successin migratingthrough the skin,causingerythematous, treatment hasnot been critically examined(31). serpiginoustracts and itching. There is usually intensepruritis, erythema,and an apapular,vesicDiagnosis ular rash at the site of larval penetration. MigraIn the past,VLM wasdiagnosedby demonstra- tion of larvae through the lungs may produce a tion of the parasite larvae in tissue,usually the Loffler-like syndromewith transitory chest sympliver. More recently, immunologicdiagnostictech- toms, diffuse opacity on X-ray, and eosinophilia. niqueshave been developed.EIA techniquesfor The major manifestationsinclude iron deficiency parasite-specificantibody are available and are anemia and chronic protein-energy malnutrition performed by some diagnosticlaboratories and (93). This syndrome of infection is known as the Centersfor DiseaseControl and Prevention cutaneouslarva migransand is causedmore often (CDC). CommercialEIA kits are under develop- by A. brasiliense than A. caninum or other lessment but are not availableat this time. Diagnosis commonhookwormspecies. This conditionis most may be confirmed by histopathologicidentifica- likely to develop following contact with sandy, tion of parasitelarvae in tissuesections.Morpho- shadysoil contaminatedwith hookwormlarvae. logic characterizationof wormsin histologicsamVery recently, it hasbeen establishedthat huples can be usedto identify the parasite species mans may rarely becomeinfected with adult A. involved (14,31,32,72). Diagnosisof roundworm caninum in the smallintestine. Infection with the infection in the final animal host is usuallv made hookwormproducesan eosinophilicenteritis with

18

GUICHON

ET AL.

abdominal pain as a common symptom (12). Affected individuals are usually treated with albendazole or ivermectin (31, 72). Diagnosis Diagnosis of infection by migrating larvae is usually made on the basis of clinical signs and history (30, 72). Initial identification of eosinophilic enteritis due to infection with adult worms may be made following endoscopic biopsy and histology (12, 76). The level of parasite-specific antibody in blood can be determined by submission of samples to CDC. These parasites do not appear to reach sexual maturity in humans. Therefore, hookworm eggs would not be present in feces. Strongyloides stercoralis

Description,natural habitat, and modeof transmission S. stercoralis is a nematodeparasiteof humans and smallanimals.The tiny (2-mm) adult worms are found in the GI tract of the definitive host. The first-stagelarvae produced either are passed out of the hostin fecesor penetratethe wall of the large bowel and reinfect the host. This latter processis referred to as “autoinfection” and is most significantin immunocompromised humans, becauseit may result in massivenumbersof larvae in their tissues.Thoselarvae that leave the host in the feces undergo a free-living period in the environmentfollowed by the production of infective parasiticlarvae. The final host isthen infected by ingestionof larvae or skin penetration by the parasite(30, 59). Human infectionsand treatment Clinical signsassociatedwith Strongyloides infection includecutaneouslarva migransfollowing skin penetration of the larvae and penetration of the intestinalmucosaby the adult worms.Approximately one-third of infected patients are asymptomatic. Infection of the skinand respiratory tract may result in a pruritic, erythematousrash and a Liiffler-like syndromewith eosinophiliaand must be differentiated from hookworm infection. The adult parasitesin the small intestine may cause diarrheawith the passage of mucus,vomiting, and epigastricpain; eosinophiliais common. Up to one-fifth of patientsmay developa generalizedor localized urticarial rash beginningperianally and extending to the buttocks, abdomen,and thighs (95). Particularly in immunosuppressed patients, massiveinvasion of the lungs and other organs by larvae may occur due to autoinfection (59). Strongyloides spp. do not appear to be common parasitesin either humansor smallanimalsin the United States, and zoonotic casesare probably limited in number. The benzimidazole anthelmintic, thiabenda-

