Cholera (Deadly Diseases and Epidemics)

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CHOLERA

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Anthrax

Meningitis

Avian Flu

Mononucleosis

Botulism Campylobacteriosis

Pelvic Inflammatory Disease

Cholera

Plague

Ebola

Polio

Encephalitis

Salmonella

Escherichia coli Infections

SARS

Gonorrhea Hepatitis Herpes HIV/AIDS Influenza Leprosy Lyme Disease

Smallpox Streptococcus (Group A) Staphylococcus aureus Infections Syphilis Toxic Shock Syndrome

Mad Cow Disease Tuberculosis (Bovine Spongiform Typhoid Fever Encephalopathy) Malaria

West Nile Virus

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CHOLERA

William Coleman CONSULTING EDITOR

I. Edward Alcamo Distinguished Teaching Professor of Microbiology, SUNY Farmingdale FOREWORD BY

David Heymann World Health Organization

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Dedication We dedicate the books in the DEADLY DISEASES AND EPIDEMICS series to Ed Alcamo, whose wit, ch a rm , i n telligen ce , and com m i tm ent to bi o l ogy edu c a ti on were second to none.

Cholera Copyright © 2003 by Infobase Publishing All ri ghts re s erved. No part of this book may be reprodu ced or uti l i zed in any form or by any means, electronic or mechanical, including photocopying, record i n g, or by any inform a ti on stora ge or retri eval sys tem s , wi t h o ut permission in writing from the publisher. For information contact: Chelsea House An imprint of Infobase Publishing 132 West 31st Street New York NY 10001 ISBN-10: 0-7910-7303-3 ISBN-13: 978-0-7910-7303-2 Library of Congress Cataloging-in-Publication Data Coleman, William, 1934 – Cholera / William Coleman. p. cm. —(Deadly diseases and epidemics) Includes index. ISBN 0-7910-7303-3 1. Cholera —Juvenile literature. [1. Cholera.] I. Title. II. Series. RC126.C695 2003 616.9'32—dc21 2002155048 Chelsea House books are available at special discounts when purchased in bulk quantities for businesses, associations, institutions, or sales promotions. Please call our Special Sales Dep a rtment in New York at (212) 967-8800 or (800) 322-8755. You can find Chelsea House on the World Wide Web at http://www.chelseahouse.com Series design by Terry Mallon Cover design by Takeshi Takahashi Printed in the United States of America Bang 21C 10 9 8 7 6 5 4 3 This book is printed on acid-free paper.

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Table of Contents Foreword David Heymann, World Health Organization

6

1.

Discovering Cholera

8

2.

Properties of Vibrio cholerae

16

3.

Dr. Snow and Cholera

28

4.

Transmission and Epidemiology of Cholera

36

5.

Signs and Symptoms of Cholera

46

6.

The Virulence of Vibrio cholerae

54

7.

The Genome of Vibrio cholerae

64

8.

Treatments for Cholera

72

9.

Prevention and Vaccines

82

10. Cholera in the Future

Glossary

90

96

Bibliography

102

Further Reading

106

Websites

107

Index

108

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Foreword In the 1960s, infectious diseases—which had terrorized generations—

were tamed. Building on a century of discoveries, the leading killers of Americans both young and old were being prevented with new vaccines or cured with new medicines. The risk of death from pneumonia, tuberculosis, meningitis, influenza, whooping cough and diphtheria declined dramatically. New vaccines lifted the fear that su m m er would bring polio and a gl obal campaign was approaching the global eradication of smallpox . New pesticides l i ke DDT cleared mosquitoes from homes and fields, thus reducing the incidence of malaria which was present in the southern United States and a leading killer of children worldwide. New technologies produced safe drinking water and removed the risk of cholera and other water-borne diseases. Science seemed unstoppable. Disease seemed destined to almost disappear. But the euphoria of the 1960s has evaporated. Microbes fight back. Those causing diseases like TB and malaria evolved resistance to cheap and ef fective dru gs . The mosqu i to evolved the ability to defuse pesticides. New diseases emerged including AIDS, Legionnaires, and Lyme disease. And diseases which have not been seen in decades re-emerge, as the hantavirus did in the Navajo Nation in 1993. Technology itself actually created new health risks. The global transportation network, for example, meant that diseases like West Nile virus could spread beyond isolated regions in distant countries and quickly become global threats. Even modern public health pro tections som etimes failed, as they did in Mi lw a u kee, Wisconsin in 1993 which resulted in 400,000 cases of the digestive system illness cryptosporidiosis. And, more recently, the threat from smallpox, a disease completely eradicated, has returned along with other potential bioterrorism weapons such as anthrax. The lesson is that the fight against infectious diseases wi ll n ever en d . In this constant struggle against disease we as individuals have a weapon that does not require vaccines or drugs, the warehouse of knowledge. We learn from the history of science that “modern” beliefs can be wrong. In this series of books, for example, you will

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learn that diseases like syphilis were once thought to be caused by eating po t a toes. The inven ti on of the microscope set scien ce on the ri ght path. Th ere are more po s i tive lessons from history. For example, smallpox was eliminated by vaccinating everyone who had come in contact with an infected person. This “ring” approach to controlling smallpox is still the preferred method for confronting a smallpox outbreak should the disease be intentionally reintroduced. At the same time, we are constantly adding new drugs, new vaccines, and new information to the warehouse. Recently, the entire human genome was decoded. So too was the genome of the parasite that causes malaria. Perhaps by looking at the microbe and the vi ctim thro u gh the lens of genetics we wi ll to be able to discover new ways of fighting malaria, still the leading killer of children in many countries. Because of the knowledge gained abo ut such diseases as A I D S , en ti re new classes of a n ti - retrovi ral dru gs have been developed. But resistance to all these drugs has already been detected , so we know that AIDS drug devel opm en t must continue. Education, experimentation and the discoveries which grow out of them are the best tools to protect health. Opening this book may put you on the path of discovery. I hope so, because new vaccines, new antibiotics, new technologies and, most importantly, new scientists are needed now more than ever if we are to remain on the winning side of this struggle with microbes. David Heymann Executive Director Communicable Diseases Section World Health Organization Geneva, Switzerland

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1 Discovering Cholera What is cholera? Cholera is the term used to describe a specific

gastrointestinal disease as well as the bacterium which causes that disease. In this case, the disease was known long before the microorganism that causes it was even recognized. To understand this, it is necessary to go back through history. THE GERM THEORY OF DISEASE Mi c roorganisms were not widely recogn i zed as the causes of many diseases until late in the nineteenth cen tu ry. However, s ome early s c i en tists did propose that living or ganisms caused ill n e s s e s . Th e Italian physician Girolamo Frac a s toro (ca. 1 4 78–1553) spo ke of “s eeds” or “germ s” of d i s e a s e . Tra n s l a ti ons of Frac a s toro’s Latin wri ti n gs i n d icate that he may have su rmised that these “s eed s” were alive . This is the earliest known wri t ten record of the Germ Theory of Disease – i . e . , the con cept that microor ganisms cause some diseases. Th i s concept was neglected for many ye a rs, h owever. Over 300 years later, Agostino Bassi (1773 –1856) described a disease of silkworms known as muscardine as being the result of a fungal infection of the worms. He could see the fungus as white, powdery material on silkworm eggs. It was identified as a fungus of the genus Botrytis. A botanist of the day confirmed this identification and named the fungus Botrytis bassiana in honor of Agostino Bassi. LOUIS PASTEUR AND ROBERT KOCH In the mid-nineteenth century the famous French scientist Louis Pasteur (1822–1895) had proven that microor ganisms do not arise spon t a n eo u s ly.

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His cl a s s i c , s i m p l i s ti c , and ingenious ex peri m ent was to de s i gn a flask with an S-shaped curve in its neck. The curve trapped microor ganisms that were pre s ent in the air before they could re ach the main part of the flask. He filled the flasks with bro t h , h e a ted them , and then all owed them to coo l . In previous ex peri m ents using flasks wi t h o ut the S-shaped neck , m i c roor ganisms would grow in the bro t h . This did not occur in Pa s teur’s ex periments. Critics s t a ted that a “ vital force” h ad been rem oved from the air by heati n g, so the microor ganisms could not grow. Pa s teu r ’s flasks allowed the air to have access to the heated bro t h , defusing this argument. His coo l ed flasks did not become spoi l ed with bacterial growth because the microorganisms in the air had no means to ascend the tube leading to the broth on ce they were trapped in the dip, or curve , of the S-shaped flask. Pa s teur went on to show that wh en he bro ke the spout of the flask, t h ereby destroying the S-shaped s po ut with the dip in it, the bacteria pre s ent in the air qu i ck ly grew in the bro t h . This experi m ent establ i s h ed on ce and for all that microor ganisms do not arise spon t a n eously. Th ey can, however, be grown in laboratories just like any other living thing. In ad d i tion to this landmark experiment, Pasteur went on to make numerous contributions to understanding microor ga n i s m s . Among his many intere s t s , Pa s teur reex a mi n ed the probl ems of s i l k worm infecti on, wh i ch had been s tu d i ed by Bassi so many ye a rs before , even though he was unaw a re of Ba s s i ’s work. Pasteu r ’s efforts were sparked by his i n terest in the more gen eral qu e s ti on of the microbial origins of i n fecti on s . He stu d i ed infecti ons in higher animals as a re sult of these efforts. This occurred in the late 1800s, a go l den era in microbi o l ogy. Pa s teur, a l ong with his main com peti tor, the German scientist Robert Koch (1843 –1910), s tu d i ed nu m erous microor ganisms and the ef fects they caused, i n cluding diseases.

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Robert Koch (Figure 1.1) was the first person to show that a specific microbe can cause an infectious disease in a higher animal. He isolated the bacterium that causes tuberculosis in 1 8 8 2 . A year later, he publ i s h ed a report de s c ri bing the bacterium that causes the disease cholera. This microorganism is called Vibrio cholerae. How was Koch able to isolate this one microorganism and associate it with cases of the infection? In order to isolate and identify Vibrio cholerae, Koch had to grow this microorganism free from all other microorganisms. A microorganism grown in this manner is called a pure culture of a microorganism. In addition, Kock had to develop a growth medium, as well as a technique to separate the many thousands of microbes in a sample so that just one microbe could grow, dividing repe a tedly to form visible growth. This growth is referred to as a colony of a specific bacterium. KOCH’S TECHNIQUES FOR THE STUDY OF MICROBES Koch first made a growth m e d i u m using 2.5 to 5 percent gelatin (a pro tein obt a i n ed from the tendons of animals) in a nutrient soup. He spre ad the medium on a glass slide and a ll owed it to solidify at room temperatu re . Next, he steri li zed a metal wi re by heating it in a flame. Wh en the wi re coo l ed , Koch dipped it into the area wh ere the bacteria were loc a ted, and used the wi re to draw a line on to the solid med ium on the slide. He repe a ted this process many times, as illustra ted in Figure 1.2. Koch then placed the slide in a warm incubator. After the bacteria had grown on the medium, the slide was removed for observation. The first streaks on the slide contained many microbes. Each subsequent streak had fewer microbes. Eventually, only single cells (instead of large clumps) grew in each area. After the cells had been allowed to grow overnight, the single cells had formed colonies (individual microbes that h ave divided repeatedly to form a group of cells visible to the

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Discovering Cholera

Figure 1.1. Robert Koch, pictured here, discovered that certain microorganisms can cause specific diseases. Along with studying anthrax and cholera, his rules for proving that a microorganism causes disease has become standard in the practice of microbiology today. These rules are known as Koch’s postulates.

naked eye) about 1– 2 millimeters in diameter. Each co l ony was a clone, a pure culture of identical cells. Koch had probl ems with the use of gel a tin in this procedu re . Ma ny microor ganisms could degrade the gel a tin pro tei n , tu rning the medium to mush. In additi on , gelatin melts at

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CHOLERA

Figure 1.2. In order to study bacteria, Koch needed to isolate individual colonies in each culture. To do this, he devised a method to streak bacteria on an agar plate so that only a few colonies would grow in certain areas. He streaked his bacteria on the plate using a linear pattern, as is shown in this diagram. Each streak contained fewer and fewer bacterial cells until only a few grew in each area.

body tem peratu re , the very temperatu re which is most likely to support the growth of an infectious microorganism. A turn of events, which is, by now, a legend, occurred. The wife of one of Koch’s co - workers su gge s ted using a cooking additive for the growth medium instead of gelatin. The additive was called agar. She had learned from a Dutch friend that this substance was often used in prep a ring jellies and soups in Java (a form er Dutch co l ony and now part of Indonesia). Agar is dri ed seaweed ( G enus and spec i e s : Agar aga r) wh i ch can be ground and dissolved wh en heated in water. It remains in a liquid form for some hours at 50°C, and it solidifies bel ow 42°C. Koch recognized that agar provided a better growth med ium than gel a ti n for the isolation of pure cultures. It remains in use today.

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Discovering Cholera

Figure 1.3. The bacteria that Koch observed looked like small commas, and thus he named them “comma bacilli.” This type of bacteria is now called Vi b r i o. Because the bacterium that causes cholera is a Vi b r i o, the scientific name for the disease is Vibrio cholerae.

Another legacy of Koch’s early lab is the glass dish that was developed by an assistant of Koch, R. J. Petri. The Petri dish is standard today in the study of microorganisms. One can see colonies without removing the glass cover of the dish. The agar medium can be varied while it is still liquid, then poured into Petri dishes rather than spread onto glass slides, as Koch had done originally. The streak plate method for the isolation of pure cultures of bacteria is also standard today. These met h ods had to be developed before Koch could establish that microor ganisms caused cholera and other diseases. When he first ob s erved the microbes he isolated, t h ey appe a red as small commas, so he referred to them as “comma bacilli” (Figure 1.3). Another name for this curved rod - s h a ped bacterium is a vi b ri o (Figure 1.4). Hence, the official name for this microor ganism is Vi b rio ch ol era e.

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Figure 1.4. This electron micrograph shows the curved and spiral nature of the Vibrio bacteria. Koch found that older cultures contained m o re spiral-shaped vibrios, while newer cultures contained more commashaped bacteria.

KOCH’S POSTULATES Not content merely to observe a microbe present in an infected individual, Koch established guidelines for proving that a m i c robe causes a particular infection. These guidelines are

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Discovering Cholera

called “Koch’s Postulates” and are still used today as standards for establishing proof of infection. They are, in brief: 1. The microbe has to be present in every case of the disease. 2. The microbe has to be isolated from the patient and grown in pure culture. 3. When the purified microorganism is inoculated into a healthy susceptible host, the same disease results. 4. Once again, the same microbe must be isolated from the host infected with the microbe.

Koch’s remarkable con tributions were landmarks to the f i elds of m i c robi o l ogy, m ed i c i n e , and scien tific stu dy in general. He received the Nobel Prize in Medicine in 1905 for his work related to tuberculosis; moreover, his techniques and protocols for laboratory investigation are still in use today.

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2 Properties of Vibrio cholerae Koch discovered that Vibrio cholerae causes cholera. But how is this

organism unique? Does it re s em ble other microorganisms that cause similar diseases? How is it different? How can this information help isolate Vibrio cholerae from the thousands of other microorganisms that inhabit the human body at any given time? KOCH’S FIRST LOOK Koch headed a com m i s s i on establ i s h ed by the German govern m en t to stu dy ch o l era in Egypt and In d i a . His discovery of the “com m a b ac i llu s” in a large nu m ber of cases of the disease indicated that a b ac i llus of this same shape was prob a bly pre s ent every ti m e . In addition, Koch was able to see firsthand the transmittal of the disease and su b s equ ent infecti on by the bac i lli wh en two of his labora tory assistants became seri o u s ly ill and nearly died after drinking the t a i n ted water. What Koch saw in his stu dy of Vibrio ch olerae was a small, curved rod , ranging in length from one to two micron s , which is on ly one or t wo mill i onth of a meter. The microbes curved in va rious ways : s om e were on ly sligh t ly bent while others had spirals of one or two tu rns that loo ked like corkscrews . The bacterium was also actively motile. When Koch stained the bacteria with a special stain, he could see that each or ganism con t a i n ed a single polar flagellu m , or tail. The or ganism did not appear to form spore s . Young cultu res con t a i n ed more com m a shaped forms while the spiral forms of the microbe dom i n a ted older

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Figure 2.1. A test called the Gram stain reaction allows scientists to d i ff e rentiate between diff e rent types of bacteria. The cholera bacillus is considered Gram negative because it takes up the pink counterstain safranin. These cells are pink because they are Gram negative.

c u l tu res. It is now known that if the microbe is grown in the l a boratory and does not pass thro u gh an animal body, the microbes tend to lose their curvatu re enti rely. Koch also ob s erved that the microor ganism was deco l orized wh en using the Gram stain reaction.

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CHOLERA LOOKING AT THE CHOLERA BACILLUS TODAY Tod ay, Vibrio ch olerae is cl a s s i f i ed as Gram negative , since it decolorizes after staining with crystal violet but takes up the counterstain safranin (Figure 2.1). The cholera bacillus is fairly easy to grow in the laboratory. It wi ll grow in most com m on labora tory media su ch as nutrient broth or nutrient agar but will also grow on meat ex tract s . The or ganism prefers media that is modera tely alkaline, but this is not essential since it will also tolerate mild acidity. Sm a ll, strongly refracting yell owish-gray colonies appear after 24 hours of growth on gelatin plate s . However, the gel a tin liquifies as the or ganism con ti nues to grow. The colonies are coarsely granular with uneven edges (due to the liquefaction of the gelatin). In gel a tin stab cultu res, liquefaction begins at the top, leading to a funnel-shaped p a ttern of gelatin liquification. This ability to attack gelatin is lost in old strains of the bacterium which have been g rown artificially in the laboratory for long periods. On agar plate s , grayish and op a l e s cent co l onies appe a r within 18 to 24 hours . The fact that Vi b rio ch ol erae i s op a l e scent helps in the identification and isolation of these microor ganisms from pati en t s , because other bacteria likely to appear in feces are not opalescent. In addition, the bacillus can liquify coagulated blood serum and also grow on starch, appearing as a brownish coarse growth. The cholera bacillus grows abu n d a n t ly on alkaline peptone medium. This trait is particularly helpful when a scientist needs to isolate the microorganism from mixed samples, such as a fecal specimen. The cholera bacilli also produce a crystalline compound called indole, which also helps with identification. Vibrio species can grow at a broad temperatu re range (from 18°C to 37°C) on a va ri ety of simple med i a , aerobi c a lly or anaerobically. Therefore, the bacterium is described as aerobic and facultatively anaerobic. It grows opti m a lly at 37.5°C, wh i ch

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Properties of Vibrio cholerae

is normal human body temperature. The microorganism has a positive oxidase reaction which means that is has the enzyme cytochrome oxidase, a key enzyme in aerobic metabolism. Cholera bacteria can live about three or four days when frozen in ice. They die immediately when heated to boiling (100°C). They are killed within an hour at a temperature of 60°C. Drying will also kill the cells in a short period of time. When in impure water, in food, on cloth, or other complex environmental conditions, they may live for many days. Dilute solutions of common disinfectants destroy these bacteria after exposure for a few minutes. ISOLATING CHOLERA BACILLI FROM PATIENTS The properties of Vibrio cholerae discussed in the previous s ecti on are important for the isolati on and identificati on of the ch o l era bacillus from pati ent spec i m en s . The procedu res for examination of stool specimens (fecal material) from p a ti ents outline met h ods to test for: 1) animal para s i te s , 2) ro utine ex a m i n a ti ons for microor ga n i s m s , and finally 3) ex a m i n a ti ons for special or unu sual cases. The search for ch o l era bacilli falls into this third category, s i n ce it is not an ord i n a ry or su s pected disease in the Un i ted State s . However, in areas wh ere the bacillus is more com m on ly ob s erved, the procedu res for isolating and identifying cholera bacilli are u n do u btedly ro uti n e . If a virus is su s pected, tissue culture and el ectron micro s copy met h ods are also used in an a t tem pt to iden tify the of fending microor ganism. Sa m p l e spec i m ens from special cases are placed in cultu re medium at 22°C, i n oc u l a ted into alkaline peptone water wh i ch wi ll help to increase the nu m ber of b ac i lli in the sample, and stre a ked thiosu l f a te - c i tra te - bile salt-su c rose (TC B S ) m ed ium plate s . TCBS plates also contain a brom o t hymol blue indicator wh i ch helps to identify co l on i e s . Mi c roor ga nisms that do not ferment sucrose grow best on TCBS medium

