Plague (Deadly Diseases and Epidemics) 2nd Edition

Plague Second Edition

Antibiotic-resistant Bacteria Anthrax, Second Edition Avian Flu Botulism Campylobacteriosis Cervical Cancer Chicken Pox Cholera, Second Edition Dengue Fever and Other Hemorrhagic Viruses Ebola Encephalitis Escherichia coli Infections Gonorrhea Hantavirus Pulmonary Syndrome Helicobacter pylori Hepatitis Herpes HIV/AIDS Infectious Diseases of the Mouth Infectious Fungi Influenza, Second Edition Legionnaires’ Disease Leprosy Lung Cancer Lyme Disease Mad Cow Disease (Bovine Spongiform Encephalopathy)

Malaria, Second Edition Meningitis Mononucleosis, Second Edition Pelvic Inflammatory Disease Plague, Second Edition Polio Prostate Cancer Rabies Rocky Mountain Spotted Fever Salmonella SARS, Second Edition Smallpox Staphylococcus aureus Infections Streptococcus (Group A) Streptococcus (Group B) Syphilis, Second Edition Tetanus Toxic Shock Syndrome Trypanosomiasis Tuberculosis Tularemia Typhoid Fever West Nile Virus, Second Edition Yellow Fever

plague Second Edition

Donald Emmeluth, Ed.D. CONSULTING EDITOR

Hilary Babcock, M.D., M.P.H.,

Infectious Diseases Division, Washington University School of Medicine, Medical Director of Occupational Health (Infectious Diseases), Barnes-Jewish Hospital and St. Louis Children’s Hospital FOREWORD BY

David Heymann

World Health Organization

Plague, Second Edition Copyright © 2010 by Infobase Publishing, Inc. All rights reserved. No part of this book may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, or by any information storage or retrieval systems, without permission in writing from the publisher. For information contact: Chelsea House An imprint of Infobase Publishing 132 West 31st Street New York NY 10001 Library of Congress Cataloging-in-Publication Data Emmeluth, Donald. Plague / Donald Emmeluth ; Consulting Editor, Hilary Babcock ; Foreword by David Heymann. — 2nd ed. p. cm. — (Deadly diseases and epidemics) Includes bibliographical references and index. ISBN 978-1-60413-237-3 (hardcover : alk. paper) ISBN 978-1-4381-2877-1 (e-book) 1. Plague—Popular works. I. Title. II. Series. RA644.P7E47 2009 614.5'732—dc22 2009028502 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 Department 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 MSRF 10 9 8 7 6 5 4 3 2 1 This book is printed on acid-free paper. All links and Web addresses were checked and verified to be correct at the time of publication. Because of the dynamic nature of the Web, some addresses and links may have changed since publication and may no longer be valid.

Table of Contents

Foreword David L. Heymann, World Health Organization

1. Historical Overview

6 8

2. Causes of the Plague

19

3. Cats, Rats, Prairie Dogs, and Squirrels

32

4. Diagnosis

41

5. Treatment

51

6. Prevention

60

7. The Problems of Antibiotic Resistance

69

8. Concerns for the Future

79

9. Hopes for the Future

86





Notes

99



Glossary

102



Further Resources

108



Index

112



About the Author

120



About the Consulting Editor

120

Foreword Communicable diseases kill and cause long-term disability. The

microbial agents that cause them are dynamic, changeable, and resilient: they are responsible for more than 14 million deaths each year, mainly in developing countries. Approximately 46 percent of all deaths in the developing world are due to communicable diseases, and almost 90 percent of these deaths are from AIDS, tuberculosis, malaria, and acute diarrheal and respiratory infections of children. In addition to causing great human suffering, these high-mortality communicable diseases have become major obstacles to economic development. They are a challenge to control either because of the lack of effective vaccines, or because the drugs that are used to treat them are becoming less effective because of antimicrobial drug resistance. Millions of people, especially those who are poor and living in developing countries, are also at risk from disabling communicable diseases such as polio, leprosy, lymphatic filariasis, and onchocerciasis. In addition to human suffering and permanent disability, these communicable diseases create an economic burden—both on the work force that handicapped persons are unable to join, and on their families and society, upon which they must often depend for economic support. Finally, the entire world is at risk of the unexpected communicable diseases, those that are called emerging or reemerging infections. Infection is often unpredictable because risk factors for transmission are not understood, or because it often results from organisms that cross the species barrier from animals to humans. The cause is often viral, such as Ebola and Marburg hemorrhagic fevers and severe acute respiratory syndrome (SARS). In addition to causing human suffering and death, these infections place health workers at great risk and are costly to economies. Infections such as Bovine Spongiform Encephalopathy (BSE) and the associated new human variant of Creutzfeldt-Jakob Disease (vCJD) in Europe, and avian influenza A (H5N1) in Asia, are reminders of the seriousness of emerging and re-emerging infections. In addition, many of these infections have the potential to cause pandemics, which are a constant threat our economies and public health security. Science has given us vaccines and anti-infective drugs that have helped keep infectious diseases under control. Nothing demonstrates 

Foreword

the effectiveness of vaccines better than the successful eradication of smallpox, the decrease in polio as the eradication program continues, and the decrease in measles when routine immunization programs are supplemented by mass vaccination campaigns. Likewise, the effectiveness of anti-infective drugs is clearly demonstrated through prolonged life or better health in those infected with viral diseases such as AIDS, parasitic infections such as malaria, and bacterial infections such as tuberculosis and pneumococcal pneumonia. But current research and development is not filling the pipeline for new anti-infective drugs as rapidly as resistance is developing, nor is vaccine development providing vaccines for some of the most common and lethal communicable diseases. At the same time providing people with access to existing anti-infective drugs, vaccines, and goods such as condoms or bed nets—necessary for the control of communicable diseases in many developing countries—remains a great challenge. Education, experimentation, and the discoveries that grow from them, are the tools needed to combat high mortality infectious diseases, diseases that cause disability, or emerging and re-emerging infectious diseases. At the same time, partnerships between developing and industrialized countries can overcome many of the challenges of access to goods and technologies. This book may inspire its readers to set out on the path of drug and vaccine development, or on the path to discovering better public health technologies by applying our present understanding of the human genome and those of various infectious agents. Readers may likewise be inspired to help ensure wider access to those protective goods and technologies. Such inspiration, with pragmatic action, will keep us on the winning side of the struggle against communicable diseases. David L. Heymann Assistant Director General, Health Security and Environment Representative of the Director General for Polio Eradication World Health Organization Geneva, Switzerland



1 Historical Overview The word plague is defined as a dangerous disease that spreads rapidly

and often causes death. It is synonymous with a cause of suffering or harm. If we mention the bubonic plague or, better yet, the Black Death, the name brings to mind a series of well-known historical events that resulted in the death and suffering of millions of people. But this is not just an event of the past. Each year, in the United States, between 10 and 30 individuals are infected with the organism that causes bubonic plague, Yersinia pestis. New Mexico alone averages seven to eight cases a year. There were no human cases in New Mexico in 2004, five human cases in 2005, eight in 2006, and five in 2007; six of those infected died during that period from 2005 through 2007.1 Yersinia pestis is endemic to the American Southwest; it is always present at low levels. Bubonic plague occurs in more than 20 countries worldwide, with an average of more than 2,000 total cases per year. The following is a fictionalized version of an event that occurred in New York City in November 2002. The names of the hospital, doctors, and hospital administrators are accurate. It should be remembered that both the city and country were still extremely concerned about bioterrorism in the wake of September 11, 2001, and the anthrax attacks in the fall of the same year. The death of several people from anthrax had everyone on edge, and none more so than the medical community. The Trip

Husband and wife John Tull and Lucinda Marker had saved for months in order to make the trip. They had been looking forward to seeing New York City in the fall. Although Santa Fe, New Mexico, was a medium

Historical Overview

sized city, it lacked the character and excitement of New York City. And there was also Ground Zero. The World Trade Center destruction area was a magnet for many, and John and Lucinda were among those who felt drawn to make the pilgrimage. Both John and Lucinda came down with fevers and flu-like symptoms at the same time, shortly before their trip. They took some over-the-counter medication and drank plenty of fluids. They were determined that this little discomfort was not going to stop their trip. And so, congested and feverish, they boarded the plane and winged their way to the Big Apple. New York City in the fall can be an imposing place. Winds blowing between the buildings and down the streets create a windchill factor that shocks visitors from the Southwest. Yet, John and Lucinda did not feel terribly cold because their fevers had not abated. Unfortunately, John was feeling weak and had difficulty walking for any length of time. Lucinda felt a swelling in her groin region, and the area in her armpits were feeling tender and also seemed swollen. Both agreed that they needed to contact a doctor. Before they left Santa Fe, they had had their family doctor provide them with the name of a physician they could contact in the New York area. When they contacted Dr. Ronald Primas and described their symptoms, he made an immediate appointment to have them examined. He was concerned that they might have smallpox, West Nile virus, anthrax, possibly the plague or — he hoped — just the flu. Once Dr. Primas saw the couple and examined Lucinda, however, it was clear that she probably had bubonic plague. John’s symptoms were also consistent with that diagnosis. That initial diagnosis was strengthened when the couple mentioned that a rat on their property had tested positive for the plague the previous July. The bacteria that cause the plague are constantly present in low concentrations in fleas that live on rats, squirrels, and domesticated dogs and cats throughout the Southwest.



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Number of Plague Cases by County, 1970–2002

Figure 1.1  Although many people think of plague as something from the Middle Ages, it is actually endemic, especially in the Southwest of the United States. Today, however, plague is rarely deadly as it was in medieval times. (Centers for Disease Control and Prevention [CDC])

Dr. Primas referred the couple to Beth Israel Medical Center. The patients were placed in isolation, and the New York City Department of Health was notified. None of the doctors in the hospital had ever seen a case of plague. Though the form of plague that the couple had was not contagious, it could become so if not treated within a few days. Health Department personnel gave the couple a new diagnostic test

Historical Overview

that involved checking their blood for the presence of certain types of protective proteins called antibodies. This test was part of a series of new tests developed since the September 11 attacks. The tests came back positive for bubonic plague. It was clear that the couple had contracted the disease back home in New Mexico and that this was not an instance of bio­ terrorism. After a few days of antibiotic treatment, Lucinda was released from the hospital and John was making a slow, modest recovery. The Present-day Plague

It may seem hard to believe that a disease that presumably caused the death of millions during the fourteenth, fifteenth, and sixteenth centuries is still active in the United States in the twenty-first century. In actuality, between 10 and 30 people in the United States are infected yearly. Plague appears in about 15 states, with most cases occurring in the West (Figure 1.1). According to the Centers for Disease Control and Prevention (CDC), between 1,000 and 3,000 people worldwide are infected annually. Dr. Thomas Frieden, health commissioner of New York City, indicates that half of all cases in the United States originate in New Mexico in Santa Fe County, the county where Lucinda and John live. Fleas that attach themselves to wild animals carry the disease organism. Fleas are the vectors, or carriers of the disease. The large rodent population in Santa Fe County is the suggested cause of the large number of infections in the area. The Black Death

Historical records suggest that a plague began in Egypt in a.d. 541, affecting a significant portion of the known world. It is estimated that 50 to 60 percent of the population died from that pandemic (a worldwide disease outbreak). Researchers at the University of Sheffield in England discovered archeological evidence in March 2004 suggesting that Egypt and North Africa might be the site of origin of the plague