CUMITECH

28

zole, hasbeenthe standardtreatment for strongyloidiasisfor manyyears.However,relapsesare not uncommonwith this therapy, and newer anthelminticslike albendazoleand ivermectin are being usedwith increasingfrequency in treatment (59). Diagnosis Diagnosisof Strongyloides infection is carried out by examinationof fecesfor first-stagelarvae. It is important that a fresh fecal samplebe used, becauselarvae from hatchedhookwormeggsmay be confusedwith Strongyloides larvae in an older sample.Fresh feces can be examined using the Baermanntechnique(Table 3), which providesan opportunity for larvae to migrate out of the sample and be concentrated for examination. The Baermanntest allowsa relatively large fecal sample to be used,thereby increasingthe sensitivityof laboratory diagnosis.This is particularly important in casesof low-level infection where only smallnumbersof larvae are present in the feces. In these cases,it may be necessaryto evaluate severalsamplescollectedover a period of several daysto effectively diagnosethe infection (2, 59). Alternatively, a concentrationtest suchasZnSO 4 flotation can be performed (2, 93). Recently, an agar plate method for the diagnosisof StrongyZoides infection has been developed, and it is highly sensitive for parasite detection. Stool is sealedon agarplatesand incubatedfor 2 days.As larvae migrate through the agarthey leave bacterial trails, which can be detectedin a microscopic exam. Larvae can then be collected for positive identification (26). Echinococcus

spp.

Description, natural habitat, and modeof transmission Two speciesof the tapewormgenusEchinococcusoccur in the United States:E. ganulosus and E. multilocularis. Both worms occur as adults in the smallintestineof carnivores.The larval stages are found in healthy animalsthat prey on the definitive host. Although E. granulosus occurs throughout the world, in the United Statesit is almost exclusively confined to western states where sheepare raised. Dogs are the definitive hosts of the parasite and sheep are the most common intermediate host, although cattle, horses,pigs, and other animalsare occasionally infected. The larval parasitesare found in fluidfilled cysts (hydatid cysts) in the liver and, less frequently, in other tissuesof sheep.The definitive hostsare infected following ingestionof the hydatid cysts.The adult tapewormsin the smallintestine produce eggsthat are passedout of the host in minute tapeworm segmentsthat are rarely detected in the fecesor around the animal (86). E. multilocularis infection hasrecently received

CUMITECH

28

DIAGNOSIS

attention as a developingzoonosisin the continental United States.In the past,this parasitewas largely confined to Canadaand Alaska, but surveysnow indicate that its distribution is extending southwardinto the central United States(96). The adult tapewormsare found in the smallintestines of foxes, dogs,andcats.Smallrodentsact asintermediatehosts. Human

infections

and treatment

If the tapewormeggsare ingestedby a human, the parasite continues its life cycle and forms hydatid cystsin the liver (50 to 70% of patients), lungs (20 to 30% of patients), or other organs, including the brain, heart, and bones (~10% of patients). These cysts may persist for years or decadesand grow to a large size, containing severalliters of fluid and tapeworm larvae, and may obstructvital tissuesor result in postobstructive bacterial infection. Infected individuals also run the risk of developing a potentially fatal hypersensitivityto parasite antigensdue to cyst leakageor rupture. Cyst rupture may alsoresult in seedingof daughter cysts to other areasof the body, resulting in organ failure and significant morbidity and mortality (86). Treatment is primarily surgical removal of cysts and additional anthelmintic therapy. Control of the parasite is accomplishedprimarily by restricting accessof dogsto sheepcarcasses and treating adult worms in dogs(49). In contrast to the balloon-likecyst of 5’. granulosus, larval E. multilocularis produces alveolar cyststhat grow by budding of new compartments containing tapeworm larvae. This alveolar-type cyst hasbeencomparedin appearanceto a bunch of grapesand tends to grow invasively in tissue. Although previoushumaninfection in the United Stateshasbeen largely restricted to Alaska, the severityof the diseaseproducedand the apparent spreadof the parasitehasled to increasedmonitoring for E. multilocularis in many states(41). Casesof multilocularand unilocularhydatidosis in humansare usually treated with surgery and anthelmintics.Several benzimidazoleanthelmintics (albendazoleand mebendazole)and praziquante1havebeenusedfollowing surgeryto prevent recurrenceof the infection. In caseswhere cysts cannot be resected,anthelminticsalone are used (50).