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Figure 2.2. Scientists grow bacteria such as Vibrio cholerae on agar plates. Agar is a gelatin-like substance that solidifies at 42°C. In this picture, a scientist streaks cells on an agar plate using a method similar to the once that was pioneered by Robert Koch. The cells will grow on the media and become colonies that are visible to the naked eye.

and produce blue-green colonies. Because Vibrio cholerae does ferment sucrose, it will produce tallow-colored colonies on this m ed ium. The fermentati on process invo lves the formati on of acid from su c ro s e , and the acid re acts with brom o t hymol blu e , re su l ting in a co l or ch a n ge . The bile salts in this m ed ium and the high pH (8.6) prevent the growth of o t h er b acteria assoc i a ted with the gastroi n te s tinal tract. Med i c a lly

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Properties of Vibrio cholerae

i m portant microor ganisms wh i ch do not ferment su c ro s e i n clude Vibrio pa ra h a emolyticus, Aeromonas spp. (species) and Plesiomonas s pp. These are the non - ch o l era food poi s oning vi bri o s , wh i ch are rel a ted to Vi b rio ch ol era, yet d i s ti n ct . Ch o l era bac i lli in fresh stool samples wi ll also h ave a ch a racteri s tic darting mobi l i ty, wh i ch aids in the i dentificati on of the microor ganism. Having proceeded this far, one might think that the search for the cholera bacillus in a patient sample is complete. This is true, yet it is still necessary to identify what type of strain is present, as many different strains of the bacillus exist. The strains can be identified by matching them to antibodies that are formed against each different type. In order to understand this process, one must examine the structural variations which define each of the strains. THE CHOLERA BACILLUS AS A PROKARYOTE OF THE DOMAIN BACTERIA All life forms are either prokaryotes or eukaryotes. Eukaryotic cells contain a nucleus and other membrane-bound organelles. The DNA of the cell is contained on chromosomes which are located within the nucleus. Animal, plant, and fungal cells are eukaryotes. Prokaryotes, on the other hand, are generally smaller in size, do not have a nucleus or membrane-bound organelles, and the DNA resides within the cytoplasm. Bacteria are prokaryotes. Using special techniques to obtain the sequence of nucleic acid bases in the ribosomal RNA, scientists discovered that t h ere are three disti n ct types of l i fe forms on Earth. Two of these cater gori e s , Arch aea and Bacteri a , a re pro k a ryo te s . Organisms in the Archaea group are thought to be the oldest l i fe forms that still exist on earth. These microorganisms l ive in extreme places in our envi ronment su ch as regi on s of h i gh salt, h i gh tem pera tu re , and places wh ere met h a n e

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can be formed. For this reason, Archaea microor ganisms are often referred to as extremophiles. For example, the hot geyser pools in Yellowstone National Park, Wyoming, have yielded numerous high temperature-growing microorganisms (thermophiles). Microorganisms in the group Archaea differ from those in the group Bacteria in several ways. Archaea have a unique RNA sequence and a different cell wall composition, consisting of ether bonds in the lipids rather than ester bonds. Due to this cell wall difference, antibiotics that work against Arch aea are different from those that wi ll work aga i n s t Bacteria. Often the antibiotics that act on eukaryotes will also act on Archaea, but not on Bacteria. Scientists have discovered that Vibrio organisms are part of the domain Bacteria. The Gram stain procedure utilizes a primary stain, crystal vi o l et , and a secon d a ry stain, safranin. Cells are first tre a ted with crystal violet, and then rinsed. Next, safranin is ad ded. Cells that are considered Gram positive will retain the crystal vi o l et dye even after they are rinsed. Gram negative cells will not retain the primary stain. Recall that Koch and others determ i n ed that Vibrio cholera e was Gram negative . Ne a rly 50 years later, scientists discovered that chemical differences i n the cell walls of d i f ferent cells leads to the disti n cti on in staining characteristics. Gram nega tive bacteria have an ad d i ti onal outer cell m em brane layer. This outer layer is out s i de the cell wall po lym er of N - acetyl glu cose and N-acetyl glu co s a m i n e polysaccharide chains, a thin layer overlying the cell membrane. Both the outer and inner membranes are composed of lipid bi l ayers . However, the ad d i ti onal lipid bi l ayer in Gram negative bacteria necessitates the formation of protein structures (porins) which can help transport water-soluble materials into the bacterial cel l. In contrast, Gram positive microorganisms have a thick layer of cell wall polymer above the single lipid bilayer cell membrane.

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Properties of Vibrio cholerae

The outer mem brane of a Gram negative bacterial cell contains nu m erous pro teins and lipopo lys acch a rides. E ach el em ent has an important functi on. Lipid A is attach ed to the outermost layer of the mem brane. A core po lys acch a ride is attached to Lipid A . E ach core polys acch a ri de has a special s i de chain wh i ch va ries from one species of Gram negative

THE GRAM STAIN REACTION The Gram stain was developed by Christian Gram. This staining procedure differentiates bacteria into two categories, Gram positive and Gram negative. First, the microorganisms are attached to a glass slide and then stained with crystal violet dye. An iodine solution consisting of iodine and iodide ion is then applied to the bacteria. The crystal violet reacts with the iodine solution inside the bacterial cells. A complex of c rystal violet and iodine forms. Next, the cells are tre a t e d with 95 percent ethyl alcohol solution. Some cells will retain the purple color after the alcohol treatment and are designated as Gram positive bacteria. Other cells will lose the purple color of the dye (the crystal violet-iodine complex washes out) and are considered Gram negative. Gram negative bacteria are then stained with safranin, which give them a contrasting light red color. Cholera bacilli are Gram negative. Years later, it was discovered that there are fundamental structural diff e rences between Gram negative and Gram positive bacteria. Gram positive bacteria have a thick cell wall surrounding the cell membrane. Gram negative bacteria have a cell membrane, a thin cell wall over that, and an additional lipid bilayer membrane outside the cell wall and facing the exterior of the cell. This additional lipid bilayer contains components that are unique for each species of Gram negative bacteria. Gram positive and Gram negative bacteria have different properties and characteristics because of these distinctly different cell structures.

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b acteria to another. This en tire com p l ex (the core po lys acch a ride and the side chain) is called a lipopo lys acch a ride (LPS ). The LPS plus Lipid A is call ed an endotoxin because the com p l ex is toxic to many different animals. Gra m n egative bacteria containing LPS without the Lipid A portions are non - toxic. ANTIBODIES AS A RESPONSE TO THE OUTER STRUCTURES OF CHOLERA BACILLI Like nearly all foreign invaders, the ch o l era bacilli will invo ke an immune response from its host. Any substance that invo kes an immune re s ponse is call ed an antigen and alerts the host or ganism to form anti bod i e s . An a n t i b o d y is a protein that attacks a specific foreign body. Early inve s ti gati ons by German microbiologists recognized two major types of anti bodies that form ed in re s ponse to bacteria like the cholera bac i llus. One type of a n ti body could recogn i ze the pro teins of the flagella. The Germans used their word for film (“Ha u ch”) to describe these anti bod i e s . These microbes can move and were observed to form films across the surf aces of media. These became known as H anti gens or flagellar a n ti gens. The anti bodies that formed against the rest of the cell were said to be “ without film” (“ohne Ha u ch”) and thu s were de s i gn a ted O anti gens. These are also referred to as s om a tic or cellular anti gens, indicating that these are part of the main body or structu re of the bacterial cells. The polar f l a gellum and fimbria of Vibrio ch olerae can be seen using an el ectron micro s cope (Figure 2.3). Using these tech n i qu e s , s c i en tists have discovered abo ut 150 different strains of Vibrio ch olerae. In additi on to the classical ch o l era form, four important strains that will be discussed are El Tor, Ogawa, Inaba, and Hikojima. The choleracausing bacteria are classified in serotype O1. Classical cholera possessing this som a tic anti gen was the on ly type with this

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Figure 2-3. This electron micrograph shows the flagellum and fimbria of a Vibrio cholerae bacterium. The flagellum is a tail-like projection that helps the cell to move.

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a n ti gen until the discovery of the strain call ed El Tor. The El Tor strain was isolated from pilgrims at El Tor in the Si n a i peninsula. This strain is capable of a ggluti n a ting red bl ood cells from ch i cks, and it is resistant to anti bi o tics genera lly u s ed to treat classical ch o l era. Th ere are three different po lys acch a ri des that have been found as part of the O1 anti gen structu re . These are i n d i c a ted as types A, B, and C. The Ogawa strain has types A and B, the Inaba strain has types A and C, and a ra re type has been found wh i ch has all three of these polys accharides ( Hi kojima strain). Th ey can be disti n g u i s h ed by using antibodies specific to each strain and by comparing reacti on s to the different bacterial stra i n s . For ex a m p l e , a n ti bod i e s to the Ogawa strain would re act best wh en mixed wi t h a n tibodies to the Ogawa bac i llus. However, there would be s ome re acti on with the Inaba strain, s i n ce it shares the A po lys acch a ride in the O1 anti gen. The re acti on of anti gens and anti bodies is usu a lly ob s erved by mixing anti gens and a n ti bodies on slides and ob s erving aggluti n a ti on (clumping) due to the large anti gen-anti body com p l exes that form and prec i p i t a te from soluti on . Pati ents re act differen t ly to the classical and the El Tor s trains. Com p a red to the classical strain, diarrhea is shortl ived in the El Tor strains. Pati ents infected with the El Tor s train of ten do not ex press sym ptoms but become carriers of the bacteria. Because of these and other biological differences, these strains which have the som a tic anti gen O1 are called biotypes. Strains that are distinguished by different immunological reactions (Ogawa, Inaba) are called serotypes. In 1992, a new sero type de s i gn a ted O139 syn . Ben ga l was isol a ted in India and Ba n gl adesh. Now, the iden ti f i c a tion of the ch o l era bacillus is comp l ete. Performing slide agglutination tests on the microbes isolated from the stool specimen by growth on TCBS aga r

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Properties of Vibrio cholerae

can indicate the specific sero type fo u n d . This is parti c u l a rly i m portant for tracking a specific type of ch o l era bacillu s du ring ep i demics. However, one can not assume that all strains of Vibrio cholerae have been discovered. It is possible that new strains can form if current forms of the organism mut a te. Should that happen, there are methods to identify, characterize, and hopefully develop appropriate treatments for the new strains.

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3 Dr. Snow and Cholera A CASE OF SUSPECTED CHOLERA: Miners in Victorian England In 1831 in Newcastle-upon-Tyne, England, a group of coal miners who had

been perfectly fit and healthy in the morning returned from the mines profusely defecating (eliminating solid wastes) with abundant diarrhea (loose, watery, solid wastes). They were in a stat e of near exhaustion. Granted, their work in the mines was hard and the conditions grueling, but this could not explain the fact that soon most collapsed and died. John Snow (Figure 3.1), then 18 years old and in his second year as apprentice to the local doctor at Newcastle-upon-Tyne, was sent to give the miners and their families any medical assistance that he could. Although Dr. Snow knew at once that the men were ill with cholera, there was little he could do for them. Cholera was familiar to the British from their visits to India, where it was common. The miners were in the same condition as people believed to be suffering from cholera in India. Af ter seven more years as an appren ti ce to several physicians, John Snow passed the medical examinations. At the age of 25, he set up practi ce as a physician and sold dru gs and other medicines. He could have con ti nued to practi ce medicine with no furt h er edu c a ti on or medical degree s . For him, though, his pre s ent knowl ed ge abo ut infectious diseases was not en o u gh . Most physicians of the day bel i eved that ch o l era and other su ch infectious diseases were carri ed thro u gh “b ad air.” Dr. Snow did not agree . Furthermore , he was intrigued by many unanswered qu e s ti ons in the medical field and thus dec i ded to stu dy medicine furt h er. He attended the co ll ege and the newly open ed medical sch ool at the Univers i ty of London wh en he was 30 years old and gradu a ted in 1844.

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Figure 3.1. John Snow discovered the path of cholera transmission through his careful observation of the 1854 cholera epidemic in London. He was the first scientist to propose that cholera was transmitted through contaminated water.

Dr. Snow was aware of documented cases of cholera in India that dated as far back as 1789. However, it is likely that cholera existed in India before Europeans went there for regular visits. As travel between India and Europe increased, it is very likely that cholera fo ll owed them hom e . Because the firs t

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cases of cholera in England were thought to have originated in India, the disease was often called Asiatic cholera. The following case study is presented to give an example of observations that piqued Dr. Snow’s curiosity. Rather than draw conclusions from one such case, Dr. Snow accumulated information from many such case observations. This case and others helped him to develop his hypothesis that cholera is a communicable disease spread through water. DR. SNOW’S DATA COLLECTION Dr. Snow began to take a close look at case studies of cholera. One case, that of John Harnold, a seaman who in the autumn of 1848 arrived in London by steamer from Hamburg, G ermany, was quite intriguing. Cholera was raging in Hamburg, and John Harnold was ill. He died of cholera within a few hours a f ter the first symptoms appe a red. Eight days later, a man who rented the room that Mr. Harnold had used also died of cholera. The doctor attending him, however, did not. Dr. Snow wanted to know why two men had died, yet the third had not. Many learned men of the day thought that diseases such as cholera were carried from one person to another through the air. Dr. Snow wanted to find out for sure. Dr. Snow knew of many cases in which it seemed clear that the disease was carried from a sick person to a healthy person. He began to collect this information and keep careful records of these cases. Many of his medical colleagues also kept recods. From all his experien ces both in treating patients and in ex a minations after death, he observed that the main part of the body affected by cholera was the alimentary canal, the part of the body re s pon s i ble for dige s ti on . He began to think that ch o l era might be spre ad thro u gh drinking water. Snow bel i eved that this was the method of disease communication. Dr. Snow form ed the hypothesis that ch o l era is a communicable disease spread through water. Following the sci en tific method for probl em solving, he continu ed his clinical observa tions,

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c a refully collecting data wh i ch might prove his preliminary i de a . However, the many clinical cases in wh i ch ch o l era had p a s s ed to a healthy person from a person who had been in contact with an infected individual did not prove or disprove his hypothesis. In some of these cases, the persons who bec a m e ill were near to but not in contact with the patient at all. Yet, some got cholera and some did not. Why was this so? One outbreak was parti c u l a rly telling. Rows of small co ttages inhabited by poor people were separated by a single street in Horsleytown, England. The north side of the cottages was called Surrey Buildings, and the south side of the cottages was called Truscott’s Court. During 1849, there were many cases of cholera in the Surrey Buildings but only two cases in Tru s co t t’s Court. Ho u s ehold wastewater was poured into a channel in front of the houses in both cases, The wastewater from the residents of the Surrey Buildings (and not Truscott’s Court) reached the well which the residents used for drinking water. Furthermore, the two sets of buildings received the well water from different water companies. Dr. Sn ow did not draw con clu s i ons from any one case. Ra t h er, he ex a m i n ed many cases and their ex tent, a ppeara n ce , and geographical loc a ti on before formu l a ting any con clusions. In short, he was the first scientist to use methods of epidemiology, wh i ch are em p l oyed to this day. DR. SNOW USES MAPS TO HELP TRACK THE DISEASE Outbreaks of cholera occurred regularly during the nineteenth century. In 1854, Dr. Snow reported one of the most severe outbreaks to date . In a period of t h ree days , 127 people in the area of Broad Street in Lon don died from the disease. Dr. Snow kept records of this epidemic by marking a street map of the area with the location of each of the cholera patients (Figures 3.2 and 3.3). He saw that there were more cases closer to the water pump, and the numbers of cases diminished at

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Figure 3.2. Snow tracked cholera cases on a map of the Broad Street area of London, during the 1854 outbreak. He placed a mark on the map, shown above, for each cholera case. This map helped him to hypothesize about the method of cholera transmission.

d i s t a n ces from the water pump. Sanitary con d i ti ons were s i milar in other areas of the neighborhood, but there were few or no cases of cholera in the vicinity of other water pumps. This convinced Dr. Snow that this cholera outbreak was the result of drinking water from the Broad Street water pump.

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Figure 3.3. Using his map of cholera deaths, John Snow also mapped out the location of water pumps. He discovered that many of the cholera cases centered around one particular pump. On the map pictured above, cholera cases are represented by dots, and each water pump is designated by a square.

Dr. Snow also realized that some people who lived far from the city and Broad Street were dying of cholera even though they most likely did not use water from the Broad Street pump. In order to prove his theory that cholera was not “in the air,” but in the water, he investigated one such case. A lady from

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Hampste ad , a su burb of the city of London , had died of ch o l era . Her son told Dr. Sn ow that she had not been in the vicinity of Broad Street for many months. This was truly puzzling. The water in the household was clear and had never been in contact with sewage. Dr. Snow began questioning members of the household, including the staff. One servant told him that the woman had a fondness for water drawn from the pump at Broad Street in London. The servant was sent to the Broad Street neighborhood every week to draw water f rom the pump and had done so during the time of the cholera epidemic. The woman, in turn, drank this water. This was the connection that Dr. Snow was looking for. The mystery surrounding cholera transmission had been solved. Dr. Snow also re a l i zed that there were fewer cases of cholera in the area just east of the Broad Street pump (see Figure 3.2 and 3.3). Dr. Snow talked to the own ers of a brewery located in this area and found that the workers at the brewery did not dri n k from the Broad Street pump because the own er of the brewery su pp l i ed beer for his workers . He assured Dr. Snow that these men drank beer and not water! This hel ped explain the lower numbers of cholera cases in that area of the city. Dr. Snow realized that something had to be done to reduce the tra n s m i s s i on of ch o l era in the are a . He discussed the problem with the Board of Guardians of St. James’s Parish, which had jurisdiction over the area of the Broad Street water pump. Snow told them that the pump was the source of the cholera in this latest epidemic, and he recommended that they shut it down.Very reluctantly, they heeded his advice to do so, and the pump handle was removed. Al t h o u gh this particular ep i demic may have alre ady re ach ed its peak and was beginning to wane by the ti m e D r. Snow convinced St. James’s Parish to remove the pump handle, it is certain that his research prevented future infecti ons from the Broad Street water pump. In later ye a rs , this s et of ob s erva ti ons has been referred to as the “Gra n d

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Ex periment” of Dr. Snow and has entered the realm of folklore in the history of medicine. THE BEGINNINGS OF EPIDEMIOLOGY Th ro u gh his careful stu dy of the Broad Street ch o l era epidemic as well as many other studies and observations, one can su rmise that Dr. Sn ow fo u n ded the scien ce of ep idem i o l ogy. Ep i dem i o l ogy is the stu dy of disease tra n smission, its incidence in a population, and methods of con trol and preven ti on . Even tod ay, an important aspect of s tu dying an ep i demic is the acc u ra te reporting of t h e l oc a tion of infected individuals. Dr. Sn ow ’s contributi ons to the scien ce of m i c robi o l ogy not on ly inclu de an understanding of the way ch o l era is tra n s m i t ted , but also i nva lu a ble tools for understanding infectious diseases within a pop u l a ti on. His work led to the stu dy of p u bl i c health, and to this day we benefit from his legacy.