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bacteria. Dr. Eva Panagiotakopulu and her colleagues found that the flea that is the major carrier of the plague organism was native to the Nile Valley. This flea is a parasite of the Nile rat. Around 3500 b.c., people began to build cities near the Nile, thus disturbing the Nile rats and providing a new place for the rats to visit—the newly constructed homes. Egyptian writings mention outbreaks of a disease with symptoms similar to the plague. The Ebers Papyrus, a medical text from 1500 b.c., identifies a disease that causes a swollen area, known as a bubo, filled with pus. It is not difficult to envision that as sea trade developed between Egypt and places such as India, rats on those seafaring ships picked up plague-loaded fleas and carried them to Europe and beyond.2 The second great plague pandemic began in 1346. It became known as the Black Death, or the Great Pestilence, because of the distinctive symptoms that the disease produced. Symptoms included a swelling of regional lymph nodes in the groin and under the arms. These swellings are called buboes. Ulcerations were common on the skin, but the most distinctive feature was the dark color beneath the skin where blood vessels had ruptured, turning the skin black in those areas. Hence, the disease was given the name the “Black Death.” Within five years of the 1346 outbreak, more than 13 million people had died in China, and, in Europe, the disease may have killed 3 out of every 4 people it infected. The plague continued over the next 300 years, killing between 20 and 30 million people in Europe. (It seemed to disappear around 1670.) Recent work in England suggests that victims of the Black Death in England between 1347 and 1351 were more often the weak rather than the strong. Evidence suggests that the victims were already in bad health, often from poor diets.3 Historical records indicate that a third pandemic began in China in the mid-1850s. Its distribution seems to have paralleled the expansion of the Chinese Empire. As Chinese troops moved into regions of Burma, India, and Hong Kong,

Historical Overview

the plague organism moved with them. It continued to spread aboard steamships that went to European and American ports. This pandemic ended around 1910. Whenever and however the plague organism became established in the United States, it spread into the western and southwestern portions of the country. Most human cases occur in northern New Mexico, northern Arizona, southern Colorado, California, southern Oregon, and far western Nevada. Between 1989 and 2003, 38,310 cases of plague were reported to the World Health Organization. These figures included 2,845 deaths from 25 countries.4 In 2002, the total number of reported plague cases in 13 countries was 1,925, of which 177 were fatal. In 2003, nine countries reported 2,118 cases including 182 deaths. These figures indicate a decrease when compared with the annual average figures (2,895 cases, 206 deaths) for the previous 10 years (1992-2001), when 28,956 cases with 2,064 deaths were reported from 22 countries. Cases of plague have been reported nearly every year during that same period from eight countries: Democratic Republic of the Congo, Madagascar, United Republic of Tanzania, Peru, United States, China, Mongolia, and Vietnam. Since 1994, there have been three new outbreaks of human plague that occurred in three countries after 30 to 50 years without reports of any new cases. The first was in India in 1994, where more than 5,000 cases were reported, resulting in disruption to travel and international trade, with devastating economic effects. A smaller outbreak of bubonic plague involving six cases occurred in Indonesia in 1997. The third was an outbreak in Algeria in 2003, where 10 confirmed cases and one probable case were reported.5 Bubonic plague continues to be endemic (constantly present, though with few cases) in many areas of the world. The Cause

The causative agent of bubonic plague was identified by Alexandre Yersin in 1894. He recognized that it was

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caused by a bacterium that ultimately was named after him, Yersinia pestis. Yersin, who worked for the Pasteur Institute in France, developed a treatment that was marginally effective in combating the disease and was the first to suggest that rats and fleas were responsible for transmission of the organism during the epidemic of 1894 in Hong Kong.

Figure 1.2  Alexandre Yersin, who identified the causative agent of the bubonic plague in 1894. (© SPL/Photo Researchers, Inc.)

Historical Overview

Figure 1.3  Alexandre Yersin in front of his hut in Hong Kong, where he isolated the plague germ. (CDC)

In an attempt to produce a vaccine to protect people against bubonic plague, Yersin inoculated horses with increasing doses of the plague organism that had been killed by heat. Yersin’s team of assistants then had to remove blood from the horses. They separated the fluid portion (the plasma) from the blood cells and inactivated the various proteins that cause blood to clot. The plasma with the clotting factors removed is known as the serum and is the portion of the blood that contains the antibodies against the plague organisms. These antibodies are designed to neutralize the protein toxins released by the bacteria. The vaccine varied in quality, and horses frequently died from the process. Yersin had hoped to produce more than 7,000 doses of the vaccine but was persuaded to go to India with only his available vaccine doses.

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In 1897, Alexander Yersin left the Pasteur Institute and entered Bombay Harbor with 700 doses of his antiplague serum. After two months of treating patients, nearly half had died. A new batch of serum from France increased the survival rate to 80 percent. However, the Bombay government began to restrict Yersin’s access to patients. He was allowed to inject only those patients who were already too far advanced in their illness to be helped. When the Pasteur Institute recalled him, Yersin left Bombay without regret. The symptoms of bubonic plague usually begin to emerge within one to seven days after a person is bitten by an infected flea. Symptoms usually include fever and swelling in the regional lymph nodes in the groin, armpit, or neck regions. There are many additional symptoms that may occur. The disease organism may spread to the lungs or bloodstream and the central nervous system. The fatality rate when this happens is nearly 100 percent. Black Death Was Not Necessarily Bubonic Plague

Not everyone believes that the bubonic plague organism caused the plague known as the Black Death. Two scientists from the Liverpool University School of Biological Sciences in England have published a book that places Europe’s plague in a new historical, geographical, and demographic perspective. Using parish burial registers, Susan Scott and Christopher Duncan have shown that, although many of the symptoms match the bubonic plague, other factors, such as incubation times and transmission agents, do not. In their book, The Biology of Plagues, they show that the incubation period is too long and that the type of rats most likely to carry the infected fleas did not migrate into England until 50 years after the plague had ended in 1670. Quarantine measures are ineffective against bubonic plague, but they were successful against the Black Death. Scott and Duncan concluded after

Historical Overview

Simond Says Dr. Paul-Louis Simond picked up plague research where Alexander Yersin left off. The prevailing sentiment of the day was that transmission of the plague was human to human. Yersin had hypothesized that rats were the main vectors. In spite of the prevailing wisdom, Simond began to uncover the evidence that would show that the fleas carried by rats were the main vectors of the disease. He developed a deceptively simple experiment that showed that the fleas killed the rat and then sought a new warm host—a human being. Simond published his work with the simple recommendation that the key to controlling plague was to keep rat populations in check. His work was met with silence or scorn. He remains a forgotten hero.6

an exhaustive review of described symptoms and primitive autopsy results that the Black Death was probably caused by a virus distantly related to Ebola. James Wood, professor of anthropology and demography at Penn State University, shares the opinion that bubonic plague was not the cause of the Black Death. Wood and several colleagues and graduate students have analyzed bishops’ records of the replacement of priests during the time period of 1349 to 1350. From these records, Wood concluded that the disease spread much too rapidly among humans without being established in a wild rodent population first. Additionally, there were no records of widespread die-offs of rats in the streets or countryside. The disease spread rapidly along roadways and rivers, and was not slowed by the geographical barriers that normally would have stopped or restricted the movement of rodents. Wood and his colleagues have not ruled out the possibility that the causative agent might be an ancestor of the modern plague organism that mutated into the present form.

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plague The French and English Continue to Disagree

In another twist, researchers from Oxford University and Barts and London Hospital attempted to confirm reports of a French team that claimed to have isolated bits of Yersinia pestis DNA from the teeth of disinterred plague victims. The English team presented their results to the Society of General Microbiology at a conference in Manchester, England, in September 2003. They were unable to replicate the results of the French team. They found no Yersinia pestis DNA in any of the teeth of the victims they examined. They suggested that one future possibility would be to find a plague victim buried in the permafrost with enough preserved DNA to make a final determination. Whether or not the Black Death and the bubonic plague are one and the same, the bubonic plague has caused enormous human devastation and has resulted in many changes in health and medical practices.

2 Causes of the Plague Donner Memorial State Park near Truckee, California, closed on Tuesday,

August 27, 2002. It would not reopen again until the spring of 2003. This would not seem to be a terribly unusual event except that the park normally stayed open much longer. The immediate cause of this closing was confirmation of the plague in two squirrels, and the appearance of plague symptoms in a cat from a nearby park campground. Chipmunks and squirrels in the area had previously tested positive for the bubonic plague organism. “The Truckee area has a history of infected animals,” said Vicki Kramer, chief of vector-borne diseases for the California health department. It has been quiet in the Donner Pass region recently, but other sections of the West and Southwest have picked up the slack. In 2006, a campground at Natural Bridges National Monument in Utah was closed for three weeks after the plague organism was detected in chipmunks and mice in the area. Colorado had several cases of plague involving cats and squirrels. The cats were thought to have become infected by eating infected rodents. In 2007, a monkey at the Denver zoo died of bubonic plague. It was thought the monkey contracted the disease by eating an infected squirrel. Five other squirrels and a rabbit were found dead of the plague on the grounds of the zoo. Arizona saw its first case of human plague since 2000 in September of 2007. Nearly two months later Eric York, a wildlife biologist working at Grand Canyon National Monument, was found dead at his home. Test results showed he had died of the plague. Evidence suggests he probably contracted the disease as the result of performing an autopsy on a dead mountain lion.1 In 2008, a Boy Scout from Connecticut was diagnosed with the plague. He and other scouts had been doing service projects in Yellowstone, 19

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Bridger-Teton National Forest, and other sites in Wyoming. Before that, the last case of human plague in Wyoming had occurred in 2004. In Santa Fe County, New Mexico, the plague capital of the United States, a cat contracted the plague in its pneumonic form in January 2008. This respiratory form is considered the most deadly. The cat coughed constantly, and it was feared it might have sprayed family members with the deadly organism. The family members were treated with antibiotics, and all survived. Unfortunately, the cat died.2 Origin of the Plague Organism

There are three human pathogenic, or disease-causing, species of the genus Yersinia: Yersinia pestis, which is the causative agent of the plague; Yersinia pseudotuberculosis; and Yersinia enterocolitica. The latter two species cause intestinal problems. Y. enterocolitica (for simplicity, genus names are often abbreviated), the most common of the three species and a very common form of foodborne sickness, causes inflammation of the intestines, a condition known as gastroenteritis. Y. pseudotuberculosis, the least common of the three, causes fever and abdominal pain that may mimic the symptoms of appendicitis. Many scientists suggest that the bubonic plague organism has been around for millions of years. Dr. Victor Suntsov, of the Severtsov Institute of Ecology and Evolution in the Russian Academy of Sciences, suggests that the plague organism originated between 15,000 and 20,000 years ago in Mongolian marmots (small rodents similar to squirrels). Suntsov contends that the plague microorganism evolved as a mutant form of Yersinia pseudotuberculosis in a population of marmots in Mongolia, Manchuria, and Transbailkal. What Are Bacteria?

Living things on this planet currently are placed into five large categories called kingdoms. These are the Kingdoms

Causes of the Plague

Plantae (plants), Animalia (animals), Fungi, Protista, and Prokaryotae (bacteria). The cells of all living organisms are organized in one of two ways. Cells from the plant, animal, fungi, and protista kingdoms all contain compartments constructed from internal cellular membranes. These compartments, which help separate chemicals and other materials from the interior of the cell, are called organelles, or miniature organs. Organelles include the nucleus, the lysosome, and the mitochondria. This type of cellular organization, with clearly defined and identifiable organelles, is described as the eukaryotic type of cellular organization. The term eukaryotic means “true nucleus” and comes from the Greek eu, meaning “true,” and karyon, meaning “nucleus.” Therefore, cells of plants, animals, fungi, and protozoa are known as eukaryotic cells, or eukaryotes, because of their internal cellular organization. The bacteria, or Prokaryotae, kingdom includes cells with a different type of internal organization. Bacterial cells lack membrane-defined organelles, are normally smaller than eukaryotic cells, and have few clearly defined internal structures. Because bacteria lack organelles such as the nucleus, they are described as being prokaryotic cells. The word prokaryotic comes from the Greek pro, for “before,” and karyon, meaning “nucleus.” Because bacterial cells do not have a nucleus, the genetic information (a single circular DNA molecule) resides in the cell with no membrane structure surrounding it. This type of cellular organization has served bacteria well for over 3.5 billion years. Important Bacterial Structures

Bacteria were originally classified as very small plants because, like plants, they have a cell wall protecting their cell membrane and the interior of the cell. However, bacterial cell walls are made of molecules that are different from the molecules in plant cell walls. Bacterial cell walls are made of

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Archaea — A Third Form of Life Another group of microorganisms also has a prokaryotic type of cellular organization. They are called the archaebacteria, or simply the archaea, and they resemble bacteria in size and prokaryotic organization. However, the archaea are quite different genetically from both eukaryotic and prokaryotic types of cells. Archaea contain genetic information similar to the eukaryotes and also genetic information similar to bacteria. In addition, more than 40 percent of their genetic information is totally unique, resembling neither eukaryotes nor bacteria. The archaea live in extreme environments, earning them the name “extremophiles.” Extreme environments include the absence of oxygen or places with very high heat, such as inside volcanoes.

a molecule called peptidoglycan that contains amino acids, which are building blocks made of proteins (peptido) and carbohydrates, which include simple sugars such as glucose (glycan). Because this unique molecule is not found among eukaryotes (including humans), the human immune system tries to remove or destroy it. Bacteria have differing amounts of peptidoglycan in their cell walls. When treated with different dyes, these differences in the amount of peptidoglycan and other factors cause bacterial cells to retain or lose the color of specific dyes (Figure 2.1). One very famous and important staining reaction used to differentiate between bacteria is the Gram’s stain process. In this process, bacteria are treated with a series of differentcolored dyes. If the bacteria retain the first dye color (crystal violet) throughout the entire staining procedure, they are called gram-positive bacteria. Other bacteria lose the first dye in the process and take on the color of the last dye (called the counterstain). These bacteria look pink or light red and are called gram-negative bacteria.