OF ZOONOTIC

INFECTIONS

19

infection with this parasitein the United Statesis low, few laboratories are equipped to test for Echinococcus spp.At the CDC, serumsamplesare screened initially by indirect hemagglutination assay,and all positive samplesare then analyzed by immunoblotassayfor an 8-kDa antigen,which is highly specificfor Echinococcus spp. Diagnosis of infection due to E. multilocularis is also made by serologicaltests.Initial screeningcan be undertaken with tests used for E. granulosus. Specific testing for E. multilocularis can be performed at CDC. Diagnosis of Echinococcus infections in the smallanimaldefinitive host is difficult. Tapeworm segmentsare too smallto be easilyvisible, andthe eggsthey contain are identical in appearanceto thoseof other membersof the Taeniad family of tapeworms,which are commonparasitesof small animals. In past campaignsaimed at parasite eradicationthroughout the world, purgative drugs have been usedto causeelimination of the parasites,which can then be recovered and identified in the feces.Any canineor feline fecal samplethat is suspectedof containing Echinococcus eggs shouldbe handledwith extreme caution (30). Dipylidium

caninum

Description, natural habitat, and mode of transmission D. caninum is the most commontapeworm of

dogsand catsin the United States.This parasiteis of little clinical significancein small animals,although ownersdislikeseeingthe motile tapeworm segmentson their pets or in the house.When the tapeworm eggsin the segmentsthat passout of the host are ingestedby flea larvae in the environment, a cysticercoiddevelops.If this infected flea, in turn, is eaten by a dog or cat, the adult tapeworm will develop. Occasionallyyoung children becomeinfected with D. caninum, either by direct ingestionof fleasor from contact with a pet that hasjust nipped at a flea and haslarvae around its mouth. Human infections and treatment In humans,D. caninum may develop into an

adult tapeworm(10). The symptomsproducedare usually mild, but as segmentsare passedin the feces, parents will become understandablyconcerned and submit them to physiciansfor identification. Infected children can be treated sucDiagnosis Hydatid diseaseisusuallydiagnosedby imaging cessfullywith niclosamideor other anticestodal ultrasound,computerizedaxial topography(CAT) compounds(10). scan,magneticresonanceimaging,or by serology. Severaldifferent tests,suchasEIA, indirect hem- Diagnosis agglutination,and immunoblotting,are available Dipylidium segments are cucumberseed-shaped and showvariousdegreesof sensitivity depending and are about 0.5in. (1 in. = 2.54cm) in length. If on the characteristicsof the case,including cyst squashed,they may releasepacketsof 2 to 20 eggs. location and vitality. Becausethe prevalence of Each eggis about 25 pm in diameterand contains

20

GUICHON

ET AL.

the 6 embryonic hooks typical of common tapeworms. These hooks are often difficult to see when eggs are clumped together in packets. Sterile segments can usually be identified by the presence of many calcareous corpuscles scattered throughout the segment (109). Dipylidium segmentscan be distinguishedfrom those of the human tapewormsTaenia solium and Taenia saginata. In DipyZidium spp.,there is a genital pore on eachsideof the segment,whereas in the Taenia parasites, there is a singlegenital openingper segment(10). APPENDIX 1

CUMITECH

28

b. Screw-top vials of 3- to 5-ml capacity containing 1 to 2 ml of virus transport medium(e.g.,Eagle’sminimalessential mediumwith Earle’ssaltsplusgentamitin [50 &ml] and 2% bovine serumif refrigeration is available, Hanks’ buffered salt solution with gentamicin in the absenceof refrigeration, or medium recommendedby the testing laboratory of choice) 3. Copy of institutional or facility standard operatingproceduresand forms for reporting injuries; names,mailing addresses, and telephone numbersof reference laboratories; and names,addresses,and telephone numbersof local physicianor health professionalsto contact for follow-up evaluation.