TRACKING EPIDEMICS Did you know that satellites can track worldwide epidemics? Ocean height, turbidity, and sea surface temperature can be observed and photographed from above and have often been linked to emerging epidemics. By examining photographs from past years as well as the data giving the numbers of cholera cases during those years, it has been shown that there is an i n c rease in the numbers of cholera cases when the sea surface temperature is elevated and the ocean height is high. NASA scientists relate that this information connects changes in climate such as the El Niño effect to cholera outbreaks. They are continuing to gather information about increased growth of algae in seas and possible relationships to cholera epidemics. http://geo.arc.nasa.gov/sge

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4 Transmission and Epidemiology of Cholera THE TRANSMISSION OF CHOLERA As learned in Chapter 3, Dr. Snow successfully described the connection

between water supply and cholera infections. He did this through careful ob s ervati on of the locati on of disease su f ferers and by adhering to the s c i en tific met h od . This was the gro u n dwork for many studies of ep i demics and the spre ad of diseases. It is rem a rk a ble that Dr. Sn ow’s insights came before scientists knew, or even accepted the fact, that cholera is caused by a microorganism. It is known that ch o l era infecti on is a result of transmission from envi ronmental contamination to an individual, or from one individual to another. Human bei n gs are the only known natu ral host of Vibrio ch ol era e, the microor ganism wh i ch causes ch o l era. Poor sanitation m et h ods lead to the con t a m i n a ti on of s oi l , food , or water with Vibrio cholera e bacilli f rom feces. The cycle of tra n s m i s s i on is com p l ete wh en a pers on becomes a carri er. Indivi duals who are recovering from the disease may feel bet ter but they sti ll carry the microor ganism within their bodies. These people are known as co nva l e scent carri ers. People who harbor the microorganism but do not yet show signs and sym ptoms of the disease are call ed i n c u ba to ry carriers. They are also important in the transmission cycle of cholera. Experiments using volunteers have shown that a dose of 103 (1000) Vibrio cholerae cells are required to infect a person. These bacteria may come from con t a m i n a ted water or from con t a m i n a ted food , such as vegetables grown in human waste fertilizer. Cholera may also be spread directly from person to person. This often occurs when a cholera patient

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is being treated at home or wh en a ch o l era victim is being prepared for bu rial. Vibrio ch olerae can survive on the body or clothing of a victim; thus the bacilli may be passed to a nyone in close con t act with the body. Because the disease causes profuse, watery diarrhea, cholera patients excrete many liters of fluid e ach day, and this fluid contains abo ut 106 to 1 08 ch o l era bacteria per milliliter. Therefore, contact with cholera patients poses a considerable risk of infection. Ch o l era bacteria can be kill ed by heat, but they su rvive in the co l d . Th ey can live for two or more weeks in food su ch as milk, coo ked ri ce , and seafood . Th ey are sen s i tive to ac i d i ty, but su rvive in alkaline envi ron m en t s . Thu s , t h ere are many opportu n i ties for infecti on thro u gh food and drink. THE HISTORY OF CHOLERA S ch o l a rs do not entirely agree on the origin of the word “cholera.” It has been suggested that the word cholera is derived from the Greek word for bile (cholera) and flow (rein). Others su ggest that in Greek the word “cholera” (which can also be i n terpreted as “roof gut ter”) probably indicates symptoms of w a ter flow like that after a heavy rain. Ch o l era has been doc u m en ted several times thro u ghout history. A disease with sym ptoms similar to those of ch o l era is described in Sanskrit documents dating from about 500 B.C. to 400 B.C. Ch o l era was described early in the sixteenth cen tu ry by European arrivals to India. It was documented by a staff member of the explorer Vasco da Gama that 20,000 men died of cholera in the early 1500s. Since 1817, research ers have documented cholera ep i demics a ll ac ross the worl d . A worl dwi de ep i demic is call ed a pandemic. Other patterns of disease spread have also been observed for cholera. If the disease is present at a low, persistent level in a population, it is said to be endemic. The first cholera pandemic occurred as a result of wars between Persia and Turkey when soldiers were traveling between their native lands and could unknowi n gly carry the disease with them . The second pandemic

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is thought to have ori gi n a ted in Russia and spre ad to the Americas, reaching New York on June 23, 1832. The disease traveled to Philadelphia, and eventually to New Orleans. It was during this second pandemic, when it passed through London, that Dr. Snow made his observations that would ultimately connect water to cholera transmission. Cholera first appeared in Chicago, Illinois in 1849 when it was brought to the city via a boat carrying immigrants. From this point on, cholera epidemics occurred regularly in the city. City officials attempted to improve sanitation by using water from nearby Lake Michigan rather than local wells which were often contaminated by sewage from the Chicago River. In 1867, the city opened a two-mile-long tunnel that carried water from the lake into the city. This further reduced the amount of sewage from the river into the local water supply. NEW YORK: The 1892 Cholera Panic By the 1890s, p u blic awareness of cholera had grown sign i f icantly. In August 1892, em i grants from Hamburg, G ermany, arrived in New York Ci ty. At that time, ch o l era was ra ging in Eu rope. Upon arriva l , these ships were qu a ra n ti n ed and steera ge p a s s en gers were sent to special quarantine hospitals. Cabin p a ss en gers traveling first- and secon d - class were not all owed off the ship for 20 days. The passen gers were not happy with this dec i s i on . Th ey wanted to disembark ri ght aw ay because some of the workers on the ship were ill with ch o l era, and the passen gers feared infecti on . The govern or of New York dec i ded to buy an unused hotel and the su rrounding 120 ac res of land in Babyl on and Islip Town on Long Island for the qu a rantined passen gers . The local re s i dents felt threaten ed because they were afraid that t h ey would catch the disease too. Th ey took up weapons and threaten ed ars on and other forms of violence if the govern or allowed the boat passen gers to stay in their town s . Wh en the ship tri ed to dock , an angry mob of a bo ut 400 people stood waiti n g. Th ey had sailed ac ross the bay to the island wh ere the hotel was

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loc a ted to confront the authori ties. A State Su preme Court ju d ge issued an inju n ction against the landing, and the mob went back h om e , satisfied. The National Gu a rd and Naval Re s erve were c a ll ed in to keep pe ace. Even tually, a few of the quara n ti n ed passen gers were all owed to disem b a rk wi t h o ut inciden t . This incident shows how the fear of infection can cause hys teria. Remember that this was during a time wh en little was known about this disease except that it made people very sick and could kill. The town residents were afraid because they did not know how they could protect themselves from cholera, and at that time, a cure did not exist. In c i den t a lly, the property that was purch a s ed by the governor of New York remained in the hands of the state. The state legislature turned it into a park in 1908. It was the first state park on Long Island, and it still exists today as part of the Robert Moses State Park. EPIDEMICS IN THE 1900s Cholera epidemics appeared at regular intervals throughout the year 1923. Many people thought that improved sanitation would have prevented the recurrence of cholera by this point. However, pandemics con ti nu ed to occur sporad i c a lly. The 1961 pandemic was the result of a new biovar (variety of the virus), dubbed the El Tor type. This biovar does not cause as severe an infection as the classic Vibrio cholerae O1 and is still present to this day on six continents. In 1992, a new serogroup of cholera appeared. Labeled Serogroup 0139, it is nearly identical to the El Tor biovar but possesses an ad d i ti onal layer su rrounding the LPS layer call ed a capsule. Scientists have discovered that 22 kilobases of DNA are missing from chromosome 01, and a new 35 kilobase segment of DNA has been added to the new capsule genes. For the first time, there is ev idence that a new strain has developed by obtaining genes from another source and incorporating them into the genes of the cholera bacillus.

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As technology improved, the documentation of cholera i m proved as well. Through o ut history, documentation has developed from vague descriptions of cholera-like diseases to clinical documentation, and finally to iden tification by serology (study of blood serum) and DNA technologies. THE STUDY OF EPIDEMICS AND PANDEMICS Before researchers can study epidemics and pandemics, they must agree on how to define death from cholera (as opposed to death from other causes). The Pan-American Health Organization defines this as “death within one week of onset of diarrhea in a person with confirmed or clinically defined cholera.”1 While not every agency agrees with this definition, there has been an international effort to describe mortality data, i.e., the number of people who died from a specific disease. The World Health Organization collects surveillance data about diseases from many countries. Cholera was the first disease for which this kind of international surveillance was organized. The number of cases reported is less than the actual nu mber of cases that occur. This is due to the fact that the def initi on of “case” (a pers on who actually has the disease) va ries considerably. Because there are many causes for diarrhea, the issue is even more unclear. The morbidity rate (the number of cases of infection) should be confined to those cases for which there has been a positive labora tory diagnosis, but this does not alw ays happen. Re s e a rchers believe that there are ten times more actual cases of cholera worldwide than the number of cases reported. Therefore, morbidity rate data are suspect without proper definition. The incidence of infections, or new cases, with cholera is depen dent on the condition of the area, the opportunities for transmitting the bacterium, and the nu m bers of those immune in 1. R. Tauxe. “Cholera” in Bacterial Infections in Humans: Epidemiology and Control. 3rd ed. Edited by Evans, A. and Brockman, P. New York: Plenum, 1998.

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Figure 4.1. Cholera spread throughout Latin America in 1991. This map shows the route of disease transmission. The epidemic was thought to be the result of ballast water exchange from ships in the area, but this was never proved.

the population. Most people who harbor the cholera bacillus within their body do not show sym ptoms, and are thus label ed as asymptomatic. One estimate is that just two percent of n ew cases are severe, five percent are modera te , and 18 percent have mild sym ptoms. Up to 75 percent of cases are asym ptomatic.1,2 Since such a large number of bacteria are necessary for infection, cholera is usually not transmitted without food or water contamination. Research ers have observed that caregivers usually do not become infected. This shows that sanitary 2. R. Stock. “Cholera in Africa.” African Environment Special Report 3, International Af rican Institute, Lon don, England, 1976.

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Figure 4.2. In the year 2000, cholera cases were reported in over 40 countries. The map above shows which countries had reported cholera cases and which of those had cases that were due to an outside source, such as imported contaminated food, or travel into the country by a person a l ready infected by the disease.

precauti ons are important in the control of the disease. O f ten it is possible to trace the source of an ep i demic, but not alw ays . In one example, the Latin America epidemic of 1991 (Figure 4.1) is thought to have come from ballast water, but scientists could not prove this theory. Scientists stu dy the geographic distribution of disease i n fections because this wi ll help them to bet ter understand the

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disease itself (Figure 4.2). In 1993, the inciden ce of ch o l era was highest in Cen tral and South America, Africa, and the sub-Asian continent (India, Pakistan, Bangladesh, and China). Cholera is a seasonal disease; its occ u rren ce is ra re in cooler months. The peak incidence in India is before the monsoon season in Calcutta and after the monsoon season in Mad ras (fart h er south). In Bangl adesh, the peak season for El Tor ch o l era is in the fall , wh i l e the peak for the classical bi o type is from Decem ber to Janu a ry. Age and sex of a person have no effect on whether or not they will become infected if t h ey do not have immu n i ty to the disease. It has been ob s erved that individuals with Type O bl ood are m ore likely to become infected and wi ll have a more severe case of the disease than people with other bl ood types. Th e reason(s) for this are unknown. Ot h er set ti n gs wh ere ch o l era occ u rs inclu de rel i gi o u s migrations and at refugee camps due to the close proximity of all the inhabitants. Cholera is also associated with extreme poverty. Since cholera bacilli are sensitive to gastric acid, any impairment of the formation of gastric acid will increase the possibility of cholera infection. This can include stomach su rgery or use of antacids or anti-ulcer medications. CHOLERA IN THE UNITED STATES TODAY Al t h o u gh ra re , cases of ch o l era do occ a s i on a lly occur in the United States (Figure 4.3). In 1991, t h ree cases of ch o l era were found in Ma ryland. They were assoc i a ted with the consu m pti on of frozen coconut milk imported from Asia. The affected individuals had not traveled out s i de the United S t a te s . Th ey had not eaten raw shellfish in the preceding m on t h . However, all of the affected individuals had atten ded the same priva te party wh ere they ate crabs and ri ce pudding with coconut milk. Un opened pack a ges of the same brand of coconut milk (but a different shipm ent) wh i ch had been i m ported from Asia were ex a m i n ed . Ch o l era bacteria were found within the food .

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Figure 4.3. The number of cholera cases in the United States has increased slightly since the 1960s. However, many of the cases were due either to food imported from other countries or travel outside the United States.

In this same year, 16 other cases occurred in the United States. All of the individuals affected had recently traveled to ether South America or to Asia. Two of these were infected with the same biotype as those infec ted in Maryland from the coconut milk imported from Asia. Other outbreaks included isolation of Vibrio cholerae O1 from oysters in Mobile Bay, Alabama in 1991–1992. International travel has led to an increased number of cholera cases in the United States. In 1992, about one case of cholera was being reported each week. Here is an example of such a case, as was discovered by the Connecticut Department of Health. A 43-year-old woman traveled with her two teenage daughters to Ecuador over the Christmas holidays. The mother ate raw clams and one of the daughters ate shrimp. The next evening, the mother ate cooked crab and lobster, and the same

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teenage daugh ter ate cooked crab. The other daughter ate no seafood at all during the trip. The mother had onset of vom i ting, cramps, and diarrhea 16 hours after the secon d meal. Twelve hours later the older teenage daughter developed similar sym ptom s . The yo u n ger daugh ter did not get sick . Both the mother and the older daughter were tre a ted with i n travenous fluids and anti m i c robial med i c a ti on . Toxic El Tor vi bri o bacteria were isolated from both individuals. Although ch o l era is ra re in the United State s , it is still po s s i ble to con tract the disease. The most com m on methods of transmission, especially for people r esiding in developed co u n tries with good sanitati on methods, is by eating contaminated seafood and/or traveling to regions where cholera is common. However, despite the few recent outbreaks, cholera is not an immediate public health problem in the United States as it is in many undeveloped countries. RECENT DISCOVERIES Detecti on of ch o l era bacteria has improved with modern technologies. Scientists can attach special fluore s cent dye s to monoclonal antibodies, which bind to Vibrio cholerae and allow the bacteria to be visualized. DNA techniques are highly selective and allow detection of very few cells in water samples. Microorganisms that cannot be grown in the laboratory can also be isolated by utilizing special techniques. Scientists have also discovered that cholera outbreaks are related to the El Ninõ effect. The El Ninõ phenomenon is a warming of surface waters in the Central Pacific. Recently, it has been observed that the surface temperature and the cholera case numbers in Bangladesh are correlated, and this has been con f i rmed using satell i te rem o te sensing of these are a s . A variety of sciences—ecology, epidemiology, oceanography, marine biology, astronomy, and medicine —are being used to gain new insights into the ways that cholera epidemics may occur and how they can be tracked.

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5 Signs and Symptoms of Cholera THE GENERAL PATTERN OF CLASSIC CHOLERA After cholera bacilli infect and establish themselves in an individual, there

is a period of time from one to three days (called the incubation period) before symptoms appear. The first symptoms of classic Vibrio cholerae i n fecti on are the rapid but painless on s et of w a tery diarrhea and vomiting. Loss of fluids thro u gh the stool can be cop i o u s . As mu ch as one liter of f luid can be lost per hour, even though it is of ten much less. Along with water, the pati ent will also lose essential salts su ch as sodium and potassium (Figure 5.1). Diarrhea is caused by adherence of Vibrio cholerae bacteria to the epithelium of the upper small bowel. The microbes do not appear to invade the intestinal cells and tissues, and there is no visible damage to the mucosal cells of the intestine. In addition, the bacilli do not usually enter the bloodstream, which would induce a condition known as bacteremia. The watery stools of ten contain wh i te flecks of sloughed of f ti s sue and wh i te blood cells, and are referred to as “ri ce water stoo l s” because of this appeara n ce . This loss of f luid leads to an intense thirst wh i ch begins on ce the amount of lost fluid equals two to three percent of the pati en t’s body weight. Severe dehydra ti on can be preven ted with rehydra ti on t h era py, d i s c u s s ed in more detail in Ch a pter 8.

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Figure 5.1. Cholera affects many areas of the body from its time of entry through the mouth until it exits the body through watery diarrhea. As is shown in this diagram, after cholera enters through the mouth (location 1) it can cause hypovolemic shock, vomiting, diarrhea, and muscle cramps.

The first sign of dehydration is the loss of skin elasticity. This is due to the loss of fluid from the subcutaneous tissues ( ti s sue ri ght underneath the skin). The skin wi ll lose its el a s ti city when five to ten percent of the patient’s body weight

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has been lost in fluid. If one picks up a fold of skin, it does not fall quickly back into place. This is loss of skin turgor. This is most easily seen in healthy individuals who show loose skin over the abdomen. Wrinkling of the skin on the fingers is also commonly observed. Eyes often appear sunken as a result of loss of skin turgor, too. However, sunken eyes as a result of a loss of skin turgor may not be a sign of dehydration in starving children or in the elderly because it is also a sign of starvation. Wh en the amount of lost fluid re aches more than ten percent of the body wei gh t , bl ood vo lume levels drop, s pecifically in the serum. This is called hypovolemia . Th ere is a serious loss of fluid in the extracellular compartments of the body and also in the volume of circulating blood. The lost electro lyte s , in additi on to sodium and potassium, i n clude bi c a rbon a te and chloride. Bl ood pre s su re drops and the pulse ra te may be gre a ter than 100 beats per minute . Th e a rm pulse is low, som etimes not even detectable. Pulses at the fem oral artery in the leg region and the caro tid artery in the neck regi on are usu a lly sti ll pre s en t , h owever. The arms and the legs become cold. The rectal temperatu re is of ten el eva ted . The fingers become shrivel ed and wri n k l ed , a ph enomen on call ed “washer wom en’s hands.” The tips of the tongue and lips may be blu e , the mouth is dry, and the eye s are su n ken into their sockets. The voi ce is hoa rs e . Pa ti en t s complain abo ut pains and cramps in the arms and legs , and s om etimes even in the mu s cles of the abdom en. Breathing is labored, the rate of respiration is up to about 35 breaths per minute, and often patients breathe with deep gasps. Bowel sounds occur and they may vary from infrequent and somewhat mild to frequent and active . The abdomen is not usually tender. Patients with severe cholera usually remain conscious but occasionally coma occurs. The loss of el ectro lytes in watery stools leads to other signs of cholera, if untreated. There is concentration of blood

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in the serum. Oliguria, which refers to urinating less than usual, occurs. A c i d o s i s , a lowering of the pH of blood plasma, occ u rs as well. Acidosis is re s pon s i ble for the labored and erra tic bre a t h i n g. An o t h er re sult of hypovo l emia is po ssible renal failu re (failu re of the kidneys ) . Hypoglycemia, the lowering of the bl ood glu cose levels, occ u rs as a com p l i c a ti on of many diarrheal diseases, inclu ding ch o l era. Half of ch i l d ren with cholera are hypoglycemic. Forty percent of children who re ach this stage of the disease wi ll die. The re a s on for this com p l i c a ti on in some but not all severe cholera patients is not known. It may reflect a d i f feren ce in nutri ti on , f a s ti n g, or en z yme failu re s . Th ere is som etimes ob s erved edema (swelling) of lung ti s su e . Erratic heartbeats som etimes occur. Loss of potassium ions will cause paralysis and abdominal ex ten s i on . This is most com m on ly seen in children. Seizures of u n k n own origin sometimes occur in children, also. The fetus of pregnant m o t h ers with cholera will die about 50 percent of the time during the third trimester. Most deaths from cholera occur within the first 24 hours of i n fections. However, nearly all of these horrid signs and symptoms can be avoi ded with proper and ti m ely treatment. VARIOUS MANIFESTATIONS OF CHOLERA Cases of ch o l era may va ry in severity. Some cases are mild, and the body is able to recover in three to four days . In this case, the disease is called a self-limiting infection. More s evere cases can re sult in death in 50 percent of the cases if the pati ents are not tre a ted. Death is mainly the re sult of s evere dehyd ration. If the pati ent receives proper tre a tment, the fatality ra te is less than one percen t . Most pati en t s who recover from the disease get rid of the bacteria in t h eir bodies in two weeks or less. However, some do not,

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and these people become c a rri ers. Th ey harbor microbe s in bile du cts and shed them into their fecal materi a l . In this way, they can supply cholera bacilli to others in the pop u l a ti on , even though they them s elves do not have symptoms of the disease. THE HOST-PARASITE RELATIONSHIP The relationship between a host and a parasite is a com p l ex and del i c a te balancing act. A good analogy is that the host and parasite are children on a see - s aw, with the host on one side and the parasite (microbe) on the other. The balance can be ti pped to favor either the host or the microbe. If the m i c robe is particularly stron g, or “virulent,” or exists in parti c u l a rly high nu m bers, it may get the upper hand, l owering the patient’s resistance, thus increasing the ch a n ces of illness. If the patient has lost his ability to combat infections, the microbe is stron gly favored. On the other hand, if the i m mune sys tem is strong and healthy or if the patient receives tre a tment, the balance may be re s tored, the patient gets the upper hand, and the illness wanes. Obvi o u s ly this rel a ti onship is not that simple, s i n ce so many factors come into play. For instance , m i c robe s m ay possess the abi l i ty to form toxins, they may have a very rapid growth ra te , t h ey may be inva s ive , t h ey may avoi d the p h a g ocytic cells of the immune defense sys tem, or they may be able to ad h ere to a specific site in the pati en t’s ti s su e . Som etimes the gen etic makeup of the microor ga n i s m ch a n ges and it becomes more virulent. In con trast, human factors that play a role in this relationship and may be characteristics of the host inclu de general health, a ge , the quality of nutriti on , the normal flora, (bacteria that normally re s i de in the host) a strong immune sys tem , and the tre a tments the host might receive .