Causes of the Plague

Figure 2.1  One way to test for the presence of the plague bacterium, Yersinia pestis, is with a procedure known as a Wright’s stain, which takes a blood sample from a suspected plague patient and uses dye to detect bacteria in the blood. The darkened stains that indicated that the person has Yersinia pestis can be clearly seen on this sample of a Wright’s stain. (Centers for Disease Control and Prevention [CDC])

It is also important to know that bacteria can move on their own if they possess a structure called a flagellum (a whip-like tail). A flagellum (plural is flagella) helps the bacterium move toward areas where there is food and security and away from harmful areas. Flagella are made from a unique protein called flagellin. Because humans do not produce this protein, when the human immune system recognizes flagellin, it works to destroy it. The unique molecular structure of bacterial cell walls and flagella is important to our understanding of the vaccines and drugs used against bubonic plague. For example, the unique structure of the peptidoglycan molecule makes it a target for anti­biotics, such as penicillin, that have been very successful in killing the bacteria that cause many illnesses, including bubonic plague.

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plague Characteristics of Yersinia pestis

Bacteria come in three basic shapes. A single bacterium shaped like a rod or a pencil is known as a bacillus (plural is bacilli). A bacterium shaped like a circle or a sphere is known as a coccus (plural is cocci). A bacterium that is shaped like a spiral or the letter “c,” or is wound tightly like a spring, is known as a spirillum (plural is spirilli). The bacterium responsible for bubonic plague is called Yersinia pestis. It is named after Alexandre Yersin, who discovered it in 1894 while investigating the plague in Hong Kong. Yersin had originally named the organism Pasteurella pestis in honor of his mentor, Louis Pasteur. It is a small, stubby, gram-negative, oval- to rod-shaped bacterium. It is a facultative anaerobe, an organism that uses oxygen when it is present but can also live and reproduce in an anaerobic (oxygen-free) environment. Yersinia pestis tends to grow slowly in culture and produces small colonies. When treated with certain dyes and staining techniques, Yersinia pestis creates an unusual staining pattern called bipolar staining, which mimics a closed safety pin. Though this bipolar staining is not unique to Yersinia pestis, it is one of its many distinctive diagnostic characteristics. Factors Involved in Yersinia’s Virulence

Yersinia pestis can multiply within a wide range of temperatures (–2°C to 45°C; 28.4°F to 113°F) and pH values (5.0 to 9.6), but optimal growth occurs at 28°C (82.4°F) and at a pH of about 7.4. The bacterium is nonencapsulated, or lacking a capsule or envelope, when it grows at its optimal growth temperature of 28°C. When grown at temperatures higher than 28°C, the organisms produce an envelope glycoprotein, called fraction 1 (F1). The genes for production of this glycoprotein are encoded in a plasmid. The F1 glycoprotein serves as an antiphagocytic capsule and allows the bacterium to obtain iron from its host. It also plays a role in the survival of the bacteria within the gut of a flea.

Causes of the Plague

The bubonic plague organism, like other members of its family, the Enterobacteriaceae, is gram-negative. This is the same family that includes the common intestinal bacterium Escherichia coli and the Salmonella organisms, including the one that causes typhoid fever. The cell walls of gram-negative bacteria contain less peptidoglycan than those of gram-positive bacteria. The outermost layer of the cell wall of gramnegative bacteria also contains lipopolysaccharide (LPS) and proteins. Because LPS is toxic to mammals, it is called an endotoxin. When the bacterium dies, the LPS becomes free in the serum. Blood is divided into the formed elements (red and white blood cells and platelets) and the fluid portion, known as the plasma. When clotting factors, such as various proteins, are removed or neutralized, the light yellow fluid that is left is the serum. The serum contains the immune proteins known as antibodies; when free LPS interacts with these antibodies, a number of problems arise in humans, including fever, changing blood cell counts, and leaking blood vessels, which can lead to shock. This outer layer, also called the outer membrane, has an inside and an outside. The outside of the membrane contains the LPS. Part of this polysaccharide chain is a series of repeating sugar units known as O antigen. The name is derived from the fact that the polysaccharide is exposed to the outer environment. Host defenses can hone in on these sugars; however, bacteria can change the makeup of the O antigen to confuse the human host’s immune system. The outermost region of the cell wall is made of an inner phospholipid layer and an outer lipopolysaccharide layer. Just outside the cell membrane is a layer of peptidoglycan. Yersinia’s cell wall is unlike that of other members of the Enterobacteriaceae family due to lack of O antigen sidechains. These O antigen side-chains are missing because a group of the bacteria’s genes is disrupted. The V (virulence) and W antigens are protein-lipoprotein complexes in the cell wall. These antigens make it difficult for white blood cells to engulf the bacteria. One important reason

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that Yersinia pestis is so virulent is its ability to survive and multiply within cells of the immune system. Antibodies produced against the V antigen provide protection against the plague organisms. Although it is not clear exactly how the V antigen works, it is obvious that it does multiple things. Any plague vaccine would include V antigen as an important component. A protein called invasin on the outer membrane aids the bacterium in attachment to human cells. Another protein called plasminogen activator is an enzyme that aids in the spread of the bacteria. This enzyme prevents fibrin molecules from creating a clot formation that would trap the bacteria at the site of the fleabite. It has been suggested that a single gene mutation may be responsible for the level of virulence in Yersinia pestis. Writing in the May 2008 issue of the journal Microbiology, Professor Robert Brubaker of the University of Chicago explained that the organism needs calcium to be able to grow at body temperature. When calcium is not available to the organism, it produces an excessive amount of the amino acid, aspartic acid. Most bacteria produce the enzyme aspartase, which breaks down aspartic acid; Yersinia pestis lacks the ability to produce this enzyme. The subsequent buildup of aspartic acid in the patient causes an imbalance in the amino acid pool, which seems to increase virulence and lethality. Brubaker suggests that developing a therapy that would reduce the aspartic acid load would reduce the death rates.3 Needling the Host Cell

How do bacteria deliver toxins into a cell? The answer for a number of human pathogens, including Salmonella, Shigella, Chlamydia, Escherichia coli 0157:H7, and Yersinia, is a molecular machine called the type III secretion system. The structure looks like a thumbtack or pushpin that you might put into a bulletin board (Figure 2.2). This molecular machine is made of 29 proteins that, when assembled, “function as a syringe-like organelle spanning the cell

Causes of the Plague

Figure 2.2 Plague has a unique way of infecting itself into a host cell. Once the bacterium’s virulence genes have been activated, Yersinia pestis clamps on to a cell and acts almost like a syringe to force itself past the cell membrane. This process is sometimes referred to as “Yersinia’s deadly kiss.”

membrane and anchored in the inner and outer membranes” of the cell wall, as explained by Dr. Susan Straley from the University of Kentucky. Straley further explains how the needle-like molecular machine works: “When the needle—the part that sticks out—comes in contact with a host cell, it punctures the cell and samples the cytoplasm. In response to the low calcium environment inside the cell, the bug’s [bacteria’s] secretion mechanism is activated, and it sends toxins right into the host cytoplasm.”4 The toxins Straley mentioned are Yops (Yersinia outer p roteins). Eleven of these proteins have been identified. They are structurally and functionally diverse, and include protein kinases, protein phosphatases, proteases, and GAP proteins.

27

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plague

These proteins collectively overcome the host’s natural defenses and make it difficult for the phagocytic white blood cells to become aware of the bacteria’s presence. Once inside the host cell, they disrupt the cytoskeleton of the host cell, allowing the bacteria to multiply and spread within the host cell. “These Yops mimic key cell biological reactions and modify them in a way that is advantageous to the bacteria,” Straley says. “One of the bug’s early high-priority activities is to prevent being engulfed and destroyed by macrophages and PMNs (polymorphonucleated neutrophils); three of these Yops participate in that.” To summarize, the Yersinia pestis organism reproduces most rapidly at 27 to 28°C (80.6–82.4°F) but does not produce the protein toxins that are responsible for human symptoms at that temperature. When the microbe encounters a human cell with a temperature range of 36 to 38°C (96.8–100.4°F), its rate of reproduction slows, but it begins to produce the apparatus needed to inject these proteins into its new human host. Also, at the higher temperatures, the microbes produce a capsule that makes it difficult for white blood cells to engulf them. The Life of the Flea

Fleas survive best in a climate that is warm and moist—15 to 20°C (59–68°F) and 90 to 95 percent humidity. Changes in the seasons lead to temperature and humidity fluctuations that affect the life span and the level of activity of fleas. In most flea species, Yersinia pestis reproduces most rapidly at temperatures in the range of 26 to 28°C (78.8–82.4°F). A flea acquires the disease by biting an infected host such as a rat or squirrel. After the microbe enters the flea’s body, it reproduces rapidly and creates a plug of bacteria in the region between the esophagus and the stomach. This plug prevents effective digestion by the flea. When the flea bites a human, the plug of bacteria is regurgitated into the new host’s wound. The plague bacteria now begin to reproduce in the host’s blood, causing the typical symptoms of plague and, possibly, the death of the host.

Causes of the Plague

Figure 2.3 Some pathogens, such as the plague-causing bacterium Yersinia pestis, escape phagocytosis and thus destruction by a host cell by commanding the cell to commit suicide. By releasing a protein into the host cell (blue cell at top), Yersinia activates host-cell proteins called caspases. Once activated, caspases trigger events in the cell that break down the DNA in the nucleus, switch off and disintegrate the mitochondria (the energy-producing cell organells), and precipitate a rearrangement of the cell’s surrounding plasma membrane. This chain of events finally leads to decomposition of the cell into smaller pieces and release of the bacterium into the tissue. The green cells are macrophages, which have ingested the cell pieces that have undergone apoptosis.

diFFereNT maNiFesTaTioNs oF The plague

After the flea has found its new mammalian host, it takes a bite and injects some of the plague bacilli into the host’s bloodstream. Some of the host’s immune system cells—large, modified white blood cells called macrophages—are invaded by the plague organisms before the immune system recognizes

29

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the danger. Ironically, the bacteria find a safe place to live inside those cells whose job is supposed to be to destroy foreign organisms. As the immune system becomes aware of the invasion, phagocytic white blood cells called polymorphonucleated neutrophils (PMNs) begin to gear up for action. The PMNs destroy many of the bacteria but are overwhelmed by the sheer numbers of the microbes. Additionally, the microbes begin to produce capsules and other proteins that prevent destruction by the PMNs. At this point, the microbes may enter the lymphatic system. This fluid-filled system runs parallel to the circulatory system and has a series of collection regions known as lymph nodes. The lymph nodes have large accumulations of PMNs, macrophages, and other chemical agents. The bacteria that enter the lymphatic system are filtered through the lymph nodes, and a battle is joined between the bacteria and the immune system cells and chemicals. This battle causes an inflammatory reaction that signals additional cells to join the fight. It also results in a swollen lymph node known as a bubo. This feature is diagnostic of the bubonic form of the plague. It usually takes from 2 to 7 days for a swollen lymph node to develop after the fleabite. The lymph carries the bacteria back into the bloodstream, where they enter the liver and the spleen. Many bacteria are undoubtedly killed by immune system cells in these organs, but their sheer number tends to overwhelm the body’s defenses. Some bacteria can wind up in the lungs, producing a highly contagious and extremely dangerous form of plague. Known as pneumonic plague, the bacteria can be spread from person to person by coughing. According to Dr. Susan Straley, inhaling as few as 100 of the bacteria can cause pneumonic plague. Given the fact that scientists estimate each square centimeter of our skin holds an average of 100,000 bacteria, these 100 bugs pack an extraordinary punch.