ReferenceLaboratoriesConducting Herpesvirus B Identification and SerologicalTesting Julia K. Hilliard, Ph.D. Department of Virology and Immunology APPENDIX 3 SouthwestFoundation for BiomedicalResearch 7620NorthwestLoop 410 Flotation Techniques:Sheather’sSugar SanAntonio, Texas78228 and 33% Zinc Sulfate Telephone:(210) 674-1410x280 or (210) 673-3269 Pager: l-800-443-7243ID011422 Sheather’ssugarflotation solution David Brown, M.R.C.Path. 454 g of sucrose Virus ReferenceDivision 355 ml of tap water Central Public Health Laboratory 61 ColindaleAvenue 6 ml of formaldehyde,U.S.P. grade London NW9 5HT, England Telephone:081-200-4400 Dissolvethe sugarin the water by stirring over indirect heat. After the sugaris dissolvedand the SeymourS. Kalter, Ph.D. solutionhascooledto room temperature,add the Virus ReferenceLaboratory formaldehydeto prevent microbial growth. 7540Louis PasteurDrive, Suite 202 SanAntonio, Texas78229 Zinc sulfate solution Telephone:512-614-7350 Add enoughwater to 330 g of zinc sulfate to bring the total volume to 1 liter. Mix until disAPPENDIX 2 solved.The specificgravity shouldbe 1.18.If zinc SuggestedContentsof Primate Exposure Kit sulfate is usedto concentrateparasitesin formafor Usein Primate Facilities lin-fixed samples,the amount of zinc sulfate shouldbe increasedto 400g per liter of solution. 1. Cleansingmaterials a. Detergent or soap:povidone-iodineor Sugar or zinc sulfate flotation procedure chlorhexidinescrubconcentrate b. Sterile surgicalscrubbrushes 1. If the sampleappearsto contain a large c. Sterile basinfor soakinglarge wounds amount of fat or mucus,perform an initial water d. Sterile 4-in. by 4-in. gauze pads for wash.Mix approximately 2 to 3 g of feceswith a soakingand dressingof wounds smallamount of water. For most fecal samplesof e. Sterile saline solution I-L bottles for normal appearance,the water washcan be elimiirrigation of contaminated eyes, nose, nated and the sameamount of fecescanbe mixed or mouth directly with flotation solution. f. Sterile rubber syringe for salineirriga2. Strain the mixture through double-layered cheeseclothor a tea strainer. tion of mucosa 3. Pour the mixture in a 15-mlcentrifugetube. g- Paper or cloth tape for dressing of wounds Spin for about 5 min. h. Needles and syringesfor blood with4. If the initial spinis a water wash,discardthe drawal as well as agentsfor chemical supernatant,resuspendthe sedimentwith flotarestraint tion solution,andrepeatthe centrifugation step.If 2. Specimencollection and culture materials the first spin is with flotation fluid, collect the a. Sterile cotton or Dacron swabs;do not uppermostlayer of fluid and placeit on a slideand usemetal shafts. examineit at x400 or x1,000.Experiencedtechnol-

CUMITECH

28

DIAGNOSIS

ogistswill easilysee Cryptosporidium oocysts.Oocysts will be present in the layer of material immediatelyunder the coverslip. It is helpful to raisethe lens out of focus and refocus. Parasites will be present in the first layer that comesinto focus. Use of the oil immersion lens may be necessary,but focusing is often difficult since oocystsand fluid will move under the pressureof the lens.

REFERENCES 1. Andrews, B. E., R. Major, and S. R. Palmer. 1981. Ornithosis in poultry workers. Lancet 8221:632-634. 2. Ash, L. R., and T. C. Orihel. 1987. Parasites: a Guide to Laboratory Procedures and Identification. ASCP Press, Chicago. 3. Barnes, R. 1989. Laboratory diagnosis of human chlamydial infection. Chn. Microbial. Rev. 2:119-136. 4. Bazala, E., and J. Renda. 1992. Latent infection caused by chlamydia which result in health problems for pig, cattle, and sheep breeders. Berl. Muench. Tieraerztl. Wochenschr. 105:145-149.