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Signs and Symptoms of Cholera CASE STUDIES In 2001, case reports from three pati ents with cholera were presented in the Centers for Disease Control and Prevention’s online journal, Emerging Infectious Diseases.3 It appeared that a new type of cholera had emerged. The case involved two twin boys. Both boys died despite being treated with antibiotics (penicillin and gentamicin). The first boy died within three days of his birth and Vibrio cholera O1 was isolated from his blood. The second boy followed the same course of the illness and died two days after his brother. The second child’s blood sample was negative. The mother did not have any diarrhea and doctors were unable to collect a stool sample. In another case, a 65-year-old woman fell ill with profuse watery diarrhea and vi s i ted a rural health cen ter. She was fed i n travenously but was not given anti bi o ti c s . Her diarrh e a s topped and she was sent home. However over the next t h ree days , she devel oped anu ria (inabi l i ty to uri n a te ) , confusion, and ch i ll s . Wh en she was ad m i t ted to a larger h o s p i t a l , she had no fever, but was dehyd rated, con f u s ed, and in shock. She was given intravenous rehyd ration and anti bi o tics (chloramphenicol and gentamicin). Blood te s t s revealed elevated wh i te bl ood cells, lowered sod ium and potassium, and elevated urea. Vibrio cholerae was grown from her blood sample. This strain was sensitive to erythromycin, but resistant to many other antibiotics. The antibiotic therapy was changed. After treatment with eryt h romycin, the bl ood cultures, rectal swab, and urine culture were negative. She was rehyd rated and had good urine output but remained in renal failure. She died 14 days after being admitted to the hospital.

3. Gordon , Melita A., et al. “Th ree Cases of Bacteremia Ca u s ed by Vibrio cholerae O1 in Bl a n tyre, Malawi.” Em erging In fe ctious Di se a se s, vol. 7, no 6. (2001).

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Finally, a 45-year-old woman with profuse, watery diarrh e a was admitted to the hospital. She was dehyd ra ted and had no fever but had an erratic heart be a t . She was given oral rehydration therapy. When her diarrhea became bl oody, doctors took a cultu re and began anti bi o tic thera py. Th e d i a rrhea s topped over the next 36 hours and she co u l d m ove abo ut , but on day four she su d denly collapsed and died. After her death, Vi b rio cholerae O1 was isolated from her blood. A s tool sample was not co ll ected. As shown in the previous ex a m p l e s , not all pati en t s su f fering from ch o l era have the same sym ptom s . These cases are the first in which b a c t e re m i a (bacteria present in the blood) was observed. Each case was different from the others.

CHOLERA AND THE BODY Symptoms of cholera are similar to other infections by microbes that cause diarrhea. These diseases are also caused by ingesting water, food, or any other material contaminated by the feces of a cholera victim. We will probably never know the effects of cholera throughout history. We can speculate that impact of cholera and cholera-like infections must have been considerable in historical times. Historians have estimated that crusaders of the eleventh to t h i rteenth centuries were defeated by bacteria more than by the Saracens. Napoleon’s soldiers retreating from Russia were decimated by infectious diseases which gave them diarrhea. Researchers have documented that President James K. Polk died of cholera in 1849. Another death from cholera is more controversial. The Russian composer Peter Ilych Tchaikovsky (Swan Lake, Sleeping Beauty, The Nutcracker) died of cholera. A troubled man, some have suggested that he drank contaminated water intentionally, thus committing suicide.

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One pati ent did not su f fer from diarrhea, one su f fered from on ly short-lived bl oody diarrhea, and another su f fered renal f a i lu re after tre a tm en t . These cases remain mys teri e s . We do not know why the same microbe wi ll act so differen t ly in d i f ferent peop l e , and why the bacteria that was isolated did not re s em ble the type of ch o l era bacteria that has been isolated from patients in the past. It is possible that cholera is tru ly an em er ging infecti on , and the bacteria have fo u n d n ew w ays to overcome the host and to de s troy the del i c a te h o s t - p a ra s i te balance.

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6 The Virulence of Vibrio cholerae THE DEFINITION OF VIRULENCE The virulence of a microorganism describes its ability to cause severe

disease. A disease or the state of having a disease is a condition whereby the phys i o l ogy of the body is disti n ct from a norm a l , h e a l t hy state . P a t h o g e n i c refers to a microor ga n i s m’s abi l i ty to cause su ch disease symptoms. A microbe can be pathogenic but not virulent if it causes a mild form of a disease. This would be a form of the disease with less harmful or uncomfortable symptoms. General features of pathogenic or viru l ent microorganisms are (1) the abi l i ty to attach to host cells, (2) the ability to escape host defenses, (3) the ability to obtain essential nutrients, and (4) the ability to produce symptoms. WHAT ABOUT THE CHOLERA VIBRIO? Vibrio ch olerae b acteria adhere to the villi which line the small inte s ti n e ( F i g u res 6.1a and 6.1b). The bacteria have special filaments that recogn i ze carbo hydra te receptors on the su rf ace of the villi. The ch o l era bacillus produ ces a toxin (abbrevi a ted CT, for ch o l era toxin) wh i ch binds to cell receptors (gangliosides ) made of the glycolipids . These gangl i o s i de receptors are call ed GM1 to identify them as a specific kind of gangl i o s i de with a known ch emical stru ctu re . One part of the toxin produced by the cholera bacilli is an en z ym e . When the en tire toxin binds to the receptors , the enzyme porti on is removed and en ters the host cell . In s i de the cell , CT causes an increase in a ch emical called cyclic AMP (cAMP).

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Figure 6.1a. Vibrio cholerae attaches to the wall of the small intestine and causes increased mucous production. This electron micrograph shows a section of the intestinal wall.

THE WORKINGS OF CYCLIC AMP IN CHOLERA BACILLI The ch emical cAMP appe a rs in small amounts in cells in order to sti mu l a te various pro teins that re s pond to out s i de signals such as hormones. It is formed from ATP, the common energy-carrying molecule in cells, and can be quickly removed by conversion to AMP after an enzyme attack. However, while the cAMP is pre s en t , it can sti mu l a te pro teins in cells. For

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Figure 6.1b. Microvilli in the small intestine increase surface area, which is ideal for nutrient absorption under normal circumstances. However, microvilli also provide an ideal place for the cholera bacilli to attach.

example, it can stimulate cells to degrade more sugars and to form more ATP. A sudden stimulus, such as the need to move rapidly in response to a threat, can request cells to supply a source of ch emical en er gy ra p i dly. This is often referred to as the “fight or flight” response in animals. AMP serves to stimulate the breakdown of glycogen and the form a ti on of glu co s e , which is used as a source of quick energy. In gut cells, the response to cAMP is to change ion transport. The body must activa te the enzyme adenyl cyclase in order to form cAMP from ATP. The cAMP is then degraded by

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another enzyme, phosphodiesterase, to form AMP. AMP can be recycled in cells to form more ATP at another time as needed. If the cAMP remains high, the cell will be stimulated to excess. The reactions can be summarized in this way: ATP

cAMP adenyl cyclase

AMP phosphodiesterase

Adenyl cyclase is membrane-bound; it is activated by neighboring receptor sites that have received some signal in the form of a molecule binding to that receptor site. This changes the shape and activity of the bound adenyl cyclase so that it can form cAMP from the ATP that is usually found in most cells. Phosphodiesterase can be inhibited by ch emicals that are similar to cAMP in structu re . One su ch ch emical is caffeine, a major com pound in cof fee . Wh en too mu ch caffeine is consumed, the phosphodiesterase can be blocked and cAMP accumulates. This can result it too much stimulation. That is what coffee nerves are all about! Cyclic AMP inside inte s tinal cells over- s timulates the sodium pumps located in the cell membranes of intestinal cells. There is first an outpouring of sodium ions (Na+), and then of chloride ions (Cl-). This creates an imbalance of ions across the cell membrane. In order to correct this imbalance, water flows across the membrane from the cells into the lumen, the intestinal tract space. This is the source of the diarrhea in cholera infections. WHAT OTHER PROPERTIES HELP MAKE CHOLERA BACILLI VIRULENT? In addition to toxin, cholera bacilli have pili, the short hair-like a ppen d a ges on bacterial cells that are similar in stru ctu re to f l agella, but much shorter. They serve to help bacteria adhere to a su rf ace , and they may be sites for the attach m ent of

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bacterial viruses in some cells. It is an important virulence factor for the cholera bacillus since it helps attach the bacteria to intestinal cells. The cholera bacillus has a sheathed flagellum at only one end, meaning it is single-polar. This can help the microbe to escape cells that would digest it, as well as to move towards s o u rces of nutrients. The microbe produces proteins which can clump red blood cells, hemagglutinins, and mucinase, an enzyme that can break down mucin, a protective cell material. The mucinase helps the microor ganism to break down a protective layer surrounding the intestinal cells, thus aiding its penetration into a cell. The role of hemagglutinins in virulence is less clear. THE TOXIN Of all the virulence factors, the formation of the adhering pili and the formation of toxin are most essential for the pathogenicity of the cholera bacillus, i.e., its ability to make people ill. The toxin is essential for the major symptoms of cholera. This toxin is oligomeric, meaning that it is composed of several proteins. It is secreted across the outer bacterial membrane of the Gram negative cholera bacilli into the external environm ent wh i ch su rrounds the bacterial cells. Two pro teins form a structu re called the A su bunit. Five other proteins form a porti on of the toxin called the B su bunit. Each pro tein in the B subunit is identical and forms a pentagonal (five - s i ded) structure with a hole in the center and is therefore referred to as a “donut.” There are two parts to the A protein, subunit A1 and subunit A2. These are connected by a disulfide bond. Subunit A 2 is a long protein docked within the “donut” of the B portion of the proteins. It has a structure called an α-helix (alpha-helix). This structure is a coiled configuration of the protein chains; each third amino acid in the sequence is held by hydrogen bonds to the first amino acid in a chain. It is often found in proteins as part of their structural architecture. This

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Figure 6.2. Nutrients enter and exit the intestine through specialized pores in the intestinal wall. Some pores will only allow specific ions, such as chloride or sodium, to pass through, while others are less selective.

p a rt of the A pro tein anch ors it to the B pro teins. The A1 su bunit is potentially an enzyme once it has been freed from the A2 protein anchor. The B pro tein pentamers bind to the receptor site of the i n te s tinal cells. A1 is then cut off f rom the rest of the pro tein and enters into the host (intestinal) cell. It is now an enzyme. The remaining toxin pro teins now en ter into the cell ( F i g u res 6.2 and 6.3).

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Figure 6.3. The cholera toxin enters the intestine by binding to the cell wall. This process involves the binding of the entire complex (both A and B subunits) and then the splitting of the individual subunits to form reagents that will help it to cause damage to its host.

THE STRUCTURE OF THE TOXIN The ch emistry and stru ctu re of the ch o l era toxin are well k n own. The pro tein has a mass of 85 kilod a l tons and is comprised of 755 amino ac i d s . The A su bunit is 27,234 daltons in mass, while the B su bunits are 11,677 daltons each . F ive of

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these su bunits com prise the total B su bunit, 58,307 dalton s in mass. The en ti re toxin, or holotoxin, is 85,620 daltons. Th e A su bunit is made of 240 amino ac i d s . It is cut by an enzyme that attacks pepti de bonds in the center of amino acid s equ en ces in pro teins (endopeptidase) bet ween amino acids nu m ber 193 and 195 to form the A1 and A 2 subunits. The A1 pro tein is wed ge - s h a ped and is enzymatic. The B pro tein is more stable to ch a n ges in the envi ron m ent than is the A pro tein, which is more loo s ely fo l ded. The B pro teins bind to the carbohyd rate chain on the ganglioside GM1. There are five carbohydrate sugars in this chain, and each one binds to a B protein. There is little change in the B protein after it is bound to the ganglioside. THE FUNCTION OF THE TOXIN A1 is an enzyme. It is an ADP ribosyl transferase. The substrate of this enzyme is NAD (nicotinamide adenine dinucleotide). This is a major electron carrier in cells, vital for energy metabolism. Therefore, it is produced in rather significant amounts in the cells. The A1 enzyme splits the NAD into two portions of this molecule: nicotinamide and ADP-ribose. The ADP-ribose portion is then attached to a portion of the receptor protein on the intestinal cells, thereby altering its activity. This receptor protein is called a G protein, because it uses GTP for its activity and control. The G protein is composed of three subunits called beta (β), alpha (α), and gamma (γ). After a hormone binds to a cell, this protein complex binds to GTP, and then splits into a portion composed of beta and gamma, and another portion composed of alpha and GTP. These may recombine, lose a phosphate group, then release the GDP thus formed, and return to the hormone receptor site for reuse. The alpha protein with GTP stimulates the adenyl cyclase so that cAMP is formed. When the cholera toxin is present, it attaches the ADPribose from NAD to the alpha-GTP protein, preventing it from

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recycling by release of a phosphate group. This inactivated alpha-GTP protein promotes dissociation of the G protein complex and inactivates an enzyme that can convert GTP to GDP (GTPase). The result is that cAMP formation continues, while the ability of the cell to turn off the formation of cAMP is lost. The G protein can no longer be recycled and the formation of cAMP continues. After toxin binding, it takes about 15 minutes before there is a rise in cAMP. This may be the time that the A protein is transported across the membrane and is split to form the free A1 subunit. The alpha subunit of the G receptor protein binds to GTP, and this sti mu l a tes the sod ium pump in gut cells. When it is inactivated by the ch o l era tox i n , the alpha-GTP protein is stabilized while the GTPase activity decreases. The result is that the amount of cAMP increases. GTP and Mg++ are needed for the action of the toxin. GTP is needed on the complex, and the magnesium is required by enzymes that can synthesize GTP. THE CHOLERA TOXIN AS A REAGENT Since the mechanism and structure of the cholera toxin are well known, the toxin has been used as a reagent. A reagent is a substance used to test for the presence of other substances in a solution. In the case of cholera toxin, these uses exploit the ability of the B protein to bind to specific cells and to deliver a protein to those cells. If the A protein is removed and another protein substituted, then this B protein can be used as a reagent to deliver proteins other than the A1 protein subunit to the interior of cells. It has been used to study ganglioside receptor sites, which are found in high concentration in membranes of neu ron s . S c i en tists have discovered that another protei n , myelin basic protein, can be ribosylated by cholera toxin. This protein is a major part of myelin, which is found associated with nerve cells. Cholera toxin can be used to study myelin and myelin defects in nerve cells. Thus, a good understanding of

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this protein has made it useful for the study of other areas of the biological sciences. Once the cholera toxin binds to gut cells, it sets in motion a chain of events which disrupts normal cell functions. Cyclic AMP is a vital intermediate for hormones to instruct cells to perform specific tasks. The control of levels of cyclic AMP in intestinal cells by cholera toxin interferes with the levels of cyclic AMP formed, as well as the control of that formation.

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7 The Genome of Vibrio cholerae THE KNOWLEDGE PROVIDED BY AN ENTIRE GENOME In the August 3, 2000, issue of the journal Nature, a research team at The

Institute for Genomic Research (TIGR) in Maryland published the entire nucleic acid sequence of the genome of Vibrio cholerae. Determining the complete genetic code for any organisms is a major research breakthrough for understanding that or ga n i s m . It of fers the blu eprint for all of the products that the organism can produce. More often, scientists will discover completely new genes in the process. In many cases, the functions of these genes are not yet known. The data of the nu cl eic acid sequ en ce can be ex a m i n ed in a vari ety of w ays . One way is to com p a re sequ en ces with those from rel a ted organisms, which may allow possible amino acid sequences of unknown pro teins to be determ i n ed . An important goal is to de s c ri be the annotated sequence for the gen om e , a de s c ri pti on of the functi on s for the genes of an or ga n i s m . In this way, s c i en tists can discern i n formation abo ut likely structu ral fe a tu res of the proteins that can be formed by the or ganism. For example, some nu cl eic acid sequ en ce p a t terns stron gly indicate that a regi on of a pro tein may be fo l ded into a α- h elix s tructu re . The nu cl eic acid sequ en ce data can be ex a m i n ed for po tential evo luti on a ry origins of genes. THE UNIQUENESS OF THE GENOME OF VIBRIO CHOLERAE The gen ome of the ch o l era bac i llus is com pri s ed of t wo circ u l a r chromosomes. This is unusual, since most bacteria have a single circular

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ch rom o s om e . Ch rom o s ome 1 has 2,961,146 nu cl eic ac i d base pairs, and Chromosome 2 has 1,072,914 base pairs. These base pairs comprise a total of 3,885 open reading frames (ORFs). An open reading frame is a coding sequ ence bet ween an initiator and a terminator codon. It is necessary to find the sequence of bases which is the site for ribosome binding and which precedes the initi a tor codon . Open re ading frames all ow scientists to locate numbers of gene groups in an organism. HOW ARE GENES DISTRIBUTED BETWEEN THE TWO CHROMOSOMES? On Ch rom o s ome 1, 58 percent of the 962 ORFs code for pro teins that are known, and six percent code for pro teins that are known but for wh i ch there are no known functi on s . Seven teen percent of the ORFs contain sequ en ces similar to other known ORFs, but scien tists do not yet know if cholera bacillus actually makes these gene products. Nineteen of the ORFs on Ch rom o s ome 1 code for pro teins that are com p l etely unknown. Forty - t wo percent of Chromosome 2 ORFs code for known pro tei n s , and six percent code for pro teins with no known functi ons. F i f teen percent of Chrom o s ome 2 sequen ces are similar to those of other ORFs, but products of these genes have not been observed in this microorganism to date. Thirtyeight of the ORFs of Ch romosome 2 code for completely unknown gene products. G enes requ i red for growth and vi a bi l i ty are mostly l oc a ted on Ch rom o s ome 1, while genes coding for som e ribosomal pro teins are found on Ch rom o s ome 2. Ch rom osome 2 also codes for some metabolic pathway intermediates. Ch romosome 2 has a DNA coding sequ en ce for a segment called an integron island. This is a sys tem of pro teins wh i ch a ll ows the captu re of forei gn gen e s . G enes found here inclu de those for drug re s i s t a n ce , for potential viru l en ce genes (hem a gglutinin and lipopro tei n ) , and for gen e

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products used by plasmids wh i ch allow them to su rvive in host cells without damage . P l a s m i d s a re ex tra - ch rom o s omal gen etic el em ents in b acterial cell s . In some cases, D NA from the plasmid can become integra ted into the DNA of the bacterial cell ch rom o s om e . In formati on on plasmids can give new characteristics to a bacterial cell . These may inclu de drug re s i s t a n ce and en z ymes for degrad a ti on of certain su b s t a n ces in the envi ron m en t . Chromosome 1 contains genes for DNA replication and repair, transcription, translation, cell wall biosynthesis, and a variety of metabolic pathw ays. Most genes that are known to be required for pathogenesis are located on Chromosome 1. Chromosome 2 contains more genes of unknown function. Ma ny of these genes are loc a ted in the integron island of the chromosome. The cholera toxin gene is similar in structure and function to a toxin gene from pathogenic strains of Escheri chia coli. However, the cholera toxin can be transported outside of the bacterial cell while the toxin from E. coli cannot. Re s e a rchers have su gge s ted that Ch rom o s ome 2 was origi n a lly a large plasmid. Th ere are plasmid-type sequ en ce s and sequ en ces unlike those from similar bacteria loc a ted on Ch rom o s ome 2. The integron island loc a ted there is similar in sequence to those often found on plasmids. One possibility is that this early plasmid acqu i red genes from other species, but did not integra te those plasmid genes into the DNA of Ch rom o s ome 1. S c i en tists have speculated that uneven segregati on at cell divi s i on could form cells with Ch rom o s om e 2, but not Ch rom o s ome 1. Such cells could not replicate, but they could have metabolic activity and be a source of viable cells that cannot be cultu red in the labora tory. Such cells might form and su rvive within b i o f i l m s . Genes for regulati on pathw ays are divided abo ut equally between the two ch romosomes.