Causes of the Plague

Because the inhaled bacteria have already adapted to the body of the first person, they arrive fully armed with their toxin-delivery mechanism. Straley says this gives the bacteria a head start in the race against the body’s defenses. As in bubonic plague, the bacteria first encounter macrophages — in this case, alveolar macrophages, the lungs’ defense cells. “Some of the bacteria go into macrophages, you get an influx of PMNs and then inflammation. Part of inflammation is fluid influx, so your lungs fill up, and you can’t breathe,” says Straley. “The bacteria also spread to the lymph nodes and the blood, so the basic process is the same as in bubonic plague, but we really know very little about the specifics. As little as one day after inhalation you start showing severe symptoms of pneumonia, and the next day you could be dead.” Straley adds that this is a very narrow window for correct diagnosis and treatment. Septicemic plague may occur as a complication of either bubonic or pneumonic plague. It is not spread from person to person but is characterized by the presence of bacteria in the bloodstream. Of the three forms, pneumonic plague is the most deadly and the fastest killer.

31

3 Cats, Rats, Prairie Dogs, and Squirrels Jim was going to spend a few days with a friend in Alberquerque,

New Mexico. Jim had met Zachary in India that previous summer at the All India Institute as part of a program to learn about the health-care system of a large urban city. When their time in India ended, the new friends exchanged e-mail addresses and promised to visit each other.1 Zachary lived south of Santa Fe, about 25 miles (40 km) away from Albuquerque. He was a student at the University of New Mexico in Albuquerque. Mid-April in Albuquerque and Santa Fe meant high temperatures ranging between 60 and 70°F (15.6–21.1°C) and low temperatures between 35 and 45°F (1.7–7.2°C). The previous year had been wetter than normal. First Stop—The Hospital?

When Jim stepped off the plane, Zachary was waiting to greet him. They got into Zachary’s car and headed out of the airport. “The university is along the way, so we’ll stop there and you’ll also get a chance to meet my dad,” proclaimed Zachary. “That’s great,” said Jim. “Does your father work at the university?” asked Jim. “No,” said Zachary, “he’s in the university hospital.” “What’s wrong with him?” asked Jim, hoping he had not made the trip at the wrong time. “He’s got bubonic plague,” stated Zachary, showing little emotion in his voice. 32

Cats, Rats, Prairie Dogs, and Squirrels

“bubonic plague!” shouted Jim. “Apparently you didn’t do your homework before you came to this area of New Mexico,” said Zachary. “If you had, you would have known that New Mexico has an average of seven to eight cases of plague each year. The Santa Fe area has become somewhat famous in the last few years for plague cases. Have you heard of John Tull and Lucinda Marker?” asked Zachary. “Can’t say that I have,” replied Jim. Zachary continued: “They are a married couple who became infected by the plague organism from the fleas of a dead rat on their property near Santa Fe. They went to New York for a vacation and wound up in the hospital there. Caused quite a stir at the hospital, since none of the doctors had ever seen a plague case before, and now they had two patients at once. John was in bad shape and spent a long time recovering.” Zachary explained that John became so ill that the doctors had to amputate both of his legs below the knee. Zachary’s car pulled up next to a building with a sign that said Health Sciences Center. “Here’s where I spend most of my time on campus. We can walk over to the university hospital after I take you on a quick tour of the facilities.” Jim was happy to get out and stretch his legs again, but he was flabbergasted at this turn of events. He had never seen a person who had bubonic plague. If he remembered his microbiology correctly, the plague was also called the Black Death. What would Zachary’s father look like? Would his extremities be all black and gangrenous? Zachary didn’t seem that concerned. As a matter of fact, Zachary appeared to be no more concerned than if his father had contracted a cold. As they walked, Zachary explained how his father had contracted the disease. Weekend at Camp

Three weeks before, Zachary’s father, Al, had visited a friend in an area near Bluewater Lake in northwestern New Mexico, not far from Albuquerque. They were going to do some repairs on the friend’s hunting cabin. Rodents had gnawed through some

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of the wooden planks, and the cabin was open to the environment. Among the unwanted guests in the cabin were a number of dead rock squirrels. Since it had been a tough winter, the dead squirrels did not seem unusual. Al and his friend used gloves and carefully removed the squirrels and burned their bodies in a large bonfire. They noticed that the squirrels had acquired large numbers of fleas. Some had crawled onto Al’s arms and bitten him. His friend was also bitten by the fleas. They used a pesticide spray to kill the fleas. Unfortunately for them, the damage had already been done. After a weekend of repair work on the cabin, Al headed for home. He was beginning to feel like he was coming down with the flu. His throat was sore, his body ached, and he felt alternately feverish and then chilled. He would have dismissed all of these symptoms except for the fact that he was starting to feel weak and had itchy fleabites on his arms and legs. The regional lymph nodes under his arms and in his groin region were swollen and tender to the touch. Al called home on his cell phone and spoke to Zachary. He told Zachary he was going directly to the hospital and would call after he had checked in. Al had lived in New Mexico long enough to know that symptoms like his could easily mean bubonic plague. His friend joined him in the hospital the following day. Al had indeed contracted bubonic plague. Going directly to the hospital had saved his family from having to take antibiotics. If he had come home and interacted with the family, all of the members would have to take antibiotics as a precaution against contracting the disease. Without prompt treatment, the first symptoms would be flulike—fever and chills, headache, tiredness, and constipation or diarrhea. The most obvious sign would be an enlarged lymph node, called a bubo, in the area adjacent to the flea bite. Incubation takes one to six days, when sepsis sets in. Abdominal involvement may lead to muscle tenderness and potential vomiting, seizures, or bleeding. Depending on the area of the bite, there may be gangrene as well.

Cats, Rats, Prairie Dogs, and Squirrels

As Zachary and Jim got to the hospital room, Al greeted a somewhat hesitant Jim with a big smile and the reassurance that he was not contagious. Al was responding nicely to the antibiotic therapy and was looking quite well. He would soon be coming home. If Jim had not known of the illness, he would never have guessed that Al had bubonic plague. He now understood why Zachary did not appear to be concerned about his father’s health. What Types of Animals Carry the Plague Organism?

As is true with many diseases, humans are not the primary host of the disease organism Yersinia pestis. Human infection is accidental or incidental, and depends on the chance contact

Figure 3.1  Tree squirrels are a known carrier of Yersinia pestis. (© Anatoli Dubkov/shutterstock)

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between humans and the infected fleas of infected rodents. In other words, Zachary’s dad was in the wrong place at the wrong time. Animal hosts, such as the rock squirrels, usually begin to die off in increasing numbers before humans are likely to be infected, because the fleas of the dying rodents will be seeking new hosts. Zachary’s dad did not see or recognize the increase in the number of dead squirrels. He only saw the dead squirrels in the cabin. Different Levels of Infection

Some animal groups are fairly resistant to the impact of the plague organisms. Mice of the genus Microtus or the genus Peromyscus are considered enzootic reservoirs of the infection. This means that the frequency of the plague organism is maintained at a low level in the population of host organisms (the mice), resulting in low mortality rates. This difference in

Figure 3.2  The Norway rat, shown here, is another carrier of the plague bacterium Yersinia pestis. (Centers for Disease Control and Prevention [CDC])

Cats, Rats, Prairie Dogs, and Squirrels

Figure 3.3  Plague is spread through the bite of infected fleas, which acquire the bacterium from rats and other rodents that are carriers of Yersinia pestis. This is an oriental rat flea, or Xenopsylla cheopis. The dark mass in its lower body, which scientists call a proventricular plague mass, is evidence that the flea is infected with plague. (CDC)

resistance to the plague organism varies from one geographical region to another. In contrast, there are a number of animal species that are highly susceptible to the Yersinia pestis organism. These species seem to be clustered in the western part of the United States and represent the greatest threat to the human population. The hosts are mostly rodents that die in large numbers, causing the fleas to change hosts. This group of highly susceptible animals is called an epizootic reservoir. Included in this group are urban and domestic rats, ground squirrels, rock squirrels, prairie dogs, gerbils, voles, chipmunks, marmots, guinea pigs, and kangaroo rats. Although rodents get most of the blame, larger mammals can also introduce infected fleas to humans or to other animal species. Coyotes, badgers, rabbits and hares, deer, antelope, goats,

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PLAGuE

sylvaTiC aNd urBaN plague When plague organisms are found mainly in the fleas of rodents, such as squirrels, in areas away from where humans live, the disease is called sylvatic plague. Infection is accidental and incidental, and is usually not the cause of epidemics. When plague organisms are found in the fleas of rats in and around densely populated cities, the disease is called urban plague. This kind of plague has traditionally been the cause of epidemics. Sylvatic Cycle

Urban Cycle

Figure 3.4 Although humans most often get plague from the bite of an infected flea, this diagram shows some of the other possible ways to catch the disease.

Cats, Rats, Prairie Dogs, and Squirrels

39

Table 3.1 Plague Testing by Species, Colorado 2007 Species

Positive

Negative

Total Tested

Cat

8

70

78

Prairie Dog

4

22

26

Flea

13

17

30

Rabbit

3

44

47

Rock Squirrel

0

3

3

13-Liner Squirrel

0

6

6

Tree Squirrel

50

251

301

Monkey

1

0

1

Bear

0

1

1

Mouse

0

5

5

Rat

0

1

1

Dog

0

5

5

Beaver

0

1

1

Fox

0

6

6

Lynx

2

1

3

Raccoon

0

2

2

Coyote

17

95

112

Total

98

530

628

Source: Colorado Department of Public Health and Environment, “Plague Testing by Species, Colorado 2007, as of 08/31/07,” http://www.cdphe.state.co.us/dc/zoonosis/plague/Plague_ cases_07.pdf.

camels, cats, and sometimes dogs can all transmit the fleas to human beings. As humans and their animal pets continue to expand their living quarters into the habitats of wild mammals and rodents, increased contact is inevitable. With this increased contact comes the likelihood of transmitting disease organisms between species. An international group of scientific researchers working in Kazakhstan have linked climatic changes to epidemics and large-scale outbreaks of bubonic plague among humans. Evidence shows that as the spring weather became warmer and the summers wetter the organisms causing the plague became

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more ubiquitous. It seems that the fleas that carry the plague bacterium become more active when temperatures rise above 50°F (10°C). Warm, frost-free spring weather allowed the fleas to begin breeding earlier, thus allowing the population to increase in size. If this was followed by a moist, humid summer the flea population continued to grow. The study showed that a two-degree rise in spring temperatures led to an almost 60 percent increase in the incidence of the disease. The researchers used tree-ring analysis to show that the weather conditions at the time of the major plague pandemics were both warmer and wetter.2