5. Beaman, M. H., R. E. McCabe, S.-Y. Wong, and J. S. Remington. 1995. Toxoplasma gondii, p. 2455-2475. In G. L. Mandell, J. E. Bennett, and R. Dolin (ed.), Principles and Practice of Infectious Diseases, 4th ed. Churchill Livingstone, New York. 6. Bullock, W., M. Meir, B. Willingham, and R. K. Sikes. 1984. Lymphocytic choriomeningitis virus infection-Georgia. Morbid. Mortal. Weekly Rep. 33~298-299. 7. Casals, J. 1979. Arenaviruses, p. 815-841. In E. H. Lennette and N. J. Schmidt (ed.), Diagnostic Procedures for viral, Rickettsial and Chlamydial Infections, 5th ed. American Public Health Association, Washington, D.C. 8. Centers for Disease Control. 1987. Guidelines for prevention of herpesvirus simiae (B virus) infection in monkey handlers. Morbid. Mortal. Weekly Rep. 36:680-682, 687-689.

INFECTIONS

21

9. Centers for Disease Control

and Prevention. 1990. Update: ebola-related filo-virus infection in non human primates and interim guidelines for handling nonhuman primates during transit and quarantine. Morbid. Mortal. Weekly Rep.

39~22-24,

29 -30.

10. Chappell,

C. L., J. P. Enos, and H. M. Penn. 1990. caninum, an under-recognized infection in infants and children. Pediatr. Infect. Dis. J. 9:745-747. Chernesky, M. A., J. B. Mahony, S. Castriciano, M. Mores, I. 0. Stewart, S. F. Landis, W. Seidelman, E. J. Sargeant, and C. Leman. 1986. Detection of Chlamydia trachomatis antigens by enzyme immunoassays and immunofluorescence in genital specimens from symptomatic and asymptomatic men and women. J Infect. Dis. 154:141-148. Croese, J., A. Lo&as, J. Opdebeeck, and P. Prociv. 1994. Occult enteric infection by Ancylostoma caninum: a previously unrecognized zoonosis. Gastroenterology 106:3-12. Current, W. L. 1990. Techniques and laboratory maintenance, p. 31-50. In J. P. Dubey, C. A. Speer, and R. Fayer (ed.), Cryptosporidiosis of Man and Animals. CRC Press, Boca Raton, Fla. Cypess, R. H. 1982. Visceral larva migrans, p. 205-212. In J. H. Steele (ed.), Crc Handbook Series in Zoonoses. CRC Press, Boca Raton, Fla. Decker, C. F., and C. U. Tuazon. 1993. Toxoplasmosis: an update on clinical and therapeutic aspects. Prog. Clin.

Dipylidium

11.

APPENDIX 4 Baermannexam The Baermann exam requires the use of a funnel with a short piece of tubing at the end, which can be clampedtightly. Alternatively, the Baermannexamcanbe carriedout in a disposable plastic champagneor wine glasswith a hollow stem(93). 1. Placeat least 10 g of the fecal specimenon a piece of double-layer cheesecloth.Gather the corners of the cheeseclothtogether, fasten, and placethe samplein the bowl of the funnel. 2. Fill the funnel with tepid water. 3. Allow the sampleto sit for a minimumof 4 h. Unclamp the tubing and collect the first few milliliters of fluid into a petri dishand examinefor larvae by usinga dissectingmicroscope.Alternatively, fluid from the Baermanncan be collected into a centrifuge tube and spun down, and the sedimentcan be examined.If the disposableplastic glassis used,a Pasteurpipette can be usedto collect material at the bottom of the hollow stem of the glass. 4. If a sampleis negativeon first examination, allow the material to remain overnight and reexamine.

OF ZOONOTIC

12. 13.

14. 15.

Parasitol.

3~21-41.