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The Genome of Vibrio cholerae THE RELATIONSHIP BETWEEN THE BACTERIAL VIRUS AND THE GENE FOR CHOLERA TOXIN Certain viruses, called bacteriophages , i n fect bacteria and i n tegrate their genetic material into that of the bacterial cell. As the bacterial DNA replicates, the integrated bacteri oph a ge DNA is also cop i ed. On occ a s i on , the virus genome may be excised, or cut out , with enzymes from the bacterial DNA . The bacteri oph a ges may then lyse (bu rst) bacteria that they have infected and reproduced within, so that they can release the new viruses into the envi ron m en t . This can be indu ced by some ch emicals, tem peratu re changes, or even UV light. Viruses may tra n s fer DNA from the bacteria it first infected i n to the DNA of any additional cell that the virus infects. This is one way of exchanging gen etic material in bacteria, and it is called transduction. The process by which virus DNA is integrated successfully into host bacterial DNA is call ed lysogeny, and the bacterial vi ruses engaged in this process are call ed temperate b acteri ophages. If the genes from the bacteriophage introduce genes wh i ch give the new recom binant bacterium new ch a racteri s ti c s , or phenotypes, this is referred to as lysogenic conversion, since the bacteria have been converted into a new phenotype as a result of infection and lysogeny. Some bacteri oph a ges are filamen to u s . S trands of nu cl eic acid are su rro u n ded by a pro tein coa t . These ph a ge s of ten do not harm the bacterial host and are lys ogenic. They m ay bind to the host at a pilus. One su ch virus has been found in Vi b rio ch ol era e. It is the bacteri ophage CTXØ wh i ch has the genetic code for CT in its genome. This vi ru s uses a special pilus called a toxin-regulated pilus (TCP) as its receptor on the ch o l era bacillu s , s i n ce both the ch o l era toxin and the pilus are reg u l a ted by the same gene (toxR). It was ob s erved that this bacteriophage infects Vi b rio ch olerae m ore of ten within the inte s tinal tract of m i ce than it doe s u n der con d i ti ons in the labora tory. Th erefore, producti on

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Figure 7.2. Some scientists have hypothesized about a virulent aspect to cholera transmission, as is depicted in this diagram. It is possible that the cholera bacterium uses a virus to help get inside its host cell.

of ch o l era toxin by Vi b rio cholerae is a result of lysogenic conversion by phage CTXØ. It is likely that other filamentous phages may also be respon s i ble for transfer of genetic material between different strains of bacteria. (Figure 7.1). In one sen s e , it may be stated that ch o l era is caused by a virus!

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The genes invo lved in TCP form a ti on re s i de on a pathogenicity island on Chrom o s ome 1. This region also codes for other genes under con trol by the toxR regulatory pro tein and a site for ph a ge integra ti on . This may be the s i te for integra ti on into the whole ch rom o s om e , wh en this regi on of D NA was first tra n s ferred from another b acterium to the ch o l era bac i llu s . THE LOCATION OF THE GENE FOR CHOLERA TOXIN The gene for cholera toxin (ctxAB) is located on Chromosome 1 within a genome for a temperate filamentous phage CTXØ. The gene clu s ter for the pilus nece s s a ry for en try of t h i s filamen tous bacteri ophage and the regulator gene for toxR, the pro tein that regulates toxin producti on, are also located on Chromosome 1. HORIZONTAL TRANSFER Horizontal transfer is the process of gene transfer from bacterium to bacterium inste ad of transfer from bacterium to progeny (vertical transfer). In the example of the cholera b ac i llu s , tra n s fer is med i a ted by a bacterial vi ru s . This is a source of va ri a bi l i ty in pop u l a ti ons of this pathogen i c b acterium. The new strains of cholera that emerged in 1992 re su l ted after acquisition of n ew genetic material. This tells us that horizontal gene tr ansfer can create new strains of a pathogen. This could hamper the development of strains for use in vaccine preparations as well as in the use of antibiotics. Genes resistant to antibiotics can be transferred in this manner and thus arise in populations rapidly. ADDITIONAL UNIQUE FEATURES OF THE VIBRIO CHOLERAE GENOME Both pathogenic and non-pathogenic strains o f the cholera bacillus have gene sequences called PilD (also called VcpD). This sequ en ce determines a pro tein that is requ i red for

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secretion of CT as well as for the assem bly of MSHA (mannoses en s i tive hemagglutinin). MSHA is not a virulen ce factor, but it is important in the formation of biofilms. Biofilms are communities of bacteria in nature, and biofilms containing ch o l era bacilli have recen t ly been described. The abi l i ty of the bacteria to form biofilms is important for the survival of Vibrio cholerae in nature. Ch o l era vibrio live two lifestyles: one in nature and one in a host. The PilD gene connects both lifestyles, and these are equally important for understanding both pathogenesis of the microor ganism and its mode of transmission in nature. Vibrio cholerae has an unusually large number of MCP genes. MCPs are methyl-accepting chemotaxis proteins. These pro teins regulate the attracti on of the microorganism to sugars, amino acids, oxygen, and other nutrients. Chemotaxis is the directed movem ent of a microor ganism tow a rd a p a rticular ch emical in its envi ron m en t . (This is po s i tive chemotaxis; negative chemotaxis is movement away from an area in the environment.) Escherichia coli are bacteria which h ave five MCP genes. Campylobacterium jejuni, a pathogen that causes stomach ulcers, has ten MCP genes. As a result of determining the DNA sequence of the ch romosomes of this bacteriu m , s c i en tists found that Vi b rio ch ol era e h a s 43 MCP genes. These genes are distributed evenly between Chromosome 1 and Chromosome 2. The genes probably arose by gene du p l i c a ti on . The re a s on(s) for the differen ces in nu m bers is pre s ently unclear. One possibility is that each MCP protein in the cholera bacillus is specific for a specific ch em o t actic ch em i c a l , u n l i ke the MCP pro teins of o t h er b acteria which can sense more than one substrate chemical. An o t h er unique fe a tu re is the pre s en ce of a gene for a toxin call ed RT X . This toxin cross-links actin pro tei n f i l a m ents within the cell . These pro tein filaments give the cell its stru ctu re , so this tox i c i ty re sults in a dra s ti c a lly ch a n ging cell shape . This is an unex pected activi ty for a

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tox i n , and any role it may have in pathogenesis is not clear at pre s en t . The wealth of information found by revealing the genetic code of the ch o l era bac i llus has given scien tists new insight into the workings of Vibrio cholerae. The same information also gives scientists new questions to ponder.

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8 Treatments for Cholera Cholera killed ten percent of the population of St. Louis, Missouri in 1849.

More than half of those su f fering from severe diarrhea died. Nearly 150 ye a rs later, in 1991, cholera attacked more than 300,000 people in Peru, one percent of that nation’s pop u l a ti on. These people devel oped the severe diarrhea which accompanies this infection; h owever, less than one percent of the infected indivi duals died. IMPROVED TREATMENT— IMPROVED CRISIS MANAGEMENT As understanding of the bacillus has improved over the years, with it has come improved ways to treat infected individuals. Even before the cholera bacillus was discovered by Koch and was shown to be the cause of this disease, physicians recogn i zed the import a n ce of replacing fluids lost from the body as a re sult of s evere diarrh e a . Tod ay, rep l ac i n g fluids, as well as the important e l e c t ro l y t e s , the ions dissolved in the l i qu i d s , remains the key to the tre a tm ent of ch o l era . At first f l u i d replacement therapy, wh i ch rep l aces lost body fluids, was perform ed by giving patients hypertonic (as oppo s ed to hypotonic) soluti ons containing a high er amount of el ectrolytes intravenously. The mortality ra tes of cholera patients who received fluid rep l acem ent thera py dropped abo ut 30 percent compared to those who were untreated (Figures 8.1a and 8.1b). ADDITIONAL STEPS IN THE TREATMENT OF CHOLERA

Step 1: Step 2: Step 3: Step 4: 72

Fluid replacement Maintain the level of fluids in the patient Treatment with antibiotics Adequate nutrition

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Figure 8.1a. “Folded skin” often results from extreme loss of fluids associated with cholera. The boy in the picture above has folded skin on his stomach. This happens when skin loses its elasticity, due to extreme dehydration.

FLUID REPLACEMENT In the 1960s, scientists discovered that the transport of sodium and water was facilitated (helped) by the presence of glucose. This led to a simple, practical, safe, inexpensive, and effective 73

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Figure 8.1b. Rehydration therapy is an important step to curing cholera. Nutrients that are lost through diarrhea must be replaced. Special mixtures of salts and electrolytes are ideal and must be constantly administered until diarrhea has stopped. The child p i c t u red above is receiving oral rehydration therapy.

way to treat cholera called ORT (oral rehydration therapy). ORT is an important and valuable medical tool for treating diarrheal diseases, including cholera. The fluids are given orally, but if patients are unable to drink, they may be given fluids intravenously. The World Health Orga n i z a ti on (WHO) recom m en d s a soluti on for ORT wh i ch con t a i n s : s od ium ch l ori de (90 mmol/liter), potassium chloride (20 mmol/liter), glucose

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(111 mmol/liter ) , and sod ium bi c a rbon a te (30 mmol/liter) or sodium citrate (10 mmol/liter). This supplies the appropriate replacement of lost electrolytes as well as a correct pH level. These solutions are provided in sterile ORS (oral replacement solutions) packets. In emerging nations, su ch packets or o t h er sources of s terile ORS may not be re ad i ly ava i l a bl e . However, an em er gency soluti on can be prep a red using 3.5 grams of sodium chloride (abo ut 1/2 teaspoon of table salt) and 20 grams (abo ut 2 tabl e s poons) of su gar into a l i ter of w a ter. These soluti ons have saved nu m erous lives and have halted ep i demics. Al ong with el ectro lyte s , ch o l era pati ents wi ll lose bi c a rbon a te in their stools. Th erefore , it is necessary to also supply an alkaline solution to replace lost bicarbonate. Originally, sodium bi c a rbon a te was used. However, bicarbon a te solutions do not keep for long periods of time, particularly if s tored in hot and humid tropical cl i m a te s . Sod ium citra te has been found to be an excellent substitute. Scientists have compared oral replacement solutions with bicarbon a te and c i tra te , and found them to be equally effective . O ral rehyd ra ti on soluti ons prep a red with su c ro s e , wh i ch is more re ad i ly ava i l a ble than glu co s e , were also te s ted. Su c rose is broken down by enzymes in the inte s tine the body to form both fru ctose and glu co s e . However, these en z ymes may not be su f f i c i en t ly active wh en the p a ti ent has severe diarrh e a . Com p a ri s on studies have s h own that ORS-glucose therapy is slightly more effective than ORS-sucrose therapy. RECENT IMPROVEMENTS OF FLUID REPLACEMENT THERAPIES An o t h er approach to ORT has been to prepare soluti ons in which glucose is replaced by starches and proteins common to the patient’s diet. The idea is that starches and proteins will be digested in the intestine, releasing glucose, amino acids, and

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peptides. These are all organic chemicals that should help the uptake of sodium and water. Cereals, especially rice, are commonly used and should also supply nutrients without an additional loss of fluids. It was shown that ri ce - powder ORS (30 grams of rice powder per liter) was as effective as sucrose ORS. A reduction of d i a rrheal outp ut can also be accom p l i s h ed with this formulation. Other grains, which have been used successfully, are from wheat and maize. Unlike rice powder, the wheat preparation does not have to be cooked. These formulations are very useful and, depending on local conditions, may be used in place of glucose ORT. At the present, there is still a need to reduce the volume output of diarrhea caused by cholera. Some of the current efforts to improve ORS formulations include the substitution of or ganic acids other than citrate, the use of polym ers of glu co s e , and the use of amino acids. Some of these formulations seem promising in clinical trials, but the best ORT is the cereal-based formulations. In emergency situations, “suga r- s a l t” solutions can be easily prepared from commonly available materials to use in therapy. However, this solution has no base (bicarbonate) or

CHOLERA OUTBREAKS The World Health Organization estimates that there are about 100,000 cases of cholera each year. There were 89,714 re p o rted cases through July 2002. This is most likely an underestimate, since many victims without access to medical care go unreported. This fact is all the more remarkable when one realizes that cholera is an infectious disease that can be prevented. Source: World Health Organization

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potassium. A diet su pp l ement of foods rich in potassium is recommended in these cases. These solutions are incomplete and should be used as temporary measures for treatment only. PROPER CARE FOR THE PATIENT Health care workers should evaluate cholera patients carefully in order to be able to determine the proper treatment. For example, caregivers must know how much fluid the patient has lost in order to determine how much needs to be replaced. Patients should also be examined for pulse rate, skin turgor, overall comfort such as nausea and vomiting, fullness of neck veins, and weight. For patients with severe diarrhea, IV-ORT (intravenous oral replacement therapy) is preferable. Severely dehydrated patients can be rehydrated in two to four hours. The degree of dehydration in the patient determines the best re hydration t h era py to use. Dehyd ra ti on is ra ted by clinicians in fo u r groups: (1) no dehyd ration, (2) mild, (3) moderate, and (4) severe dehyd rati on . The mild category patient has lost about five percent of his body weight in fluid and may have somewhat reduced skin turgor. The moderately dehydrated patient has lost seven and a half percent of body weight, has poor skin turgor, a dry mouth and somewhat sunken eyes. Patients who have lost ten percent or more body weight are we a k , s om ewhat uncon s c i o u s , h ave very poor skin tu r gor and very su n ken eye s , and have poor pulse ra te s . Th ey are considered severely dehydrated. The weight of the patient should be noted upon admission for treatment, so that weight gain from rehydration therapy can be observed. Weight can also be used to estimate the percent of dehydration. For example, an individual weighing 50 kg and who su f fers from severe dehydra ti on, wi ll requ i re about f ive liters of f luid to replace fluid loss. (50 kg x 10% = 5 kg.). A child weighing 10 kg with mild dehydration would require 500 ml (1/2 liter) of replacement fluid.

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Figure 8.2. Cholera cots are specially designed so that caregivers can carefully monitor the amount of fluid a patient loses through the course of the disease. The cot contains a plastic sheet with a hole in the middle, which directs diarrhea to a bucket beneath the bed. This allows the caregiver to determine how much rehydration therapy is necessary to stabilize the patient.

STABILIZING THE PATIENT Once fluid rep l acem ent thera py has stabilized a pati ent, it is necessary to make su re no more fluid is lost until diarrh e a ceases. This is call ed maintenance therapy. A spec i a lly de s i gn ed cot, c a ll ed a cholera cot (Figure 8.2), may be used for this purpo s e . This cot has a plastic sheet under the patient. Th ere is an opening in the cot thro u gh wh i ch the plastic sheet directs fluids lost from diarrhea to em pty into a container placed bel ow the cot. This con t a i n eris calibra ted so that an attendant can record

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the amount of fluid lost. This will provide the information n eeded so that the caregiver can give en o u gh fluid to the patient to maintain proper hydration levels. A container for vomit is also provided, as patients lose fluid this way too. The cholera cot provides the information needed t o kee p the patient stabilized until the infection begins to wane. THE USE OF ANTIBIOTICS Antibiotics can reduce the intensity of diarrhea as well as its duration, although they will not cure the patient. Antibiotic treatment should begin from three to six hours after the start of fluid replacement therapy. The antibiotic tetracycline is given at a dose of 250 milligrams every six hours for up to f ive days. There are problems with the use of antibiotics, however. People tend to expect antibiotics to be a permanent cure in a ll cases of infection. This is not the case for cholera. Antibiotics a re ex pen s ive and may not be practical in poor nati on s . An ti biotics m ay also have side effects in many cases. These s i de effects may come at a time when the health of the patient is alre ady compromised. Use of antibiotics may help only a few, and it m ay pro l ong the time in wh i ch those that have been tre a ted can still become infected with the cholera bacillus. Since the benefits of antibiotic therapy are limited, their routine use is not recommended. However, use after maintenance therapy can hasten the healing process. An o t h er major public health probl em is anti bi o ti c re s i s t a n ce , p a rti c u l a rly because this re s i s t a n ce is of ten mu ltiple resistance. Wh en the microbe develops re s i s t a n ce to s everal different types and kinds of antibiotics at the same ti m e , the use of different types of antibiotics wh en needed in critical situ a tions is preven ted. An example of this was experienced in 1998. Up to that time, the Indian Ocean had been free of cholera for years. Then, in January, 1998, an outbreak of cholera occurred. One

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coastal city and another provi n ce were su rro u n ded by a s a n itary barricade. All individuals leaving these areas were routinely given oral doxycycline (an antibiotic). Patients with s evere diarrhea were also given the same anti bi o ti c . In spite of t h i s , ch o l era re ach ed all ten provi n ces on the islands within ten mon t h s . A su rvei ll a n ce team was establ i s h ed , and they found that the strain of Vibrio cholerae there was serogroup 01, s ero type Ogawa, bi o type El Tor. It was resistant to trimethoprim-sulfamethoxazole, sulfonamides, trimethoprim, chloramphenicol, and streptomycin as well as agent 0129, a n a tu ra lly occ u rring ch emical that norm a lly kills ch o l era b acteri a . O f a ll the strains of the ch o l era bac i llus isolated , 55 percent were found to be resistant to tetrac ycline as well as the antibiotics listed above. This pattern conti nu ed as other strains were isolated at other locations on the islands. The proporti ons of i s o l a ted bacteria that were re s i s t a n t to tetrac ycline con ti nu ed to cl i m b. Th erefore aut h ori ti e s recom m en ded that doctors and public health officials 1) not ro uti n ely use anti bi o tics for ch o l era preventi on , 2) use oral rehydrati on thera py for mild-to - m oderate cases, and 3) use antibiotics for cases of severe cholera illnesses only. They also recommended that the areas should be continually monitored for anti bi o ti c - resistant strains, so that em er gency anti bi o tic t h erapy could be used properly wh en needed. Ot h er studies have shown that the re s i s t a n ce genes in ch o l era are carri ed on conjugative plasmids, wh i ch are plasmids needed for bacteria to mate by a process called conjugation, bearing multiple resistance gene locations. Bacterial viruses that kill cholera bacilli have been tried as therapy. This was found not to be as useful as tetracycline treatment. THE ROLE OF NUTRITION The ch o l era patient should be given nutri ti o u s , age - a ppropri a te food, even before the diarrhea stops. This is the final stage of treatment, and this helps the patient return to health.

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F i n a lly, s c i en tists are attem pting to devel op spec i f i c medications to redu ce the debi l i t a ting diarrhea. They have found that chlorpromazine and nicotinic acid are useful in this regard. However, research is still in the early stages, and the mode of action of the drugs and patient responses have not been studied thoroughly. While there will always be searches for better ways of treatment, the methods described in this chapter are the best and most widely used at present.