4 Diagnosis About 20 minutes after leaving the hospital, Zachary pulled into the driveway

of his home. Zachary’s brother Garrett greeted them. “Dad called and said you were on the way and that Jim had arrived on time,” said Garrett. “I made some coffee and Mom said you could have some of the cake in the refrigerator. She’ll be home in about an hour.” Zachary thanked his brother as he poured a cup of coffee for himself and one for Jim. After bringing in Jim’s luggage and getting him settled in the guest room, the two young men sat out on the back porch and enjoyed the warmth of the coffee and the crispness of the fresh air. “You wanted to know how they figured out that my dad really had the plague. You asked the right person, so sit back and I’ll give you an overview of what happens,” said Zachary. Trying to diagnose a disease strictly on the basis of its symptoms is not very useful, Zachary explained, since many diseases have similar symptoms. Everything from the flu to various types of food poisoning may have symptoms including headache, fever, chills, body aches, and overall weakness. Diagnosis of bubonic plague requires that health-care workers carry out laboratory tests on the patient’s blood, sputum (material coughed up from the lungs), or the fluid taken from an enlarged or swollen lymph node (a bubo). Suspected plague organisms can be stained using a Gram’s stain or Wayson’s stain. Usually, a chest X-ray is taken to determine whether the plague organism is causing pneumonia. Diagnosis of the different forms of the plague involves modifications of some of the techniques. Forms of the Plague

The same bacterium can cause three different forms of plague. The bubonic form is best known because of the swollen and tender lymph nodes called

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Figure 4.1  A plague patient with a swollen lymph node, known as a bubo. (Centers for Disease Control and Prevention [CDC])

Figure 4.2  An X-ray of a plague infection involving both lungs. Rapidly developing pneumonia is one of the first signs of plague. (CDC)

Diagnosis

buboes. If the organisms invade the bloodstream, they may cause destruction of blood vessels under the skin, leading to the darkened or blackened areas that gave the Black Death its name. Usually, the patient’s high fever, over 104°F (40˚C), leads to delirium and, in more than half of all untreated cases, death. The bacterium may invade the lungs and cause a pneumonic form of the disease. This form is extremely contagious and is the most dangerous form, since it is spread from person to person through aerosol droplets. Every time the patient coughs or sneezes, he or she may contaminate people in the area. Victims of this form of plague usually develop a severe cough and eventually bloody sputum. Pneumonic plague leads to coma and death in nearly 100 percent of untreated cases. The third form of plague occurs when large numbers of the bacteria enter the bloodstream. This is known as the

Figure 4.3  One possible effect of the plague is the development of gangrene, in which the affected body part turns turn black because of insufficient blood flow. This person is suffering from gangrene of the fingers. Gangrene is extremely dangerous because it can lead to the amputation of the rotted body part. (CDC)

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septicemic form. It normally causes gangrene in various body areas, and death usually occurs within a day or two. Untreated, all infected people die from this form of plague. Diagnosis of Bubonic Plague

The incubation period for the bubonic form of the plague ranges from 2 to 10 days. Patients are often tired, have a fever as high as 105°F (40.6°C), and exhibit tender lymph nodes, particularly in the groin region or areas near the fleabite. The liver and spleen may also be tender. About one-fourth of all bubonic plague patients may have skin lesions. A presumptive diagnosis is usually made microscopically when gram-

Summary of Laboratory Diagnosis for Suspected Plague Cases Staining of Specimens • Appropriate clinical specimens include: Blood, bubo aspirates, sputum, cerebrospinal fluid (CSF) (if there are signs/symptoms of meningitis), and skin scrapings (if a lesion is present). • Gram’s stain: Polymorphonuclear leukocytes and bipolar staining, “safety pin” ovoid, gram-negative coccobacilli identified in bubo aspirate, sputum, blood, or CSF are highly suggestive of plague. • Wayson stain: Yersinia pestis appears as light blue bacilli with dark blue polar bodies on a contrasting pink ground. • Immunofluorescent staining of capsule (F1): A positive finding is diagnostic. Must use fresh specimens to avoid false negatives. This test is available only at reference laboratories.

Diagnosis

negative coccobacillary cells show a “safety pin” or bipolar staining pattern. This staining pattern is the result of granules that are localized in the ends of the cell. The sample may be taken from a lymph node, sputum, or cerebrospinal fluid. If it is available, immunofluorescent staining can also provide useful information. A definitive diagnosis is made when the organisms are cultured on various microbiological media such as blood agar, MacConkey agar, or infusion broth. Y. pestis does not have many enzymatic functions, such as adenine deaminase, aspartase, ornithine decarboxylase, glucose-6-phosphate dehydrogenase, and urease. It primarily utilizes glucose and

Bacterial cultures • Blood, bubo aspirates, sputum, CSF, and skin scrapings can be cultured. • Materials should be inoculated into blood and MacConkey agar plates and infusion broth. It generally takes two days to identify visible colonies. Rapid biochemical identification systems may not be reliable for identification due to slower growth rate of Y. pestis. Serologic Testing • Several serologic tests are available, including a passive hemagglutination test. A fourfold or greater rise is diagnostic, a single titer (concentration) of >1:16 in someone without prior immunization against plague is suggestive. Serology is not useful for rapid diagnosis. Source: Medical Treatment and Response to Suspected Plague: Information for Health Care Providers During Biologic Emergencies, New York City Department of Health, Bureau of Communicable Disease. Available online at http://www.nyc.gov/html/doh/html/cd/plaguemd.shtml.

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mannitol, and cannot ferment most carbohydrates. It grows on sheep blood agar with little or no hemolysis, and forms gray-white colonies with a shiny appearance after about 48 hours. The colony borders are irregular, and are often termed “fried-egg.” Positive cultures that display the above characteristics are considered evidence of Y. pestis infection, and are reported and forwarded, if necessary, to state authorities for further identification and confirmation. Laboratory Diagnosis and Safety

Microscopic examination of stained samples from body fluids and the use of immunofluorescent stains provide the first evidence of the possibility of Yersinia pestis infection. The diagnosis is confirmed through serology, in which characteristics of a disease are shown through the study of proteins in blood serums, but because serologic tests can take several days to perform, it is not useful if a rapid diagnosis is needed. Serologic tests check levels of antibodies to specific Yersinia antigens. These levels are low early in disease. If antibodies against the bacterial capsule (F1 antigen) are found to have increased at least fourfold a few weeks after infection in a patient with no history of plague vaccination, then this confirms the diagnosis. Handling Laboratory Specimens

Laboratory work on those suspected of having Y. pestis infection should be done in biosafety level 2 facilities, using standard and special practices, equipment, and facility specifications. For example, staff must wear surgical gloves, protective gowns, and shoe covers. Staff must make every effort to avoid splashing or creating an aerosol, and must wear protective eyewear and masks if work cannot be done in a biosafety level 2 cabinet. Yersinia pestis is classified as a select agent, one with the potential to pose a severe threat to public health and safety, and, therefore, its handling is regulated under strict federal guidelines. These provide requirements for laboratories that handle

Diagnosis

Figure 4.4  The Laboratory Response Network (LRN) was founded in 1999 by the Centers for Disease Control and Prevention to ensure an effective national bioterrorism response system. The LRN was reorganized in 2002 into sentinel, reference, and national laboratories. Sentinel (formerly Level A) laboratories include most clinical labs with at least biosafety level 2 (BSL-2) containment. These labs receive samples, rule out ordinary pathogens, and refer for additional testing. Reference (formerly Levels B and C) laboratories include mostly state or local public health labs with BSL-3 containment facilities with access to nonpublic testing protocols and chemicals. These labs perform limited confirmation testing and refer for additional testing. They also perform diagnostic identification tests, and refer for additional testing. National (formerly Level D) laboratories have BSL-4 containment facilities. They perform the definitive identification testing for the highest level of agent characterization.

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select agents such as Y. pestis, including registration, security risk assessments, safety plans, security plans, emergency response plans, training, transfers, record keeping, inspections, and notifications. The requirements went into effect on February 7, 2003. They supersede earlier government requirements for the handling and transfer of particular agents. In 1999, a network of laboratories was developed by the Centers for Disease Control and Prevention—partnered with the Federal Bureau of Investigation and the Association of Public Health Laboratories—to coordinate all clinical diagnostic testing for suspected bioterrorism events. The Laboratory Response Network (LRN) links public health laboratories to private hospital, clinical, and referral labs that normally send suspected select agents to the public health labs for confirmation. The Laboratory Response Network depends upon voluntary cooperation among laboratories. It requires that labs assess their capabilities relative to the biosafety requirements needed to perform the types of analysis normally done by labs at biosafety levels 1 and 2 (BSL-1 and BSL-2).1 The LRN was originally divided into four categories: Levels A through D, based on their capabilities. In 2002 the LRN was reorganized (see Figure 4.1). There are now only three categories of labs (Figure 4.4): sentinel (formerly level A), reference (formerly levels B and C), and national (formerly level D). Sentinel laboratories can perform the standard initial tests designed to rule out (but not definitively identify) Y. pestis. Sentinel labs include Clinical Laboratory Improvement Act (CLIA)–certified clinical laboratories with BSL-2 safety equipment and practices. The protocols for sentinel laboratories include Gram’s stains, spot tests, and other simple tests such as motility. The intent is to rule out agents of bioterrorism. The majority of these labs are hospital based, clinical institutions and commercial diagnostic laboratories. These labs play a key role in the early detection of biological agents. Reference laboratories have the ability and capacity for agent isolation and confirmatory testing and include most state public health laboratories. These laboratories are expected to

Diagnosis

49

Summary of recommended biosafety levels (BSL) for infectious agents s

BSL Agents

Practices

Safety Equip- Facilities ment (Primary (Secondary Barriers) Barriers)

1

Not known to cause disease in healthy adults

Standard microbiological practices

Not required

Open bench top sink required

2

Associated with human disease; hazard: auto- inoculation, ingestion, mucous membrane exposure

BSL-1 practice plus: limited access, biohazard warning signs, “Sharps” precautions, biosafety manual defining any needed waste decontamination or medical surveillance policies

Primary barriers: Class I or II BSCs* or other physical containment devices used for all manipulations of agents that cause splashes or aerosols of infectious materials; PPEs*: laboratory coats, gloves, face protection as needed

BSL-1 plus: autoclave available 







3

Indigenous or exotic agents with potential for aerosol transmission; disease may have serious or lethal consequences

BSL-2 practice plus: controlled access, decontamination of lab clothing before laundering, baseline serum

Primary barriers: Class I or II BSCs or other physical containment devices used for all manipulations of agents; PPEs: protective lab clothing, gloves, respiratory protection is needed

BSL-2 plus: physical separation from access corridors; self-closing, double door access; exhausted exhausted air not recirculated; negative airflow into laboratory

4

Dangerous/exotic agents that pose high risk of life-threatening disease, aerosol-transmitted lab infections, or related agents with unknown risk of transmission

BSL-3 practice plus: clothing change before entering, shower on exit, all material decontaminated on exit from facility

Primary barriers: all procedures conducted in Class III BSCs or Class I or II BSCs in combination with full-body, air-supplied, positive pressure personnel suit

BSL-3 plus: separate building, or isolated zone, dedicated supply/ exhaust, vacuum, and decon systems, other requirements outlined in the text









* BSCs = Biosafety cabinets * PPEs = Personal protection equipment View the entire CDC/NIH Biosafety Guidelines online at http://www.research.umich.edu/policies/um/committees/BRRC/BSLChartCDCNIH.html.

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perform biochemical identification of Brucella, Francisella, Yersinia and Bacillus species to rule in agents of bioterrorism. They require a minimum of biosafety level 2 and usually 3. Some of the reference laboratories have advanced capacity for rapid identification. They require BSL-4 safety equipment and practices. There are more than 100 state and local public health, military, veterinary, agriculture, food and water testing laboratories included as reference labs. In addition, there are facilities in Australia, Canada, and the United Kingdom that serve as reference laboratories outside of the United States. National laboratories have the highest level of containment (BSL-4) and expertise in the diagnosis of rare and dangerous biologic agents and include specialized federal laboratories. Specific information about LRN reference and national laboratory protocols for Y. pestis are not available to the public.2 The National Medical Society provides a very general online diagnosis of plague based on the patient’s symptoms. The Centers for Disease Control and Prevention (CDC) provides a very extensive online manual with a self-assessment at the end. This is all part of Emergency Preparedness and Response by the CDC.