16. De Souza, M., and A. L. Smith. 1989. Comparison of isolation in cell culture with conventional and modified mouse antibody production tests for detection of murine viruses. J Clin. Microbial. 27: 185-187. 17. Dilehay, D. L., N. D. Lehner, and M. J. Hue&. 1990. The effectiveness of a microisolator cage system and sentinel mice for controlling and detecting MHV and Sendai virus infections. Lab. Anim. Sci. 40:367-370. 18. Dubey, J. P. 1987. Toxoplasmosis. Vet. Clin. North Am. Small Anim. Pratt. 17: 1389 -1404. 19. Dubey, J. P., and J. L. Carpenter.

1993. Histologically confirmed clinical toxoplasmosis in cats: 100 cases (19521990). J. Am. Vet. Med. Assoc. 203:1556-1566. 20. Dykewicz, C. A., V. M. Dato, S. P. Fisher-Hoch, M. V. Howarth, G. I. Perez-Oronoz, S. M. Ostroff, H. Gray, Jr., L. B. Schonberger, and J. B. McCormick. 1992. Lymphocytic choriomeningitis outbreak associated with nude mice in a research institute. JAM4 267~1349-1353. 21. Edwards, D. D. 1993. Troubled water in Milwaukee. ASM News 59:342-345. 22. El-Ahraf, A., J. V. Tacal, Jr., M. Sobin, M. Amin,

W. Lawrence, and B. W. Wilcke. 1991. Prevalence of cryptosporidiosis in dogs and human beings in San Bernadino County, California. J. Am. Vet. Med. Assoc. 198:631-634. 23. Faergemann, J. 1984. Quantitative culture of Pityrosporum

orbiculare. Int. J Dermatol. 23~330-333. 24. Fayer, R., C. A. Speer, and J. P. Dubey. 1990. General biology of Cryptosporidium, p. l-30. In J. P. Dubey, C. A. Speer, and R. Fayer (ed.), Cryptosporidiosis of Man and Animals, CRC Press, Boca Raton, Fla. 25. Garcia, L. S., and R. Y. Shimizu. 1993. Diagnostic parasitology: an overview of topics. Lab. Med. 24:13-19. 26. Garcia, L. S., and R. Y. Shimizu. 1993. Medical parasitol-

ogy: update on diagnostic techniques and laboratory Lab.

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27. Garcia,

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24181-89.

L. S., and R. Shimizu. 1993. Diagnostic parasitology: parasitic infections and the compromised host. Lab. Med. 24:205-215. 28. Gay, F. P., and M. Holden. 1933. The herpes encephalitis problem II. J Infect. Dis. 89:287-303. 29. Genovesi, E. V., A. J. Johnson, and C. J. Peters. 1988. Susceptibility and resistance of inbred strains of Syrian Gold hamsters (Mesocricetus auratus) to wasting disease caused by lymphocytic chroiomeningitis virus: pathogenesis of lethal and non-lethal infections. J. Gen. ViroZ. 692209-2220. 30. Georgi, J. R., and M. E. Georgi. 1992. Canine clinical narasitoloev. Lea & Febiner. Philadelnhia.

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L. T. 1990. Toxocariasis and related syndromes, p. 447-455. In K. S. Warren and A. A. F. Mahmoud (ed.), Tropical and Geographical Medicine. McGraw-Hill, New York. 32. Glickman, L. T., and P. M. Schantz. 1981. Epidemiology and pathogenesis of zoonotic toxocariasis. Epidemiol. Rev. 3:230-250. 33. Gregg, M. B. 1975. Recent outbreaks of lymphocytic chroriomeningitis in the United States of America. Bull. WH.0. 52:549. 34. Grimes, J. E. 1994. Avian chlamydiosis, p. 389-402. In G. W. Beran (ed.), Handbook of Zoonoses, Section A: Bacterial, Rickettsial, Chlamydial, and Mycotic, 2nd ed. CRC Press, Boca Raton, Fla.

31. Glickman,

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J. E., D. N. Phalen,

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