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9 Prevention and Vaccines CLEAN WATER — THE BEST PREVENTION People who reside in developed nations often take clean water for granted. Yet, clean water and modern sew a ge tre a tment fac i l i ties are the main re asons that cholera is no longer a problem in nations that can afford to maintain sanitary conditions (Figure 9.1). In the United States and other nations, cholera increased as the growth of population centers increased. Even before ch o l era and other infectious diseases were shown to be a s s ociated with contaminated water supplies, many cities developed ways to provide clean water and sewage treatment facilities. Modern and we ll maintained water treatment facilities and sewage tre a tm ent plants are the best preven ti on against ch o l era outbre a k s (Figure 9.2). Where su ch facilities do not exist, as in many underdeveloped nations, water-borne infectious diseases cause problems. It is for this reason that cholera is often referred to as a disease of the poor. Crowded cities and international travel provide other conditions for the spread of cholera. In the United States and other developed nations, there are public health agencies which oversee the maintenance of clean water and sewage treatment facilities. When a cholera case appears in the United States, prompt action by public health personnel, determination and elimination of the sources of infection, and sanitation will prevent an outbreak. This includes the proper disposal of fecal waste to halt transmission of the disease. PREVENTION WITHOUT CLEAN WATER FACILITIES Where there are no clean water facilities, careful food and water handling and prompt medical treatment can still be helpful to prevent and reduce the incidence of cholera. Boiling water, for example, will kill the cholera 82

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Figure 9.1. Now that cholera can be easily identified, signs are used to warn people about contaminated water sources. Proper sanitation procedures, and avoiding contaminated water supplies, can greatly reduce transmission of the disease.

bacillus. However, it is not always easy or practical to sustain the boiling of a ll water supplies. Water is often stored in homes in poorer nations, and it may be dipped out by hand. Thus the water may be contaminated. One simple solution to this practice has been to use water containers with narrow-mouthed openings. These require pouring rather than hand scooping the water. Other efforts are the addition of chlorine to disinfect the water supply. Better attention to production of safe ice supplies is important, and this should also be monitored. Individuals can protect themselves by thoroughly cooking food before eating it. Often, street vendors sell foods such as shellfish, which may have been caught in unsafe waters and therefore be con t a m i n a ted with ch o l era bacilli. Providing cleaner vending carts and the means for their sanitation would be beneficial. All of these met h ods requ i re the education of people in areas prone to ch o l era ep i demics. Even then, the ch o l era bacillus requ i res few lapses in these routine types of sanitation procedures in order for it to start another round of infections. Recen t ly, a discovery has been made that could help con trol 83

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Figure 9.2. Sewage treatment plants prevent contaminated water from affecting the public. Most developed nations have methods to treat drinking water. However, in countries that cannot afford to do this, cholera is quite common.

ch o l era in poor econ omic areas. Dr. Rita Colwell reports that most cholera bacteria are attach ed to the gut of a copepod, a kind of zooplankton found in ponds, rivers , and other standing waters. These or ganisms, along with the cholera bacteria they carry, can be rem oved from water by using a simple filter made of old, much-washed cloth used to make sari s , a common dre s s in southern Asia. Use of this inex pen s ive and available materi a l could be a breakthro u gh in con trol of ch o l era and other rel a ted types of microor ganisms in third world countries. 4 ADDITIONAL PRECAUTIONS Should antibiotics be used as a prophylactic (preventative) measure? Studies in wh i ch family mem bers of ch o l era patients are given antibi o tics (20 doses of tetrac ycline over a five-day 4. As s oc i a ted Pre s s . “Old Sari Cloth Filters Ch o l era, S tu dy Finds.” The New York Ti m e s. Ja nuary 13, 2003. A11.

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peri od) show that there is a 13 percent dec rease in the inciden ce of infection among family mem bers . This kind of treatment may be va lu a ble in isolated envi ronments, su ch as on ships, wh ere the infection might be contained. However, antibiotic prophylaxis will not con trol ch o l era in open envi ronments. In addition, strains with antibi o tic resistance appear wh en there is wi despre ad use of antibi o tic thera py (see Chapter 8). This resistance is of ten to mu l tiple drugs, and thus redu ces the nu m ber and d i fferent kinds of antibi o tics that might have been useful for tre a ting non-resistant cholera bacteri a . The use of antibiotics also allows many people to let down their guard, wanting to bel i eve that the antibiotic treatment is a permanent cure for the infecti on . Th ey may practice good sanitation less stringen t ly. Antibiotic treatment is not a cure and not without side effects in many cases. IS THERE A PERMANENT CURE FOR CHOLERA? Vaccination is the practice of introducing a foreign substance into an organism to elicit an immune response, ideally in order to obtain permanent, or at least long-lasting, protection from the foreign agent. Vaccination against the smallpox virus was demonstrated by previous exposure to cowpox, which is closely related to the human virus. Wh en microorganisms were identi f i ed as the causes of many infectious diseases, scien tists immediately tri ed to make prep a ra ti ons of the infectious agent wh i ch would give immunity while not infecting an indivi du a l . The trick is to modify the infecting agent in su ch a way that it can call up a strong immune response in an individual while not causing the disease. To that end, scientists tried a number of ways to modify or attenuate infectious bacteri a . One attem pt to prep a re su ch an attenu a ted form of the ch o l era bac i llus was prep a red and used as a vaccine the year after Koch iden ti f i ed the ch o l era bac i llus. However, this vaccine was not su ccessful since many people who received the inoculation had a va ri ety of s i de ef fect s . Scientists su s pect that these “attenuated bacterial prepar ations” contained microorganisms other than the ch o l era bac i llus.

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Louis Pasteur thought that live microorganisms made the best vaccines. He encouraged a colleague to develop such a live vaccine. It had been observed that once someone survived a cholera infection, he or she was immune to reinfection. One of Pasteur’s colleagues attenuated cholera bacteria by growing it at 39°C with oxygen aeration. He also prepared a more highly infectious strain by passing it through guinea pigs repeatedly. The microbe would be injected into the animals, then isolated , and then injected into other animals. In this way, a more infectious strain was sel ected. He injected the attenuated preparation and after a short wait, challenged the immunity of the “vaccinated” individuals who had received the attenuated bacteria by giving them a dose of the more infectious bacteria. The results were not promising because the volunteers developed many side effect s .In other words, they were sicker from the vaccination than from the disease! Other investigators tri ed prep a ring vaccines by growi n g cholera bac i lli on agar and then heating them so that they were no longer alive. This process is call ed heat inactiva ti on and is of ten used to attenu a te bacteria. This vaccine was first tri ed in Japan in 1902 with limited success. Sti ll , other researchers attem pted to inactiva te the ch o l era bac i lli using bile. These prepara ti ons were call ed bilivaccines. Wh en used in tablet form they provi ded protection for 82 percent of a population. However, this method failed because of reactions to the bile in the prep a ra ti ons. It was not until the 1960s that interest was revived in the prep a ra ti on of a ch o l era vacc i n e . Investigators tri ed using whole b acterial cells as well as the ch o l era toxin or com pon ents of the toxin for vaccine prep a ra ti on s . S train JBK 70, def i c i ent in both the A and B toxin su bunits (tox A- tox B-) indu ced the form a tion of a n ti bodies that kill the ch o l era bac i llus and for this reason are call ed vi b riocidal anti b od i e s. Prep a ra ti ons using whole toxins or toxin B subunit induced antitoxin in the serum, but it did not last very lon g. Such prep a ra ti ons were found to induce a sec retion of antibodies from inte s tinal cells. This meant that both whole cells and toxin prep a ra tions would be needed for an ef fective vaccine. Pre su m a bly, the antitoxin form ed from intestinal cells preven t s

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binding of the bac i llus. The role of the vi bri ocidal anti bodies in pro tecti on is less cl e a r, since the microbes are ra rely found in the bl ood stream. Prep a ra ti ons for vaccines containing the B su bunit of the toxin as well as whole ch o l era vi brio cell s that have been made inef fective give good protecti on . This prep a ra ti on is given ora lly. Su ch a preparati on is referred to as s y n e rg i s t i c , s i n ce two prep a ra ti ons (the whole bacterium and the tox i n - derived anti gen , wh i ch indu ces anti body form a tion) give bet ter pro tecti on together than each does wh en used alone. The vibriocidal antibody induced by the vaccine for the most part reacts against the lipopolysaccharide antigen. Since both the toxin subunit proteins and the proteins for the stru cture of pili are contro ll ed by the same regulatory protein (TCP-toxic co-regulated pilus), it is thought that the antibody form ed in re acti on to the toxin subunits prevents attachment by the pili. METHODS AND APPROACHES TO VACCINE PREPARATION Vaccines can be prep a red from de ad whole cell s . They may be prepared from one strain (monovalent) or from more than one s train (polyvalent). Vaccines can also be prep a red from fra gm en t s of cells, such as protein subunits of a toxin or outer membrane prep a ra ti ons ri ch in lipopo lysacch a ri des. Toxins can be ren dered into toxoids (toxins modified to remain antigenic but not toxic) by physical or ch emical treatments. Cells may be attenuated in more trad i tional met h ods, such as treatment with aldehydes (such as form a l dehyde or glutaraldehyde), alcohol, and/or heat. Mutant strains of pathogenic microor ganisms lacking genes to determine known vi ru l ence factors can also be prepared . These should remain anti genic, while also remaining non-toxic. More recently, recombinant DNA technologies have been used to prepare such modified strains. APPROACHES TO CHOLERA VACCINE PREPARATION Vaccines with dead whole cells give some protection. A polyvalent preparation for cholera was effective in a little more than

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half of the cases. Preparations of outer membranes given orally gave a good response but were not well studied in field tests. Toxoid vaccines did not provide significant protection. B toxin su bunit prepara ti ons were capable of inducing significant antibody re s pon s e , but the ef fectiveness as a vaccine alon e was not studied thoroughly. Combination oral vaccines were prepared from different sources. One, a preparation of multivalent dead whole cell preparation added to a purified B toxin protein subunit, was shown to be the best. It was effective in more than 60 percent of the time. At first, scientists attempted to use non toxic strains in vaccines. However, natura lly occ u rring and chemically induced mutant strains which lacked virulence components proved d i s a ppointing. One, a strain call ed Texas Star- S R , l acked the a bi li ty to synthesize the A toxin subunit, but could synthesize the B subunit. It was discovered by screening mutants induced by nitrosoguanidine, a mutagen. While this strain was promising, the po s s i bi l i ty of reversion of the induced and unknown mutations was a drawb ack . Scien tists needed to look for more precise methods for prep a ring mutants. New methods emerged with the advent of recombinant DNA technology. With these techniques, scientists can delete s pecific genes that determine the vi ru l en ce properties of certain cells. They can also isolate strains which cannot grow in the intestine. The virulence genes can be removed from cholera bacilli and placed into bacteria which are otherwise rendered harmless. However, these approaches have been somewhat disappointing. For example, genetically engineered strains that cannot form either the A or B subunits of the cholera toxin, and strains that cannot form A, but can form the B subunit of the toxin, were prepared. Both induced loose stools in a significant number of cases. The strain producing the B subunit also produced a hemolysin. The role of this in pathogenicity is unknown. Scientists created a new strain, which they labeled CVD 103. It is A-B+ but also lacks TCP, a colonization factor for pili attachment to intestinal cells. Fu rther, this strain

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was made resistant to mercury (Hg++), so that it can easily be distinguished from a wild type strain of Vibrio cholerae 01. This strain holds promise for future vaccine research. There was mild diarrhea in two percent of the cases tested, and there was a significant increase in vibriocidal antibodies in cases vaccinated using this strain. VACCINATIONS IN THE UNITED STATES The best vaccine prep a ra ti ons available are the oral combination using mu l tivalent de ad whole vi brios and puri f i ed B protei n su bu n i t . However, these vaccines also have drawbacks. They are not as effective in younger children as in adults. The i m mun i ty is of s h ort du ra ti on, l a s ting only six months in many cases. Th ey are expen s ive to produ ce . At least two doses at regular time intervals are requ i red (three are recommended ) . In many devel oping co u n tries it is often impractical and difficult to get a vaccination shot just on ce, let alone twi ce. In the United States, the Public Health Service does not require vaccination for travelers coming to the United States from cholera-infected areas. The World Health Organization does not recom m end ch o l era vacc i n a ti on for travel to or from cholera - i n fected regi on s . Vaccines pre s ent in the United States are prepared from a combination of phenol-inactiva ted suspensions of Inaba and Ogawa classic strains grown on agar or in broth. High risk personnel should be vaccinated, i n cluding at least one boo s ter shot within six months of the first shot. There is no gener al recommendation for United States citizens or residents. SHOULD WE BE CONCERNED? We should be concerned about the welfare of others and about the possibilities of new cholera strains reaching the United S t a te s . An ef fective vaccine will help thousands around the world, a world that is now made very small because of rapid international travel.

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10 Cholera in the Future E. COLI AND CHOLERA E. coli (Escherichia coli) is a microorganism very much in the news these

days. Meat supplies in the United States have been recalled because of contamination, beaches have been closed because of it, and people have died from eating contaminated foods. Pathogenic strains of E. coli produce a toxin similar in chemical stru cture to the cholera toxin. It is likely that this toxin came from strains of cholera bacilli by genetic transfer. Indirectly, then, the cholera toxin is very much a medical concern in the United States. IS CHOLERA AN EMERGING INFECTIOUS DISEASE? An emerging infectious disease is one wh i ch is newly recogn i zed , often as a re sult of human activity. The microorganisms in question may have been around for thousands of years wi t h o ut causing health problems, but may have recen t ly infected humans. A classic example of an em er ging disease is the microbe Legionella pneumophila which causes “Legionnaire’s disease” (a type of pneumonia) after it is inhaled. This microbe was ori gi n a lly found in soil, where it is gen era lly harmless. However, air conditi on ers with holding tanks that opened into the outside environment (as opposed to fully enclosed holding tanks) increased in usage by the 1970s. Legionella pneumophila microbes thrived in the holding tanks and could be spread to humans by droplets. Human beings had created the mechanism and opportunity for an increase in infections caused by this microorganism. Could cholera become an emerging infection in developed countries? New cases have been found in the United States as a result of travel from an endemic area, where cases are found at a constantly low level in the population. These cases have been found to be associated with importing

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and eating contaminated food, and with aging or improperly maintained water purification and sewage treatment facilities. The overuse of antibiotics has created cholera (and other) b acteria that are resistant to this kind of treatment. S c i en tists have recen t ly come to understand that vi brio bac i lli can en ter a non-cultiva t a ble state but sti ll remain infectious. This means that water supplies wh i ch may test nega tive for the presence of these bacteria may, in fact , be unsafe. A new s train of ch o l era bacillus appe a red in Bengal recen t ly. It was shown that this strain (Vi b rio ch ol erae 0139) becomes dormant within a week even at cool tem pera tu res. New strains of cholera bac i lli may be con s i dered em er ging pathogen s . Futu re human activi ties su ch as those that prom o te gl obal warming wi ll also con tri bute to the po s s i ble em ergen ce of ch o l era infections in areas wh ere it has not usu a lly been fo u n d . THE POSSIBILITIES FOR NEW T R E ATMENT METHODS Modern scien tists have a renewed respect for traditional medicines used in different cultures, and this may provide opportunities for finding new medications for treatm ent and preven tion of diseases. In Japan and China, diarrheal diseases have been con trolled by herbal medicine preparations called Kampe formulations for centuries. Recently, the chemicals within these herbal medicines have been isolated and chara cterized. These medicines have been shown to inhibit all cholera toxin activities including ADPribosylation activi ty, the el on ga tion of tissue culture cell s , and the accumulation of fluids in gut prep a ra tions. It is suggested that the most active component of these ancient medications may be added to the latest formulation for oral rep l acem ent therapy in order to help con trol the severe diarrhea of ch o l era. This may become a future treatment for ch o l era. Another novel approach uses more modern recombinant DNA technologi e s . The gene for determining ch o l era toxin has been cut out and spliced into the genes of potatoes. Mice that were fed these geneti c a lly altered potatoes became immune to

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cholera infection. The abi l i ty to incorporate these genes in plants may en a ble doctors to distri bute vaccinations simply by tell i n g their patients which foods to eat. This is another direction scientific research may take in order to improve the treatment and prevention of cholera and other infectious diseases in the future. CHOLERA ON GENE CHIPS Once scien tists had mapped out the en ti re ch o l era gen ome, they could utilize the new tech n o l ogy of gene chips. To create a gene chip, scien tists place tiny dots of DNA segm ents onto a gl a s s plate in a regular pattern . This is referred to as a m i croa rray. Nex t , genes from any source can be label ed (this is referred to as a probe and often contains a flu orescent or rad i oactive marker) and then all owed to hybridize on the microa rray. Genes that hybridize to the probe will display the label. In this way, s c i en tists can identify genes wh i ch match those of the probe (Figure 10.1). This tech n i que has been used to compare differen t strains of cholera bac i lli such as those found in en demic areas and those found du ring ep i demics. A high degree of s i m i l a ri ty was found wh en this tech n i que was used , wh i ch shows that the two strains are cl o s ely rel a ted . It does appear that cholera strains that can incorporate a cluster of genes called a TCP pathogenicity island and the filamen tous phage CTØ (and perhaps a few other antigenic proteins) can become a pathogen to humans. The TCP pathogenicity island is a group of genes that en code for TCP pili, a co l on i z a ti on factor and receptor for CTXØ (the filamentous cholera toxin phage), and toxR, an essential vi rulence regulation gene. These results need further testing and verification, but the methods used will enable very precise definition of pathogenicity in these microorganisms at the genetic level. CHOLERA AND QUORUM SENSING Scientists have shown that some bacteria communicate with each other using ch emical signals. When the population density of bacteria reaches a high en o u gh level for this communication to

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Figure 10.1. New DNA technologies have allowed scientists to better understand how cholera functions. Using a gene chip, scientists can compare genomes of diff e rent strains of Vibrio cholerae. Fluorescent p robes are used to match the DNA of an unknown micro o rganism to that of the known cholera genome. The picture above is an example of a DNA microarray.

occur, chemical signals, called autoinducers , travel from one cell and bind to another. This changes the cell’s behavior. In this way, the cell population monitors its own density. This process is c a lled quorum sensing. Gram po s i tive bacteria use short po lypeptides as autoinducers , while Gram nega tive bacteri a , including the vibrio bacilli, use a ch emicals including acyl h om o s erine

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l actone. Examples of beh avi or ch a n ges that have been observed so far inclu de : 1) the abi l i ty to form spore s , 2) the abi l i ty to form bi ofilms, 3) the abi l i ty to tra n s form (to take up DNA from another microor ganism and incorporate it into a cell), and 4) bioluminescence. A marine bacterium closely related to the cholera bacillus is Vibrio harveyi. This microbe uses a qu orum sensing sys tem to tu rn on the genes wh i ch direct the form a ti on of proteins wh i ch a ll ow it to luminesce, i.e., to emit light. Scien tists ob s erved thro u gh examination of the com p l eted gen ome sequ ence that some of the genes for this process are found in the genes of Vibrio cholerae. Genes for producing and responding to the qu orum sensing signal were present, a l t h o u gh genes wh i ch give the ability to lu m i n e s cewere not. It was found that the qu oru m sensing sys tem in the ch o l era bac i llus is invo lved in controlling the ex pre s s i on of ch o l era viru l en ce genes. However, u n l i ke o t h er bacteria wh i ch con trol viru l en ce with qu orum sensing system s , Vibrio ch ol era e repress the viru l en ce genes. Ot h er processes reg u l a ted by a qu orum sensing system in the vi bri o bac i llus inclu de motility, pro tease enzyme producti on, and the abi l i ty to form bi ofilms. A better understanding of the ch o l era bac i llus qu orum sensing system may som ed ay aid in discoveries that may redu ce or el i m i n a te the vi ru l en ce activi ti e s of this microorganism. CHOLERA AND BIOTERRORISM The Federation of Am erican Scien tists recently listed som e microor ganisms that may be used for bi o l ogical warfare. In ad d i tion to diseases su ch as anthrax, plague, and the Ebo l a virus, cholera is on this list as well. Why might cholera be considered as an agent for bi o l ogical warfare? The goals of bi o terrorists are to disru pt society and to prom o te unrest. Killing is not necessarily, and not always, a primary goal in warfare. Creating large numbers of sick, disabled persons would sorely tax any nation’s resources. Cholera could be used to contaminate

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u n g u a rded water su pplies and at safe distances from large pop u l a tions. Ma s s ive numbers of cases would tax medical resource s , po s s i bly making the soc i ety more vulnera ble to other types of attack s . Pre s ent vaccines are only about 50 percen t effective, and the immunity they provide lasts less than a year, often only about six months. Antibiotics have a limited effect. In reality, water supplies in developed countries would most likely eliminate contaminating cholera microorganisms before they could harm anyone. Developed countries practice good sanitation and treat water supplies with chlorine or other halides regularly. Cholera bacilli are susceptible to these treatments. Further, water supplies are regularly monitored for fecal contamination. Public sewage areas are also treated and monitored for fecal contamination. However, should the water treatment somehow fail, ch o l era might survive and could make thousands very sick. Obviously, terrorist are aware of this, too. A FINAL WORD Cholera bacilli are part of the estimated two to three percent of all microorganisms on earth that are known to cause disease. Free-living vibrios produce no toxin. However, when in contact with human waste, the toxin is produced. There is no known function for the cholera toxin in nature. Ordinarily, these b acteria serve to rec ycle or ganic matter in waters . Tox i n formation is not needed for its survival in this environment. The abi l i ty of this microor ganism to form a toxin changes human behaviors. Both microbes and humans will, out of necessity, continue to share this planet. Each must adapt to the other for survival. New technologies such as gene sequencing and construction of m i c roarrays are all owing scientists to examine the natu re of the pathogen i c i ty of Vi b rio ch ol era e as never before. These human activities to control cholera will be co u n tered by activities of Vi b rio ch ol erae to adapt to its changing environment. We must remain ever vigilant.