5 Treatment After dinner with Zachary’s family, Jim and Zachary sat down with another

cup of coffee and talked more about the plague. Zachary said, “It’s really amazing when you consider that an average of only 10 Yersinia pestis microorganisms need to invade the body in order to cause bubonic plague. But it’s equally amazing to me that our bodies use secretions of other bacteria to kill off the first bacteria. I know that this is a natural series of events that has been occurring for millions of years. This competition has helped to maintain a balance between microbes and larger organisms like humans. It’s hard to believe that until less than a century ago, we had few ways to aid us in the fight against bacterial diseases. You tend to take antibiotics for granted until they are needed to cure someone close to you.” Early Treatments for the Plague

Since their discovery and development in the 1940s, antibiotics have taken the lead in fighting the effects of bacterial diseases. Prior to the use of antibiotics, however, people employed a number of unique and unusual treatments. Some, such as the use of garlic and silver compounds to ward off infection by bacteria, were based on folk medicine passed down through generations. Others, such as trying to purify the air with rosemary and thyme, were based on uncritical human observations that sometimes led to false conclusions. Before the discovery and study of microorganisms such as bacteria, European medical thought was dominated by what an Italian historian named Carlo Cipolla has called the “miasmatic paradigm.” (Miasma refers to a thick, vaporous atmosphere.) Contagion was thought to 51

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arise from exposure to unhealthy, “corrupted” air, although the exact nature of this dangerous influence was unspecified. In Seville, Spain, in the 1600s, city officials made residents clean the streets, burn the clothing and bedding of the sick, and isolate people who were clearly ill. The officials recommended that people add rosemary and thyme to the air for the purpose of purification. Variants of these techniques continue to be used today in some disease settings; for example, the burning corresponds to sterilization of materials, and isolation of people with disease is obviously a method of quarantine. In today’s world, we know that disease transmission and prevention is related to sanitary conditions. A clean environment reduces the likelihood of disease organisms being spread from animals to people and between people. That is why some of the early treatments for the plague, such as bathing in human urine, wearing human excrement, and placing dead animals in homes (called “stinks”), seem so strange to us. The use of leeches (a worm-like animal that sucks blood) was a common mechanism for treating many illnesses. Drinking molten gold (gold heated until it melted) and powdered emeralds was also recommended but was usually fatal. Some suggestions, such as eating figs before 6:00 a.m., chopping up a snake every day, falling asleep on the left side of the bed, or not sleeping during the day, make no obvious medical sense and seem to be home-remedy suggestions. People from all lifestyles sought ways to protect themselves from disease. Grave robbers used garlic to protect against bubonic plague by washing themselves, their clothes, and stolen items with garlic vinegar. French priests of the Middle Ages used garlic as well as prayer to protect themselves against bubonic plague. Some people thought that coffee had powers great enough to cure the plague. During the plague of 1664–1665, Gideon Harvey, a well known human physiologist, published a book entitled Advice Against the

Treatment

Figure 5.1  During the Middle Ages (between 500 and 1500), the plague was a terrible threat to humans. It killed vast numbers of people and devastated the population of Europe. Those who could afford to do so often fled cities during warmer weather, which was considered “plague season.” This illustration depicts the plague of London in 1665, which killed tens of thousands. (© Science Source/Photo Researchers, Inc.)

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Plague, in which he advised drinking coffee in large amounts. A 1721 publication by Richard Bradley entitled The Virtue and Use of Coffee with Regard to the Plague and Other Infectious Distempers; Containing Most Remarkable Observations also promoted the notion that coffee protected from the plague. Neither Harvey nor Bradley proved to be correct, but coffee continued to grow in popularity nonetheless. Current Treatment

When left untreated, plague can result in rapid death. Approximately 14 to 17 percent of all plague cases in the United States each year are fatal. However, if treatment is received early enough, 5 out of 6 patients survive. “My father increased his chance of survival by going directly to the hospital,” said Zachary. He informed the hospital admissions personnel that he thought he had bubonic plague because of his symptoms and his contact with fleas. Because plague was suspected, Zachary’s father was immediately isolated, and local and state departments were notified. Antibiotic treatment reduces the risk of death to less than 5 percent. Streptomycin is the preferred treatment and should be given immediately upon admission to the hospital. Other possible useful anti­biotics include gentamicin, chloramphenicol, tetracycline, and trimethoprim-sulfamethoxazole. Doxycycline is also used for the treatment of plague and is approved by the Food and Drug Administration (FDA) for this indication. Rifampin, aztreonam, ceftazidime, cefotetan, and cefazolin have been shown to be ineffective and should not be used to treat plague. The Working Group on Civilian Biodefense also developed consensus-based recommendations for treatment of pneumonic plague during a bioterrorist attack. The Working Group made the recommendations outlined in Table 5.1. The patient’s doctor will determine the specific treatment for plague based on a number of factors, including

Treatment

Figure 5.2  A pharmacist creates streptomycin, an antibiotic first produced in 1943. Streptomycin is the preferred treatment for plague. (Hulton-Deutsch Collection/Corbis)

the patient’s age, overall health, and medical history. The patient’s ability to tolerate specific medications, procedures, or therapies will also be considered. In each case, the patient will be hospitalized and medically isolated and given immediate antibiotic treatment. The isolation continues for 48 hours after antibiotic therapy is begun or until cultures are negative. Gloves are worn at all times when in contact with

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the infected individual and hospital personnel must avoid contact with all bodily fluids (urine, sputum, saliva, blood, and semen). Treatment of Pneumonic Plague

Treatment for the pneumonic form of plague is different from other forms because it can be spread from person to person by droplet transmission (i.e., coughing and sneezing). When caring for patients with suspected or confirmed plague, hospital personnel must be especially cautious. Patients with pneumonic plague should be placed on strict respiratory isolation until appropriate anti­biotics have been administered for 48 hours, and the patient shows clinical improvement. Droplet precautions require that the patient be placed in a private room and that anyone entering the patient’s room wear a surgical mask, particularly when standing within three feet (one meter) of the patient. Because transmission can occur from plague skin lesions (such as draining buboes or abscesses) to contacts, if such skin lesions are present, wound and skin precautions should be followed. If a person becomes exposed to airborne or aerosolized Yersinia pestis or comes into close physical contact with a patient who has a confirmed case of pneumonic plague, he or she must receive antibiotic therapy referred to as post-exposure prophylaxis, to prevent disease. Individuals to whom this precaution applies would include people in the same household and health care workers. All antibiotic therapy should continue for seven days from the date of the last exposure to the organism or infected patient. Treating Environmental Surfaces

Since September 11, 2001, governmental officials have been concerned about the possible use of bacteria such as anthrax and plague as biological weapons by terrorists. Law enforcement officials have the right to test for the presence of these organisms if it is suspected that an environmental surface has

Treatment

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Table 5.1 Recommendations from the Working Group on Civilian

Biodefense for Antibiotic Postexposure Prophylaxis During an Outbreak of Pneumonic Plague Following a Bioterrorism Event Choices by Patient Category

Therapy Recommendations*

Adults: Preferred choices

Doxycycline, 100 mg PO [orally] twice daily for 7 days†‡ or Ciprofloxacin, 500 mg PO twice daily for 7 days‡§

Adults: Alternative choice**

Chloramphenicol, 25 mg/kg PO 4 times daily for 7 days††

Children: Preferred choices

Doxycycline: if >45 kg, give adult dosage; if 25 mcg/mL can cause reversible bone marrow suppression. The oral formulation is available only outside the United States. §§According to the Working Group, children younger than two years of age should not receive chloramphenicol. Source: Center for Infectious Disease Research & Policy, “Plague: Current, comprehensive information on pathogenesis, microbiology, epidemiology, diagnosis, and treatment,” http://www.cidrap.umn.edu/cidrap/ content/bt/plague/biofacts/plaguefactsheet.html (updated March 24, 2009)/html#five.

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NonToxic Disinfectant Kills Bubonic Plague American Biotech Labs has a disinfectant called ASAP-AGX-32 that has been approved by the Environmental Protection Agency (EPA) for use against gram-negative bacteria, including Yersinia pestis. The company was interested in determining how well the disinfectant would actually work on Y. pestis. It did its work at a biosafety level 3 (BSL-3) laboratory, testing the disinfectant against a concentration of 81 million bacteria per milliliter (mL), a dose that is 160 times greater than the 500,000 per mL that is the normal in vitro (performed in a test tube) test dosage. Even at this higher bacterial load, all bacteria were killed in less than 2 minutes. “The ASAP-AGX32 (EPA registration No. 73499-2) product has already been approved by the EPA for use against gram-negative bacteria, and Y. pestis is a gram-negative bacterium,” said Keith Moeller, vice president of American Biotech Labs. “We wanted to test the limits of the product’s capability, and we are delighted with the results.” Another unique aspect of the disinfectant is that it is odorless and colorless. “Unlike other disinfectants, the ASAP-AGX-32 can be used or sprayed around both adults and children with no toxic effect. The product is not known to irritate the skin, eyes, nose, or lungs,” noted Moeller. It would seem that this product may prove to be very useful in a variety of medical and industrial settings. “To date, this product has killed every strain of every bacterium on which it has been tested. It has received EPA approval for use against the most deadly bacteria, including gram-negative, gram-positive and even nosocomial or hospital acquired (superbug) pathogens. The product has been approved for use as a broad spectrum, general use surface disinfectant in homes, hospitals, and medical settings,” said Moeller.1

Treatment

been contaminated. Jim and Zachary would be in minimal danger, since Yersinia pestis is not known to remain viable for very long periods on most external surfaces. Some surfaces, however, are an exception. Cells of Yersinia pestis have been shown to live up to 6 hours on steel, 7 hours on glass, 24 hours on polyethylene, and 120 hours on paper. Very few commercial chemicals have been marketed specifically for destruction of this organism. These types of samples must be tested at a Reference or National LRN laboratory.

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6 Prevention There are a number of basic, commonsense methods that can be effective

in the prevention of the plague. Preventing the spread or transmission of the plague bacterium requires that the carriers or vectors of the disease must be controlled. This normally means that rodents and their fleas are the targets. The fleas must be eradicated prior to or at the same time as the rodent population is eliminated to prevent the fleas from finding a new host to feed upon. In addition, vaccination or prophylactic use of antibiotics is advised for people who may be exposed to the fleas of infected rodents. Changes in sanitary conditions that would eliminate shelter or food for potential rodent vectors are possible long-term solutions. Killing all the organisms and all the fleas carried by these organisms would not be practical or desirable. Early Means of Prevention

In the past, many of the same types of remedies that were thought to be useful in treating the plague were sometimes used to prevent it. In keeping with the belief that the disease was spread through the air, many preventive measures involved odor. It was thought that if one carried flowers or wore a strong perfume, the odors would help keep away the disease. People believed carrying a lucky charm could ward off the disease, although there is no indication what that charm should consist of. Smoke from pipes was thought to repel the disease. After the year 1350, plague patients were placed in so-called pesthouses, where people with diseases were isolated from the general population. Ships coming from plague-infested areas were quarantined for 40 days until the disease could die out.