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Glossary Acidosis —Increase of acidity in blood serum. Adenyl cyclase — An enzyme which forms cyclic AMP from ATP. Aerobic —In the presence of oxygen. Agar —A semi-solid polymer made from seaweed that is used to hold

nutrients that bacteria require for growth. Alimentary canal — The connection from mouth to anus. Alpha (α) helix —A spiral stru ctu re wh i ch gives form to some protei n s . Anaerobic — In the absence of oxygen. Annotated sequence —A sequence of DNA for which the protein product

and/or function is known. Antibody —Proteins formed in the blood serum in response to antigens. Antigen —A substance which induces the formation of antibodies. Asymptomatic —The state of an infected patient in which there are no

symptoms of evidence of that infection. Attenuate — To weaken. Bacteremia —When bacteria are present in the blood stream. Bacteriophage — A bacterial virus. Bile salt —A chemical formed by the gall bladder to help digest fats by

emulsifying them. Biofilm — Mixtures of microorganisms growing in a natural state. Biotype —S trains of b acteria wh i ch are very similar and may have ori gi n a ted

from the same strain, yet have different identifying characteristics. Biovar —A variety of a species with shared biological properties. Carrier — An infected individual who can transmit that infection to another

individual. Chemotaxis —Moving towards specific chemicals. Cholera cot —A special cot with an opening which allows fluid lost in the

form of diarrhea to be collected. 96

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Clone — An identical organism. Colony —A single microbe that has divided repeatedly to form a group of

cells which are visible to the naked eye. Conjugation — A method of bacterial recombination in which DNA is trans-

ferred from one strain to another by cell to cell contact. Conjugative plasmids —Plasmids which can be transferred between cells by

conjugation. Counterstain — A stain used to contrast another in procedures when more

than one stain is used. Communication of disease —How a disease is spread in a population. Cyclic AMP — A chemical involved in controlling cell metabolism. Defecate — To eliminate solid waste. Diarrhea — Loose or watery bowel movements. Electrolytes — Substances dissolved in solutions and which are positively or

negatively charged; for example, ions in water. Emerging infectious disease — A newly recognized infectious disease, often

a result of human activity. E n d e m ic—De s c ri bes a disease that is found in a particular place all year lon g. Endotoxin — The lipopo lysaccharide of Gram nega tive bacteria that is a factor

in their ability to cause disease. Epidemic — An increase in the number of disease cases above the normally

expected number of cases. Epidemiology — The study of disease transmission, incidence, and control. Endopeptidase — An enzyme which breaks peptide bonds in the interior of

a protein. Epithelium — The outermost layer of the skin or related tissues. Eukaryote —A cell which contains a true nucleus, (membranes surrounding

the genetic material). Facultative — Ability to live under different conditions.

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Glossary Feces —Solid waste. Filamentous virus —A virus with a linear shape. Fluid replacement therapy — Treatment of providing water and electrolytes

to replace those that are lost as a result of infection. Fungi (singular: fungus)— Eukaryotic saprophytic kingdom of organisms;

also called molds. Ganglioside — A unique type of lipid associated with cell mem brane stru cture;

glycolipids with a complex carbohydrate group. Gene chips — Genetic material for specific traits placed on slides and which

can be identified by hybridization. Germ Theory of Disease — The theory that infectious diseases are caused by

specific microorganisms. Glycolipid — A lipid containing a carbohydrate group. Gram stain reaction —A procedu re wh i ch vi su a l i zes bacteria and determ i n e s

if t h ey retain crystal vi o l et dye (Gram po s i tive) or do not (Gram nega tive). GTP — Guanosine triphosphate; a nucleotide. Hemagglutination — Clumping of red blood cells. Hemolysis —Bursting of red blood cells. Holotoxin —A toxin including its protein and all other chemical factors. Hypertonic —Solutions higher in electrolytes than a standard. Hypoglycemia — Lowering of glucose in the blood. Hypothesis — An educated guess that is testable; part of the scientific

method of problem solving. Hypotonic —Solutions lower in electrolytes when compared to a standard. Hypovolemia —Lowering of the volume of blood. Incidence — The nu m ber of n ew cases of a disease wh i ch arise over a spec i f i c

period of time. Incubation period —The time after infection and before disease symptoms

first appear. 98

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Initiator codon — The set of t h ree bases in messenger RNA wh i ch

is the first set to be translated into a po lypepti de du ring pro tein synthesis. Integron island — Genes which specify a system of proteins which

allows the capture of foreign genes. Intravenous (IV)— Inoculation into a vein. Kilobase —One thousand nucleic acid bases. Lipid A —A lipid in the outer membranes of Gram negative bacteria. LPS (lipopolysaccharide)— Lipids in the outer mem branes of Gram

nega tive bacteria. Lumen — The internal gut body cavity. Lysogeny — Incorporation of a bacterial genome into that of its bacterial

host without lysing (bursting) that host. Maintenance therapy — Tre a tment to redu ce immediate sym ptoms of a

disease and to stabilize a patient. Medium (plural: media)— Term to indicate the food used for growth of

microorganisms in culture. Microorganism — Life form requiring a microscope to see. Monoclonal antibodies —Homogeneous (identical) antibodies which react

against a single antigen. Monovalent vaccine — An antigen preparation that induces the formation

of antibodies against a single strain of bacteria. Morbidity — The number of dead plus the number of infected, but living,

individuals. Mortality — The number of deaths from an infectious disease. Motile — Ability to move, often because a microbe has flagella. Mucin — Protein in mu cous sec reti ons many of wh i ch contain po lys acch a ri de s . M u t a g en — A chemical that can damage DNA and cause mutants to be

formed. 99

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Glossary NAD (nicotinamide adenine dinucleotide)— A common enzyme cofactor

which carries hydrogen in cells; it is key to energy generation by cells. Normal flora — Microorganisms normally found in healthy individuals. Oligomeric —Molecules such as proteins made up of several subunits. Oliguria — Urinating less than usual. ORF (open reading frame) —A DNA sequence between the initiator codon

and the terminator codon. Pandemic — A global epidemic. Pathogenic — The ability of a microorganism to cause disease. Pathogenicity Island —A cluster of genes which determine characters that

make a microorganism able to cause disease. Pathogen — A microbe that can cause disease. Phagocytic cells — Cells capable of i n ge s ting other cells and other materi a l s . Phosphodiesterase—An en z yme that attacks cyclic AMP (c AMP) converting

it to AMP. Pili (singular: pillus) — Hair-like projections from some bacteria. Plasmid —A gen etic el em ent out s i de the ch romosome in the cytoplasm of cell s . Polyvalent vaccine —An antigen preparation that induces the formation of

antibodies against more than one strain of a microorganism. Porin — A protein in the outer membranes of Gram negative bacteria which

permits transport of materials across that membrane. Prokaryote — A cell without a true nucleus. Prophylactic — Preventative. Pure culture — A population of microorganisms arising from a single cell;

a clone. Quorum sensing — The ability of cells to communicate with other cells

through chemicals they produce. Recombinant DNA — DNA formed in laboratories using DNA from more

than one species. 100

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Regulatory proteins —Proteins that function to change the production or

activity of other proteins, particularly enzymes. Sanitation — Prom o ting hygi ene by reducing the nu m bers of microorganisms

in a location. Self-limiting infection —An infection that a person can ward off naturally. Serogroup — A group of distinct microorganisms that can react with one

antibody preparation. Serotype —Strains of an organism that are distinguished by different

immunological reactions. Skin turgor —The tightness of the skin. Slide agglutination — The clumping reaction of antigens and antibodies on

a glass slide. Stool specimens — Sample of fecal waste. Streak plate methods — Using sterilized wire (inoculating needles) to place

microbes on agar plates in order to isolate individual clones (colonies). Synergistic — Cooperation such that combined effects are greater than that

of either participant. Temperate virus — A virus that induces lysogeny after infecting a host. Terminator codon — The “Stop” signal in the DNA code that signals the end

of a transcription. Tissue culture —Growth of animal or plant cells, in tubes or plates, outside

of the tissues they came from. Toxoid —Mod i f i ed toxin wh i ch remains anti genic but wh i ch is no lon ger toxic. Transduction —Method for gene recombination in microorganisms in

which a virus carries DNA between cells. Transmission — The spread of a disease. Vaccine — An antigenic preparation used to prevent infection by inducing

formation of protective antibodies. Villi (singular: Villus)—A finger-like projection of cells lining the intestinal tract . Virulence —The abi l i ty of a pathogenic microor ganism to cause severe disease.

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Bibliography Boyer, R. Co n cepts in Biochemistry. 2nd ed. Pacific Grove, CA: Broo k s / Co l e , 2001. Chicago Pu blic Library. “1849-1855, 1866-1867: Early Cholera Ep i demics.” In ternet site : www.chipublib.org/004chicago / d i s a s ters / early_cholera . h tml. Co lwell, R. “Bacterial Death Revisited.” in No n c u l tu ra ble Microo rganisms in the Environment. Co lwell, R. and J. Grimes, editors. Washington, D.C.: ASM Pre s s , 2000. Co lwell, R. “G l obal Cl i m a te and In fectious Disease: The Cholera Pa radigm.” Science. 274 (1996): 2025-2031. Cron enwett, E. “Cholera and Cholera Toxin.” In ternet site : http://atti l a . s tevens-tech . edu / chembio/ecro n enw / f i n a l ~ 1 . h tm, 1997. DeWan, G. “The 1892 Ch o l era Pa n i c .” Su f folk Count Hi s torical Society. In ternet site: http://www.lihistory.com/histpast/past711.htm DiRita, V. “G enomics Ha ppens.” Science. 289: (2000) 1488 –1489. Dromigny, J., O. Rakoto-Alson, D. Rajaonatahina, R. Migliani, J. Ranjalahy, and P. Ma u cl ere, “Emer gence and Rapid Spre ading of Tetrac ycline Resistant Vibrio Cholerae Strains, Mad a ga s c a r.” Emerging In fe cti ou s Diseases. 8 (3), 2002 Dziejman, M., E. Balon, D. Boyd , C. Fraser, J. Heidelberg, and J. Me k a l a n o s . “Com p a rative Genomic Analysis of Vi b rio ch ol era e: Genes that Correlate with Cholera Endemic and Pa n demic Disease.” Pro ceedings of the Na tional Ac a d emy of Sciences, U. S . 99 (2002): 1 5 56–1561. Ew a l d , P. Evolution of In fectious Di sease. New York: Ox ford Un iversity Press, 1994. Federation of Am erican Scientists. “Biological Warf a re Agents (Pa rtial List).” In ternet site: http://www.fas.org / nu ke/intro/bw/agent.htm. F i n kel s tein, R. “ Personal Ref l ections on Cholera: the Im p act of Serendipity.” ASM News 66 (2000): 663 –667. F i n kel s tein, R. “Cholera, Vi b rio cholera e O1 and 0139, and Other Pa t h ogenic Vibrios.” In ternet site : http://www.gsbs.utm b. edu/microbook / ch 0 2 4 . h tm

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Fon t a i n e , O., S. G ore, and N. Pierce. “Ri ce - b a s ed Oral Rehyd ration So lution for Treating Diarrhea.” Na ture. 417 (2002): 6 42– 648. Glass, R. “Cholera and Non - ch o l era Vibrios.” in: Enteric In fe ctions, Mechanisms, Manifestations and Management. M. Farthing, and G. Keusch, editors. New York: Raven Press, 1989. G ordon , M., A. Walsh, S. Rogerson, K. Ma gom er, C. Machili, J. Cork i ll , and A . Ha rt. “Three Cases of Bacteremia Caused by Vi b rio ch ol erae O1 in Blantyre, Ma l awi.” Em erging Infectious Diseases 7, Nov-Dec 2001. In ternet site : www.cdc.gov / n cidod/ei d / vol7no6/go rd o n . h tm Grave s , P., J. Deeks, V. Demicheli, M. Pratt, and T. Jefferson. “Vaccines for Preventing Cholera.” The Co chrane Li b ra ry. Is sue 2, 2002, Up d a te Sof t w a re, Ox ford. Hei del berg, J., J. Ei s en, W. Nelson, R. Clayton , M. Gwinn, R, Dod s on , D. Ha f t , E . Hi ckey, J. Peters on , L . Um aya m , S . G i ll , K . Nel s on , T. Read, H. Tet telin, D. Ri chard s on , M. Ermolaeva, J. Vamathevan, S. Bass, H. Qin, I. Dragoi , P. Sellers, L. Mc Don a l d , T. Ut terback, R. Fleishmann, W. Ni erman, O. White, S. Salzberg, H. Smith, R. Co lwell, J. Mekalanos, J. Venter, C. Fraser. “ D NA Sequ en ce of Both Ch rom o s omes of the Cholera Pathogen Vi b rio ch ol era e.” Na ture 406 (2000): 477-484. Hi s s , Philip and Hans Zi n s s er. A Textbook of Bacteriology. 4th ed.. New York: App l eton , 1918. Huq, A., I. Rivera, and R. Co lwell. “ Ep i demiological Si gnificance of Viable but Non c u l turable Mi c roorganisms.” in: No n c u l tu rable Microo rganisms in the Envi ro n m en t. R. Co lwell, and J. Grimes, editors. Washington , D.C.: ASM Pre s s , 2000. Holisiti c - on l i n e . “Critical Biological Agents that May be Us ed in Bi o terrorism.” In ternet site : http://www.holisti co n l i n e . co m. Keusch, G. and M. Bennish. “Cholera.” in: Textbook of Pediatric Infectious Di se a se s. Vol. I. 3 rd ed. R. Feigin, and J. Cherry, editors. Philadelphia: Sa u n ders, 1981. Levine, M. and N. P i erce . “Immu n i tyand Vaccine Development.” in: Cholera. D. Barua, and W. Greenough, editors. New York: Plenum, 1989. Lo s i ck, R. and D. Ka i s er. “Why and How Bacteria Com mu n i c a te .” Scientific Am erican. 276 (1997): 68 –73.

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Merck Ma nual, Sec. 13, Ch. 157, Bacterial Diseases. Internet site : http://merck.com/pubs/mmanual/section13/chapter157/157d.htm. Mo rbi d i ty and Mortality Weekly Report, Com municable Disease Center, “Cholera As s oc i a ted with In ternational Travel, 1992.” In ternet site : www.cdc.gov / m mwr / preview / m mwrhtml/00017594.htm. Mo rbi d i ty and Mortality Weekly Report, Ep i demiologic No tes and Reports 42 (1993): 9 1–93. Morbidity and Mortality Weekly Report, Communicable Disease Center. “Ch o l era As s oc i a ted with Im ported Frozen Coconut Mi lk—Maryland, 1991.” Internet site: www.cdc.gov/mmwr/preview / m mwrhtml/00015726.htm. Mo rbidity and Mo rtality Weekly Reports, Com municable Disease Center. “ Recom m endation of the Im munization Practi ces Advisory Com m i t tee Cholera Vaccine.” In ternet site : http://www. cd c . gov/mmwr/ preview/mmwrh tml/00042345.htm Oi, H., D. Ma t suura, M. Miya ke , I. Takai, T. Yamamoto, M. Ku bo, J. Mo s s , and M. Noda “ Identification in Traditional Herbal Medications and Con f i rmation by Synthesis of Factors that In h i bit Cholera Tox i n indu ced Fluid Acc u mu l a tion.” in: Pro cedings of the Na tional Ac a d emy of Sciences, U. S . 99 (2002): 3 0 42– 3046. Ri ch a rdson, B.W. Sn ow on Cholera. Lon don : Ha f n er Pu blishing Com p a ny, 1965. Salyers, A. and D. Wh i t t . Mi crobi ology, Divers i ty, Di seases and the Envi ro n m ent. Bethesda, Md . : F i t z gerald Scien ce Press, 2001. Sn ow, J. On the Mode of Co m munication of C h ol era. London : Jo h n Chu rchill, 1855. Stock, R. “Cholera in Africa” Af rican Envi ro n m ent Special Report 3, In ternational African Insti tute, Lon don , England, 1976. Su m m ers, J. Soho — A History of Lo n d o n’s Most Col ourful Neighborh ood. Lon don : Bl oom s bu ry, 1989. Tauxe, R. “Cholera.” in: Bacterial In fe ctions of Humans: Ep i d em i ol o gy and Co n trol. 3rd ed. A. Evans and P. Brockman, Editors. New York: Plenu m , 1998.

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Waldor, M. and J. Me k a l a n o s . “Lysogenic Convers i on by a Filamentous P h a ge Encoding Cholera Tox i n .” Science. 272 (1996):1910 –1914. Z hu , J., M. Miller, R. Va n ce , M. Dziejman, B. Ba s s l er, and J. Me k a l a n o s . “Q u orum-sensing Regulators Con trol Viru l en ce Gene Ex pression in Vibrio ch ol era e.” Pro ce ed i n gs of the Na tional Ac a d emy of Sciences U. S . 99 (2002): 3 1 29– 3134.

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Further Reading Alcamo, E. Fundamentals of Microbiology. 6th ed. Boston: Jones and Bartlett, 2001. Ba n n i s ter, B. , N. Begg, and S. Gillespie. Infecti ous Disease. Ca m bri d ge : Bl ackwell Science, 1996. Barua, D., and W. Greenough, editors. Cholera. New York: Plenum Medical Book Company, 1992. Colwell, R., and J. Grimes, editors. Nonculturable Microorganisms in the Environment. Washington, D.C.: ASM Press, 2000. Evans, A., and P. Brockman, editors. Cholera. New York: Plenum, 1998. Ewald, P. Evolution of Infectious Disease. New York. Oxford University Press, 1993. Farthing, M. and G. Keusch, editors. En teric In fections, Me chanisms, Manifestations and Management. New York: Raven Press, 1989. Feigin, R. and J. Cherry, editors. Textbook of Pediatric In fe ctious Dise a se s. Vol I, 3rd ed. Philadelphia: Saunders, 1981. Gest, H. The World of Microbes. San Francisco: Benjamin Cummings, 1988. Goodwin, C., ed i tor. Cholera and Other Vi b rios. Melbourne, Australia: Blackwell, 1984. Mims, C., A. Nash, and J. Stephen. The Pathogenesis of Infectious Disease. San Diego: Academic Press, 2001. Mims, C., J. Playfair, I. Roitt, D. Wakelin, and R. Wi lliams. Medical Microbiology. 2nd ed. Philadelphia: Mosby, 1998. Murray, P., K. Rosenthal, G. Kobayashi, and M. Pfaller. Medical Microbiology. St. Louis: Mosby, 1997. Salyers, A. and D. Whitt, Microbiology, Disease, Diversity and the Environment. Bethesda, Md.: Fitzgerald Science Press, 2001.