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Prevention Current Means of Prevention

Almost every brochure, magazine, medical pamphlet, or Web site about the plague suggests the same type of advice regarding prevention. To reduce the risk of contracting the plague, food sources and possible nest sites used by rodents should be eliminated. Every attempt should be made to rodent-proof homes, buildings, warehouses, or feed sheds. Chemicals that kill fleas and rodents are effective but can also cause health risks to humans and, therefore, should usually be applied by trained professionals. Trained professionals who can, if necessary, fumigate cargoes should inspect ships and docks. It is imperative that individuals avoid handling wild animals such as rats, cats, rabbits, and squirrels, especially in the western part of the United States. It is a good idea to avoid handling any sick or dead animal. Hunters should always wear gloves. Pets should receive weekly flea treatments with flea powder (especially in areas where plague is present). People should stay away from areas that are known to harbor rats or seem likely to harbor rats. If people anticipate that they might be exposed to rodent fleas, they should apply a permethrin-containing repellent to clothing, shoes, and all camping gear. Permethrin is a synthetic broad-spectrum insecticide, similar to pyrethrin, a naturally occurring insecticide. Ineffective when applied to your skin, permethrin is very effective and durable on clothing and gear. Insect repellants that contain N,N-diethyl-m-toluamide (DEET) should be applied to the skin and to clothing. However, because of toxicity, only formulations containing approximately 30% DEET should be used. DEET products should not be applied to infants under two months of age or to women who are pregnant or breastfeeding. DEET is designed for direct application to human skin to repel rather than kill insects. DEET was developed by the U.S. Army in 1946 and was registered for use by the public in 1957. Approximately

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140 products containing DEET are currently registered with the EPA by about 39 different companies. Home on the Range, Where the Ferret and Prairie Dog Roam

In the spring of 2007, the CDC confirmed that black-tailed prairie dogs in the Buffalo Gap National Grasslands in southwestern South Dakota were infected with the plague organism. These prairie dogs are the prey of the endangered black-footed ferret. The ferret is one of the rarest mammals in North America, and its successful reintroduction into the area was widely hailed as an ecological success. The relationship between the ferrets and prairie dogs is important because the prairie dogs support many members of the mixed-grass

Figure 6.1  Technicians in Wind Cave National Park, South Dakota, spray prairie dog burrows with insecticide in an attempt to kill fleas and prevent the spread of plague. (National Park Service)

Prevention

prairie ecosystem in addition to the ferret. The ferrets help to keep the prairie dog population from exploding and destroying the grasslands. To help increase ferret survival during this outbreak, biologists are vaccinating ferrets with an experimental vaccine. The vaccine was originally developed for human use by the U.S. Army. More than 40 ferrets have been captured and vaccinated with an initial shot followed by a booster a month later. The USGS National Wildlife Health Center in Madison, Wisconsin, is working on an oral vaccine that can be delivered by being put into bait, thus reducing the need to handle the animals.1 Not far from Buffalo Gap is Wind Cave National Park. Because plague has been found within 25 miles of the park boundary, there is concern that hikers and wildlife can be affected. In an attempt to prevent this potential public health threat, the staff of the National Park, working with several other government agencies, dusted about 1,100 acres of prairie dog burrows. The crews used ATVs to complete the work in a 10week period. This dusting technique has shown to be effective in other areas of the country such as Montana.2 For those who have been exposed to animals infected with the plague bacterium or who must be present in an area where plague is present in the animal population, it is advisable to begin treatment with prophylactic antibiotics. Antibiotics such as doxycycline or tetracycline or any of the sulfonamides are recommended. In June 2007 an estimated 2 billion rats invaded 22 counties surrounding Dongting Lake in central China. Homes in the area had been flooded prior to the invasion. Global warming is also blamed for allowing rats to thrive in the woodlands. These events caused China’s Ministry of Health to revise an emergency response plan on the prevention of rat plague. The plan classified plague into four categories ranging from “extremely serious” to “ordinary” and requires relevant departments to set up a national plague emergency command

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Bubonic Plague Hits San Francisco 1900 –1909 On March 6, 1900, an autopsy on a dead Chinese man in San Francisco, California, revealed organisms that resembled those that cause plague. Despite public officials’ immediate concerns over the potential health threat this discovery might pose, there were political issues that hampered them from taking immediate action. At the time, anti-Chinese sentiment was running strong. Many whites disliked the Chinese immigrants, who were blamed for taking jobs away from white American workers. Upon finding plague in the dead man, San Francisco officials decided to quarantine Chinatown, the section of the city where most of the Chinese population lived. Both Chinese residents and members of the business community protested, although the businesses were worried more about the effects that the idea of plague in their city would have on business than about the rights of the Chinese. The city ended the quarantine, but instead carried out inspections of each house in Chinatown. Eventually, two more people were found to have died from plague. At this point, the city made a formal announcement that there was an outbreak of plague in San Francisco. When the governor of California refused to acknowledge the announcement, the U.S. Surgeon General obtained presidential permission to enact laws to help stop the plague. Still, the state’s officials refused to admit there was a problem, and in the meantime, additional cases of plague turned up. Only in 1903, when a new governor came to office, was a concentrated effort made to help the board of health put an end to the epidemic. By February 29, 1904, when a woman from Concord, California, died of plague, the epidemic was over—at least temporarily. The area had counted 121 cases in the city of San Francisco and 5 outside, with a total of 122 deaths. A few cases of plague were reported again in 1907, but this time, San Francisco, as well as other cities, were better

Prevention

Figure 6.2  Ratcatchers, such as this one in New York in 1908, used ferrets to catch and kill rats. (Library of Congress)

prepared. Officials offered rewards for people who killed or captured potentially infected rats. This method, which worked well, was later used by other cities and states to help control outbreaks of plague.3

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with close cooperation between different agencies when the incident is considered “extremely serious.”4 Vaccination

Unfortunately, there is no vaccine against plague currently available in the United States. A licensed vaccine was available until the manufacturer discontinued its production in 1999 for financial reasons. This vaccine had been used against bubonic plague transmitted by fleabites but did not protect against pneumonic plague. Though data about the effectiveness of this vaccine are limited, the vaccination did seem to provide protection for military personnel during the Vietnam War. The vaccine caused antibody production against the F1 capsular antigen. New vaccine development is currently aimed at using live, attenuated strains of the bacterium (Yersinia pestis) or, more commonly, developing vaccines that target specific molecules on the cell surface of the plague organism. However, in January 2003, the U.S. Defense Department granted Avant Immunotherapeutics Incorporated of Needham, Massachusetts, an $8 million contract to develop an oral vaccine against Yersinia pestis and Bacillus anthracis, the causative agent of anthrax. This vaccine is being developed to provide military personnel fast-acting protection against these two potential agents of biochemical warfare. Avant will not only design the vaccine but will also carry out all of the necessary laboratory investigations prior to human testing. In the March 2003 issue of Vaccine, a veterinary products company named Heska published research about a vaccine to prevent the spread of plague in animals. The company had previously worked with the U.S. Geological Survey’s National Wildlife Health Center on an experimental vaccine to prevent plague in prairie dogs. The new vaccine uses the same technique as the vaccine used to immunize mice against plague. The research is designed to develop a vaccine that would be delivered orally to wild animal populations by embedding it in food left as bait in areas frequented by rodents.

Prevention

Figure 6.3  Vaccine immunity.

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plague Vaccines Outside the United States

Although it is not available in the United States, a vaccine against bubonic plague is manufactured and is available in some countries, including Australia. The plague vaccine can be given to both adults and children. For adults and children older than 12 years of age, two doses of the vaccine are needed and are given one to four weeks apart. Children under 12 years of age require three doses of the vaccine spaced one to four weeks apart. To achieve the possibility of full protection, all doses must be received. It takes the body several weeks to develop a sufficient supply of antibodies to provide protection. Even then, the vaccine cannot ensure 100 percent protection against the disease. People who live in areas where plague is active and widespread should receive a vaccine booster shot every six months. Other groups of individuals who should consider vaccination include people who work in the laboratory or in the field with the plague organism, including veterinary doctors and staff working with potentially infected animals. Since 2003, a number of initiatives have been undertaken to develop vaccines against the plague. These will be discussed in Chapter 9.

7 The Problems of Antibiotic Resistance Antibiotics are chemicals produced by living organisms, usually fungi or

bacteria, that destroy or inhibit other microorganisms or their ability to reproduce. Since the mid-1950s, an increasing number of microbes have shown antibiotic resistance, and are no longer killed or inhibited by particular antibiotics. The initial relief that came from the ability to conquer disease through use of antibiotics such as penicillin and streptomycin has become muted as more microbes have found ways to neutralize or inactivate the antibiotics. Fortunately, antibiotic resistance in Yersinia pestis continues to be rare. Development of widespread antibiotic resistance would be a significant threat given that antibiotics are used for both plague treatment and for prevention of human-to-human transmission. The National Academy of Sciences has suggested that it may cost as much as $30 billion annually to treat antibiotic-resistant infections. Microorganisms have lived in close proximity to each other in the soil and water for millions of years. Often, they compete for the same scarce resources. To succeed in such a competitive environment, microorganisms find biochemical means of destroying or inactivating chemicals produced by other organisms. This ongoing process in which one organism produces destructive chemicals and another finds ways to destroy the first organism is nature’s way of maintaining a balance among the microbes in the soil and water. The two main issues to address regarding antibiotic resistance are why and how it occurs.

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plague Why Resistance Occurs

Simply put, antibiotic resistance develops due to the abuse, misuse, and overuse of antibiotics. For example, antibiotics are ineffective against viruses, yet patients often press their doctors to prescribe anti­biotics for viral maladies, including colds or influenza. If antibiotics are taken for a viral infection, they destroy some of the helpful bacteria in the intestines. Bacteria, both “good” and “bad,” are in constant competition in the intestines. Destruction of the “good” bacteria provides opportunity for the unfettered growth of “bad” bacteria. Another reason antibiotic resistance occurs is the failure of the patient to follow instructions. When antibiotics are prescribed, the patient is directed to take all of the antibiotics provided. Often, patients stop taking antibiotics as soon as they start to feel better, even though there are still bacteria in their bodies. By not completing a full course of antibiotics, only the least resistant of the offending bacteria are killed off, leaving the most resistant alive. These more resistant bacteria survive and reproduce, which means that the next time the person is infected, it will take a higher dosage or a different antibiotic to kill the same bacteria. Microorganisms also can become resistant to antibiotics when antibiotics are used in low doses in livestock and agriculture. Antibiotics are often mixed with animal feed. The amounts are too small to be used for treating a disease, but they do destroy the least hardy bacteria. The most resistant bacteria survive. Part of the rationale for giving antibiotics to livestock is to keep the animals healthy and allow them to grow rapidly without competition from internal parasites in the form of intestinal bacteria. Antibiotic residues are also often found on fruits and vegetables that have been sprayed to reduce premature decomposition. If we are not careful about washing fruits and vegetables before we eat them, we may kill off some of our useful bacteria, allowing the more resistant and harmful organisms to survive. On June 3, 2002, the New York Times documented the discovery of the

The Problems of Antibiotic Resistance

antibiotic chloramphenicol in shrimp imported from Asia. Agricultural use of this antibiotic is banned in the United States because it has been shown to cause problems such as childhood leukemia. Finally, antibiotics may be available without a prescription in some countries. If a person taking an antibiotic is not aware of the limitations and potential problems that can occur when some bacteria survive antibiotic treatment, an environment may be created in which resistant organisms not only survive a course of antibiotics, but also grow more and more resistant over time. How Resistance Develops

Microorganisms have an amazing ability to adapt very rapidly to new and changing environmental conditions. They often have a reproductive rate that doubles their numbers in minutes. Both Escherichia coli and Staphylococcus aureus can double their numbers every 12 to 20 minutes under optimal conditions. Bacteria have genetic material that is extremely flexible. It consists of a single chromosome and a number of small chunks of DNA called plasmids, which look like miniature chromosomes. Plasmids contain a limited number of genes that are often genetic codes for enzymes and other proteins that provide resistance to one or more antibiotics. There are a number of ways that bacteria can develop resistance to antibiotics. One of these methods is through mutations of their genetic information or DNA. A mutation is a change in the genetic information of a cell or virus. A single mutation can spread through a population of bacteria in a matter of hours. If that mutation provides a way for bacteria to survive in the presence of a particular antibiotic, the genetic change will soon be found in millions of newly resistant bacteria. When bacteria die, other bacteria often scavenge the DNA of the dead microbe and incorporate it into their own genetic programs. Called transformation, this

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new information may contain genetic codes for inactivating or neutralizing various antibiotics. Some bacteria can exchange copies of plasmids. A copy of a plasmid is passed through a protein tube called a pilus into another bacterium, during a process called conjugation, making it possible to exchange genetic information between live bacteria (Figure 7.1). In a sense, this was the first information highway; it has been active for millions, perhaps billions, of years. Some of this new genetic information may provide the recipient bacterium with a competitive advantage in its environment. Bacteria often are infected by specific viruses. As a virus infects one bacterium, it takes over the cell and forces the bacterium to produce and assemble new virus parts. As the new virus is constructed, it may incorporate some of the bacterial DNA into its own genetic program. When the new viruses infect other bacteria, they may leave some of the incorporated bacterial DNA in the newly infected bacterium. This process is called transduction and, like the others mentioned earlier, this method provides new genetic information to a bacterium that may allow it to become resistant to one or more antibiotics. Bacteria have evolved a number of mechanisms at the molecular level that allow them to resist antibiotics.