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Websites American Society for Microbiology www.asmusa.org

Virtual Museum of Bacteria www.bacteriamuseum.org

Family Practice Notebook www.fpnotebook.com

Medical microbiology with pronunciation station and photo gallery www.geocities.com/CapeCanaveral/3504/ Pictures of microorganisms by Dennis Kunkel Microscopy, Inc. Choose “Stock Photogra phy” at the top of the page and search wi t h o ut signing-in. www.denniskunkel.com

Online microbiology tutorials and videos www-micro.msb.le.ac.uk/Tutorials/Tutorials.html

Dr. John Snow www.ph.ucla.edu/epi/snow.html

Interactive simulation for discovering the origins of epidemiology www.sph.unc.edu/courses/course_support/case_studies/JohnSnow/

World Health Organization’s fact sheet on Cholera www.who.int/inf-fd/en/fact107.html

Cholera Toxin Project at the University of Washington www.bmsc.washington.edu/projects/toxins.html

Genetics Science Learning Center at the University of Utah http://gslc.genetics.utah.edu

Basic paper on cholera by Dr. Elizabeth Cronenwett, including descriptions of the toxin and treatment options. http://attila.stevens-tech.edu/chembio/ecronenw/final~1.htm

“Bad Bug Book” from the U.S. Food and Drug Administration http://Vm.cfsan.fda.gov/~MOW/chap7.html

Microbiology and Immunology On-line www.med.sc.edu:85/book.immunol-sta.htm

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Index Acidosis, 49 Adenyl cyclase, 56, 57, 61 Aerobic, Vi b rio ch ol erae as, 18-19 Aeromonas spp, 21 Africa, cholera in, 43 Aga r, 12, 13, 18 Agent 0129, 80 Al i m en t a ry canal, 30 Al pha-helix, 58-59 Am ericas, cholera pandemic in, 38 Amino ac i d s , in fluid replacement thera py, 76 An n o t a ted sequ ence, 64 Antibiotics, 26, 51, 52, 79-80 and Archaea, 22 and Bacteri a , 22 as prophyl actic measu re , 84-85 resistance to, 69, 79-80, 85, 91 in treatment of cholera, 26, 72, 79-80 Anti bod i e s , 21, 24, 26-27 mon oclonal, 45 vi bri ocidal, 86-87 Antigens, 24, 26 Archaea, 21-22 Asiatic cholera , 30 A subunit, 58, 59, 61, 62 A1 su bunit, 58, 59, 61, 62 A2 su bunit, 58, 59, 61 As ym ptom a tic, cholera cases as, 41 ATP, and cyclic AMP, 55-57 Attenu a ted bacterial prep a ra ti ons, in vaccines, 85-86, 87 Autoi n du cers, 93 Ba byl on , Long Island, and New York Ci ty cholera epidemic, 38-39 Bacteremia, 52

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Bacteria, cholera bac i llus as prokaryo te of domain, 21-24 Bacteri ophages, and gene for ch o l era toxin, 67-69 Ba n gl ade s h , cholera in, 26, 43, 45 Ba s s i , Ago s tino, 8, 9 Bengal, cholera in, 26, 39, 91 Bi c a rbon a te in fluid replacement t h erapy, 75 loss of with dehyd rati on , 48 Bile salts, 20 Bi l ivaccines, 86 Biofilms, 66, 70 Bioterrorism, and cholera , 94-95 Bi o type s , 26 Biova r, 39 Botrytis bassiana, 8, 9 Broad Street (London ) , cholera in, 31-35, 38 Bromothymol blue, 19, 21 B subunit, 58, 59, 60-61 and vaccine, 88, 89 Ca rri ers, 26, 36, 49-50 Cause of cholera , 9-15, 16 See also Vibrio chol erae Cereal, in fluid replacem ent thera py, 76 Chemotaxis, 70 Chicago, cholera epidemic in, 38 China, cholera in, 43 Chloramphenicol, 51, 80 Chlori de , loss of wi t h dehyd ra ti on, 48 Chlorpromazine, 81 Cholera, origin of word, 37 Ch o l era cot, 78-79 Ci tra te , in fluid replacem ent thera py, 75, 76 Clone, 11

Colony of bacterium, 10-11, 18 Combination oral vaccines, 88, 89 Communicati on of cholera , 30 See also Transmission of cholera Con juga ti on , 80 Con juga tive plasmids, 80 Con trol of cholera and detection of cholera bacteri a , 45 and sanitati on , 41-42 Convalescent carri ers, 36 Copepod , 83-84 Co u n terstain, 18, 22 Crystal vi o l et , 18, 22 CTXØ, 67, 68, 92 CVD 103, and vaccine, 88-89 Cyclic AMP, 54-57, 61, 62, 63 Cytochrome oxidase, 19 Dead whole cells, and vaccines, 87, 88, 89 Death from cholera , 28, 30, 31, 37, 40, 49, 50, 51, 72 Dehyd ra ti on and fluid replacement therapy, 72, 73-77, 80 level of , 77 and maintenance therapy, 78-79 s i gns and sym ptoms of , 46-49 Di a rrhea, 26, 28, 37, 40, 46, 51, 52, 53, 57, 72, 76, 78-79, 81, 91 Discovery of Vi b rio cholerae, and Koch, 9-15, 16-17, 22 DNA and ch o l era on gene chips, 92 and detecti on of ch o l era bacteria, 45 and lys ogeny, 67, 68

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and strains of cholera bac i llus, 39-40 and treatment of ch o l era, 91-92 Donut, 58 Doxycycline, 80 Electro lytes, loss of with dehyd ra ti on, 48, 73-75 El Niño effect, 45 El Tor strain, 24, 26, 39, 43, 45, 80 Emerging infectious disease, ch o l era as, 90-91 Endemic area, 90 Endopeptidase, 61 Endotoxin, 24 England, cholera in, 30-35, 38 Epidemics/pandemics, 3135, 37-45 in Broad Street , London , 31-35, 38 and ch o l era on gene chips, 92 and death from ch o l era, 40, 72 and detection of ch o l era bacteria, 45 in 1800s, 31-35, 37-38, 72 and El Niño effect, 45 and geographic distribution of cholera, 42-43 and incidence of cholera, 40-41, 42, 43 in 1900s, 39-40, 42-45, 72, 79-80 and nu m ber of cases reported, 40 setti n gs for, 43 tracking cholera bac i llus during, 27, 31-35 Epidemiology of cholera, 31 and Sn ow, 30-35 See also Epidemics/ pandemics E ryt h romycin, 51

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Esch eri chia coli, 66, 70, 90 Eukaryotes, 21 Fac u l t a tively anaerobi c , Vibrio cholerae as, 18-19 Feces as carri ers of cholera , 36, 50 and stool specimens, 19-21, 26-27 Federa ti on of Am erican Scientists, 94 Filamentous phages, 67-68 Fluid rep l acem ent thera py, 51, 52, 72, 73-77, 80, 91 Food thoro u gh cooking of, 83 and transmission of cholera, 36, 37, 41, 43-45, 83, 90-91 Frac a s toro, Girolamo, 8 Fungus, in silkworms, 8, 9 Future, ch o l era in, 90-95 Gangliosides, 54, 61 Gastric acid, and cholera, 43 GDP, 62 Gelatin, 10-12, 18 Gene chips, cholera on , 92 Genome of Vi b rio ch ol erae, 64-71 and bacterial vi rus and gene for cholera toxin, 67-69 and Chromosome 1, 65-66, 69, 70 and Chromosome 2, 65-66, 70 and gene chips, 92 and hori zontal transfer, 69 and loc a tion of gen e for cholera toxin, 69 nu cl eic acid sequ en ce of , 64 uniqu eness of , 64-65, 69-71

Gentamicin, 51 Germ Th eory of Disease, 8 G lu cose, in fluid replacem ent thera py, 73, 76 G lycolipids, 54 G protein, 61, 62 Gram nega tive, Vibrio cholerae as, 17-18, 22-24 Gram po s i tive, 22 Gram stain reaction, 17-18, 22 “Grand Experiment” of Dr. Snow, 30-35 GTP, 61, 62 H antigens, 24 Harnold, John, 30 Heat inactiva ti on, 86 Hemagglutinins, 58 Herbal medicines, and treatm ent of ch o l era, 91 Hikojima strain, 26 History of cholera . See Epidemics/pandemics Holotoxin, 61 Hori zontal transfer, 69 Host-parasite relationship, 50, 53 Hypertonic solutions, 72 Hypoglycemia, 49 Hypothesis, 30 Hypovolemia, 48-49 Immune system, and hostparasite rel a tionship, 50 Inaba strain, 24, 26, 89 Incidence of ch o l era, 40-41 Incubation peri od , 46 Incubatory carri ers, 36 India, ch o l era in, 26, 28-29, 37, 43, 91 Indian Ocean, cholera in, 79-80 Indole, 18 Initiator codon, 65 Institute for Genomic Research (TIGR), 64

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Integron island, 65-66 Islip Town , Long Island, and New York Ci ty cholera ep i demic, 38-39 IV-ORT (intravenous oral replacement therapy), 72, 77 Kilobases, 39 Koch, Robert, 9-15, 16-17, 22 Koch’s Postulates, 14-15 Latin Am erica, cholera in, 38, 42, 43, 72 Legionella pn eu m ophila (Legi onnaire’s disease), 90 Lipid A, 23-24 L i popo lys accharides, 23-24, 26, 87 Lumen, 57 Lysogenic conversion, 67 Lysogeny, 67, 68 Maintenance therapy, 78-79 Maize, in fluid rep l acem en t t h erapy, 76 Ma ryland, cholera in, 43 MCP genes, 70 Medium, for growth of b acteria, 10-13, 18 Mi c roa rrays, 92, 95 Mi c robiology and Koch, 9-15, 16-17 and Pasteur, 8-9 and Snow, 31-35 Mon oclonal anti bodies, 45 Monovalent strain, for vaccines, 87 Morbi d i ty, 40 Mort a l i ty, 40 See also Death from cholera MSHA (mannose-sen s i tive hemagglutinin), 70 Mucin, 58 Muscardine, 8-9

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Mut a gen, 88 Myelin, and cholera toxin, 62 NAD (nicotinamide adenine dinucleo ti de ) , 61 New Orleans, cholera pandemic in, 38 New York Ci ty, ch o l era epidemic in, 38-39 Nico tinic ac i d , 81 Non toxic strains, and vaccines, 88 Normal flora, 50 Nutri ti on , and treatm en t of ch o l era, 72, 80 O anti gen s , 24 and O1 antigen structure, 24, 26, 39, 51, 52 Ogawa strain, 24, 26, 80, 89 Ol i gom eric, toxin as, 58 Ol i g u ri a , 49 Open reading frames (ORFs), 65 ORS (oral replacement s o lutions), 75 ORT (oral rhyd ra ti on t h erapy), 73-77, 80, 91 Outer membranes, for vaccines, 88 Pakistan, cholera in, 43 Pan-American Health Organization, 40 Pandemics, 37-38 See also Epidemics/ pandemics Pa s teur, Louis, 8-9, 86 Pa t h ogen i c i ty island, 69 Pathogenic microorganisms, 54 See also Virulence of Vi b rio ch ol erae Penicillin, 51 Peptone medium, 18, 19 Persia, cholera pandemic in, 37

Peru, cholera in, 72 Petri , R. J., 13 Petri dish, 13 Phagoc ytic cells, 50 Philadelphia, cholera pandemic in, 38 Phosphodiesterase, 57 PilD (V c p D ) , 69-70 Pili, 57-58, 67 Plasmids, 66 Plesiomanas spp, 21 Po ly va l ent strain, for vaccines, 87-88 Pori n s , 22 Potassium, loss of wi t h dehyd ra ti on, 48, 49 Potassium chloride, in fluid rep l acem ent thera py, 74, 76-77 Prevention of cholera, 82-85 and antibiotics, 84-85 and clean water, 82-83, 91 and thoro u gh cooking of food , 83 and use of copepod , 83-84 See also Vaccines Prokaryo te of domain bacteri a , ch o l era bac i llus as, 21-24 Properties of Vibrio cholerae, 16-27 and anti bod i e s , 21, 24, 26-27 and de s tru cti on , 19, 37 as Gram negative, 17-18, 22-24 growth of in labora tory, 10-13, 18-19 and isolation and identification from patient specimens, 18, 19-21, 26-27, 29 and moti l i ty, 16, 21 as op a l e s cent, 18

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as prokaryo te of domain Bacteri a , 21-24 and shape , 16-17 and size, 16 and strains, 21, 24, 2627, 39, 43, 45, 69, 80, 87, 88-89, 91 and su rvival capabilities, 19, 37 Pro teins, in fluid replacement thera py, 75-76 Pu blic Health Servi ce , and vaccines, 89 Pu re culture, 10 Quorum sensing sys tem, 92-94 Re a gent, cholera toxin as, 62-63 Recom binant DNA, and vaccine, 87, 88 Rehyd ra ti on thera py. See Fluid replacem ent thera py Renal failu re, 49 Resistance genes, and antibiotics, 69, 79-80, 85, 91 Rice powder, in fluid replacem ent thera py, 76 Rice water stoo l s , 46 RTX, 70-71 Russia, cholera pandemic in, 38 Safranin, 18, 22 St. Louis, Mi s s o u ri , cholera in, 72 Salt, in fluid replacem ent thera py, 75, 76-77 Sanitation and con trol of cholera, 41-42 and transmission of ch o l era, 36 Scien tific met h od, 30

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Sel f - l i m i ting infecti on, 49 Serogroup, 39 Serogroup 0139, 26, 39, 91 Sero type s , 24, 26, 39 Sewage treatment plants, and prevention of cholera, 82, 91 Signs and sym ptoms of cholera, 46-53 and case studies, 51-53 in classic cholera, 46-49 and dehyd ra ti on, 46-49 and diarrhea, 26, 28, 37, 40, 46, 51, 52, 53, 57, 72, 76, 78-79, 81, 91 and host-para s i te rel ationship, 50, 53 and incubati on peri od, 46 and mild cases, 49 and severe cases, 49 and vomiting, 46 Silkworms, f u n gal disease of , 8, 9 Skin, and dehyd ra ti on, 47-48 Skin tu r gor, 48 Sl i de agglutination tests, 26-27 Snow, John, 28-35, 36, 38 Sod iu m in fluid rep l acem en t thera py, 73 loss of with dehyd ration, 48 Starches, in fluid replacement therapy, 75-76 Stool specimens, and isolation and iden tification of cholera bacillus, 19-21, 26-27 Strains of Vi b rio ch ol erae, 21, 24, 26-27, 39, 43, 45, 69, 80, 88-89, 91 and vaccines, 87, 88-89 Streak plate method, 10, 13 Streptomycin, 80

Sucrose, in fluid replacem ent thera py, 75 Sugar, in fluid replacem en t t h erapy, 75, 76-77 Sulfonamides, 80 Synergistic, vaccine as, 87 TCP pathogenicity island, 92 Tem pera te bacterioph a ges, 67 Term i n a tor codon , 65 Tetrac ycline, 79, 80, 84-85 Texas Star-SR, 88 Thiosulfate-citra te - bile salt-sucrose (TCBS) m ed ium plates, 19-21, 26-27 Tissue culture, and isolation and identificati on of ch o l era bac i llus, 19-21, 26-27 Toxin, 50, 54, 58-63 and human waste, 95 location of gene for, 69 and relationship bet ween bacteri a l vi rus and, 67-69 and RTX, 70-71 and vaccine, 86-87, 88 Toxin-regulated pilu s (TCP), 67 Toxoid vaccines, 87, 88 Tracking ch o l era, 27, 31-35 Tra n s du ction, 67 Transmission of cholera , 36-37 and carri ers, 26, 36, 49-50 and food , 36, 37, 41, 43-45, 83, 90-91 and Koch, 16 and nu m ber of b acteri a needed for infection, 36, 37, 41 from person to pers on, 36-37

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Index and Snow, 28-35, 36, 38 and travel, 44-45, 82, 89, 90-91 and water, 16, 30-35, 36, 38, 41, 42, 82-84, 91 Travel, and transmission of cholera , 44-45, 82, 89, 90-91 Treatment of cholera, 72-81 and antibiotics, 26, 51, 52, 72, 79-80, 91 and bacterial vi ruses, 80 and dehydra ti on level, 77 and diarrhea redu ction, 81, 91 and fatality ra te, 49 and fluid replacem en t t h erapy, 51, 52, 72, 73-77, 80, 91 and futu re , 91-92 and herbal medicines, 91 and hori zontal gene transfer, 69 and mainten a n ce therapy, 78-79 and nutri ti on, 72, 80 and proper pati en t care, 77 and resistance to antibiotics, 69, 79-80, 85, 91 and weight of patient, 77 Trimethoprim, 80 Trimethoprimsulfamethoxazole, 80

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Tuberculosis, and Koch, 10, 15 Tu rkey, cholera pandemic in, 37 United States, cholera in, 72, 82, 90-91 in 1800s, 38-39 tod ay, 43-45 and vaccines, 89 Vaccines, 85-89 and attenuated bacterial prep a ra ti ons, 85-86, 87 and bi l ivaccines, 86 and heat inactivation, 86 h i s tory of, 85-86 and hori zontal gene transfer, 69 and live microor ga nisms, 86 and plants, 91-92 prep a ra ti on of , 87-89 in United States, 89 and vi bri ocidal antibod i e s , 86-87 Vasco da Gama, 37 Vi b rio ch ol erae discovery of, 9-15, 16-17 gen ome of , 64-71, 92 properties of, 16-27 as scientific name for cholera , 13 s trains of, 21, 24, 26-27, 39, 43, 45, 69, 80, 87-89, 91 vi ru l en ce of , 54-63

Vibri ocidal anti bod i e s , in vaccine, 86-87 Vi b rio harveyi, 94 Vi b rio parahaemolyti c u s , 21 Virulence of Vi b rio cholerae, 54-63 and cyclic AMP, 54-57, 61, 62, 63 and future, 92-95 and hem a gglutinins, 58 and mu c i n , 58 and pili, 57-58, 67 and qu orum sensing, 92-94 and toxins, 54, 58-63 Vom i ti n g,46 Water and bi o terrorism, 94-95 cleanliness of and prevention of cholera , 82-84 and detection of cholera bacteri a , 45 in fluid replacem en t thera py, 73 and transmission of ch o l era, 16, 30-35, 36, 38, 41, 42, 82-84 Wheat, in fluid rep l acement thera py, 76 World Health Organization (W H O ) and ORT, 74-75 and su rveillance of ch o l era, 40 and vaccines, 89

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Picture Credits 11: © Bettmann/Corbis 13: Hulton Archive/Getty Images 14: Courtesy Dartmouth College Rippel Electron Microscope Facility 17: Courtesy CDC/Dr. William A. Clark 20: © Richard T. Nowitz/Corbis 25: Courtesy Dartmouth College Rippel Electron Microscope Facility 29: NLM/History of Medicine 32: National Geographic 33: National Geographic

41: 42: 44: 55: 56: 59: 68: 74: 83:

U.S. Department of Health/Human Services Courtesy WHO Courtesy MMWR Courtesy CDC Courtesy Dartmouth College Rippel Electron Microscope Facility © Johnjoe McFadden © Nature Vol 399, 1999 by MacMillan Publishers, Ltd. AP photo/Cobus Bodenstein AP photo/Michael Conroy

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About the Author William H. Coleman has taught microbiology to undergraduate students for more than 30 years. He earned a B.S. in Biology at Washington College and an M.S. and Ph.D. in microbiology at the University of Chicago. He was a post-doctoral fellow at the University of Colorado Health Science Center. Since then, he has been on the faculty in the College of Arts and Sciences, Department of Biology at the University of Hartford in West Hartford, Connecticut. His research has included studies on proteins from grains toxic to eu k a ryo tic pro tein synthesis sys tems and studies of the inhibi ti on of horm one re s ponses by fungi. He served on the Bloomfield, Connecticut, Board of Education for ten years. He was Chair of the Department of Biology at the University of Hartford for three years. Currently, he is Associate Dean at Charter Oak State College, a public distance learning institution. He is an active member of the Microbiology Education Group of the American Society for Microbiology. He has wri t ten and pre s en ted nu m erous arti cl e s in both basic re s e a rch in biological sciences as well as those concerning improving and instilling active learning in microbiology education. He has written study guides and test banks for several newly published texts in this field. Currently, he reviews educational materials, both written and visual, for the Edu c a tion Libra ries on the web site of the Am erican Society for Microbiology. He currently resides in South Windsor, Connecticut.

About the Editor The late I. Edward Alcamo was a Distinguished Teaching Professor of Microbiology at the State Un iversity of New York at Farmingdale. Al c a m o stu d i ed biology at Iona Co ll ege in New York and earned his M.S. and Ph.D. degrees in microbi o l ogy at St. John’s Un ivers i ty, also in New York . He taught at Fa rm i n gdale for over 30 years. In 2000, Alcamo won the Ca rski Award for Di s ti n g u i s h ed Te aching in Microbi o l ogy, the highest honor for microbi o l ogy teachers in the Un i ted States. He was a member of the American Society for Mi c robiology, the Na ti onal Associati on of Biology Teach ers, and the Am erican Medical Wri ters As s oc i a ti on . Alcamo aut h ored nu m erous books on the su bj ects of microbi o l ogy, AIDS, and DNA tech n o l ogy as well as the awardwinning tex tbook Fundamentals of Mi crobiology, now in its sixth ed i ti on.

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