• Often, an antibiotic must attach to a particular structure such as the cell wall or a protein receptor in the cell membrane. If the bacterium changes the target molecule in some way, the antibiotic will not be able to attach to the bacterium and will be ineffective.

• Some bacteria take the antibiotic into their cells and surround it with a membrane of proteins. This effectively prevents the antibiotic from interfering with a biochemical process or locking on to a receptor molecule.

• Certain bacteria produce chemicals such as enzymes that inactivate or destroy the drug. One example is gonorrhea

The Problems of Antibiotic Resistance

Figure 7.1 Bacteria can transfer antibiotic resistance in three ways. In the process of conjugation (a), a bacterium injects its mutated genes into another bacterium through a special connection. A virus can also give its resistance genes to a new bacterium in a process known as transduction (b). Resistance genes can also be integrated into a new bacterium when another bacterium dies, in a process called transformation (c).

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plague microbes that produce a chemical called penicillinase, which neutralizes penicillin. These microbes are designated penicillinase-producing Neisseria gonorrhea (PPNG). • Other species of bacteria may contain structures, such as a capsule, which keep the antibiotic from penetrating into the cell. • Some bacteria have molecular pumps called efflux systems that actively remove antibiotics or other unwanted chemicals from the cell. Current Research

A group of researchers at Old Dominion University in Norfolk, Virginia, has developed a way to study the efflux mechanisms of specific gram-negative bacteria. Efflux systems are designed to pump out of the bacterial cell substances such as antibiotics that the cell considers harmful. Dr. X. Nancy Xu and her colleagues from the department of chemistry and biochemistry combined two types of microscopy with a fluorescent dye called ethidium bromide. Their method allows for real-time study of individual live cells for an extended period. The group found that individual cells vary greatly in the rate at which they pump out the antibiotics. Since the usual study method is to look at what happens with the majority of cells, being able to see individual cell differences will allow for earlier detection of the resistance. The short-term goal of the group is to understand the nature of multidrug resistance in the bacteria so that new therapies and drugs that target the pump mechanisms may be developed. “The purpose of this understanding of multidrug resistance is to be able to use a very low dose of drugs, for fewer side effects,” said Dr. Xu. Xu suggested that the group also hopes to use this knowledge to construct and bioengineer an efflux pump that could possibly sense and deliver drugs.1

The Problems of Antibiotic Resistance

The National Institute of Allergy and Infectious Diseases (NIAID) is a component of the National Institutes of Health (NIH). A major part of its job is to support research efforts to prevent, diagnose, and treat infectious and immune-mediated illnesses. NIAID supports research to study the molecular mechanisms responsible for drug resistance and to develop new chemical interventions for disease treatment and prevention. NIAID spends $200 million annually on antimicrobial resistance research.2 Research using gene-sequencing techniques can identify the critical molecules involved in microbial reproduction. These molecules may then serve as targets for new drugs. Research conducted so far in the area of drug resistance has led to: • The discovery of methods that will make it possible to reverse resistance to antibiotics.

• An understanding of the genetic reasons why some parasites become resistant and the ability to recognize the organisms that are, in fact, resistant.

• New molecular level mechanisms and tools that make it easier to recognize those species that are resistant to specific drugs.

• New drugs being created to combat the drug-resistant strains of Plasmodium, the organism responsible for the disease malaria.

An editorial by David Dennis and James Hughes, both from the CDC, appeared in the September 4, 1997, issue of the New England Journal of Medicine. In the editorial, Dennis and Hughes addressed the problems associated with emerging infections, particularly with respect to growing antibiotic resistance in the plague bacillus and other bacteria carried by a variety of vectors such as fleas, ticks, flies, and mosquitoes. They suggest that the first line of defense against these emerging

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An Antibiotic-resistant Plague Organism In 1995, a 16-year-old boy in Madagascar was diagnosed with bubonic plague. The particular strain isolated from the boy was identified as 17/95 biotype orientalis. This particular strain was resistant to eight different antibiotics, including ampicillin, chloramphenicol, kanamycin, streptomycin, spectinomycin, the sulfonamides, tetracycline, and minocycline. Resistance to most of these antibiotics was the result of the bacteria being able to produce chemicals that neutralized or inactivated the antibiotics. Genetic analysis of the plasmid DNA found in the 17/95 strain showed that the plasmid contained fragments of another plasmid normally found in Escherichia coli, a normal intestinal bacillus. This raises the question of how and where the Y. pestis bacteria acquired the E. coli plasmid. There are a number of places where both microbes would be found together, including inside and outside the mammalian host. Of greater concern is the fact that plasmids are transferable between species, increasing the likelihood that more multidrug-resistant Y. pestis will appear again. In October 2002, the question of how and where Yersinia acquired the E. coli plasmid appeared to be answered. A group of researchers showed that Yersinia acquires antibiotic-resistant plasmids from E. coli by horizontal transfer within the midgut of the flea. It is clear from the article that close physical contact between the E. coli and the Y. pestis plasmids leads to highfrequency conjugative genetic exchange. The article suggests that this may have been the source of the antibiotic-resistant strain isolated from the boy in Madagascar.

diseases would be a system of domestic and global surveillance and response. Dennis and Hughes further indicated that there are four basic requirements for such a system. The first would

The Problems of Antibiotic Resistance

be the ability to detect clusters of new and unusual diseases and syndromes. Second would be the capability to identify and characterize the infectious agents in a laboratory setting. Third is the ability to analyze and distribute information quickly and globally. Fourth would be the development of feedback systems that would cause both investigative and control responses. Hughes and Dennis suggest that each of these four components is “critical in confronting the emergence of plague and the potential for the emergence of drug-resistant disease.” The editorial was written in response to another article also appearing in the same issue. The article, entitled “Multidrug Resistance in Yersinia pestis Mediated by a Transferable Plasmid,” was written by Marc Garlimand and six of his colleagues. The article points out that plague is now considered a reemerging disease and, since 1994, has appeared in epidemic numbers in at least three countries. The plague organism is normally susceptible to a number of antibiotics including streptomycin, chloramphenicol, tetracycline, and the sulfonamides. As explained at the beginning of this chapter, microbes live in close proximity to each other in a variety of habitats including the intestinal areas of many animal species. Since 2002, there has been a rapid worldwide emergence of MDR (multiple drug-resistant) bacterial pathogens in a number of animals used as food sources by humans. The most common symptom caused by eating these contaminated food sources is a self-limited gastroenteritis that normally resolves itself without the use of antibiotics. Because multiple drug resistance information is located on mobile plasmids, there is an excellent chance that MDR genetic information can be passed from foodborne pathogens to more virulent human pathogens, including Y. pestis. The mechanism of that transfer could be any of those mentioned in the chapter. Such genetic transfer would constitute a serious public health threat. The plasmid-positive strains were found in bacteria associated with beef, chicken, turkey, and pork, and were found in

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samples from the following states: California, Colorado, Connecticut, Georgia, Maryland, Minnesota, New Mexico, New York, and Oregon. MDR Salmonella and E. coli have been found in droppings from wild geese, raising the possibility that wild animals might be able to spread MDR far beyond the livestock where it originated.

8 Concerns for the Future There are two major interrelated concerns that seem to override all others

when it comes to the plague: (1) the concern that new, antibiotic-resistant strains of the organism will become common; and (2) the concern that the plague organism might be used as a weapon of bioterrorism. Evolution of Resistant Strains

Increasing antibiotic resistance is a major problem in dealing with disease organisms such as Yersinia pestis. One of the major reasons that Y. pestis survives in the environment of the flea’s midgut is because it has an enzyme called phospholipase D (PLD). This enzyme, formerly called Yersinia murine toxin, gives the microbe resistance to a product in the blood plasma of the flea that would otherwise destroy the bacterium. It is still not known exactly what molecular mechanisms provide this protection. Yersinia pestis acquired the gene for production of PLD from either an unrelated bacterium or a simple nucleated organism such as a protozoan. Once Y. pestis had acquired this gene, it had a new means of transmission. The bacteria transformed from an organism that caused mild stomach illness through contaminated food and water to a flea-borne carrier of a life-threatening disease. Research at Rockefeller University in New York has provided at least a partial answer as to how Yersinia protects itself from the host’s immune system. The researchers found that Yersinia contains a protein—whose shape is similar to that of an apparently unrelated mammalian enzyme—that blocks a host cell’s ability to change shape and move. Since many of the immune system cells carry out phagocytosis, which requires both movement and shape changes, a loss of these capabilities would impair the ability of cells of

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Global Outbreak Alert and Response Network In April 2000, the World Health Organization (WHO) set up a meeting between representatives of technical institutions, various organizations, and networks involved in surveillance and response to global epidemics. The participants recognized the need to provide a global network to identify and deal with the threats posed by epidemic-prone and emerging diseases. This newly formed network developed a set of Guiding Principles for International Outbreak Alert and Response. In addition, they have developed laboratory rules and regulations that provide standardization in various areas such as research, communications, security, and clinical management. The Global Outbreak Alert and Response Network uses technical and other resources from the scientific groups within WHO. In addition, it has partnered with United Nations organizations such as UNICEF, the Red Cross, and international humanitarian organizations worldwide. This network works towards providing security to global health by: • Combating the international spread of outbreaks. • Ensuring that appropriate technical assistance reaches affected states rapidly. • Contributing to long-term epidemic preparedness and capacity building. Since its inception, the network has been involved in projects in at least 40 different countries from Afghanistan to Uganda, dealing with diseases including yellow fever, influenza, Ebola fever, and Rift Valley fever.

the immune system to track down and “eat” foreign invaders, the researchers explained. These results provide insights that could lead to new strategies for fighting the organism.

Concerns for the Future

“Yersinia injects several virulence factors into its host,” said C. Erec Stebbins of Rockefeller University. “If we can discover which ones are critical, we might identify the pathogen’s Achilles heel—an attractive target for antibacterial or antivirulence compounds. It is becoming increasingly clear that a common strategy used by bacterial pathogens to manipulate host cell biology is the mimicry of their own biochemical processes,” Stebbins said.1 History of Plague as a Weapon

It has been suggested that plague served as the original biological weapon in wartime. History records that the Tatars were attempting to capture the Italian-controlled city of Kaffa on the shore of the Black Sea in 1347. Plague broke out in the Tatar camp. The defenders of Kaffa resisted until the Tatars began to hurl the bodies of their own dead over the walls and into the city. The Tatars had been dying of the plague by the thousands, and now, so did the defenders of the city. Those who eventually escaped the city took to their ships and headed for ports in the Mediterranean, carrying the plague with them. The city of Kaffa became uninhabitable, and the Black Death quickly spread throughout Europe. During World War II, the Japanese were known to have dropped fleas infected with plague organisms over populated areas of China. A number of outbreaks of plague resulted. After World War II, both the United States and the Soviet Union used their biological weapons programs to find new techniques and delivery methods for a variety of microbial agents, including plague. Both countries were successful in finding ways to aerosolize Yersinia pestis, which increased its usefulness as a potential biological weapon. Obviously, this ability to spread the organism in an aerosol form has led to concern that terrorist groups or nations might acquire and use it. Scientists who defected from the former Soviet Union suggested that an antibiotic-resistant strain of Yersinia pestis had been developed.

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In 2001, the complete genome of the plague microbe was sequenced. Julian Parkhill, leader of the sequencing team, suggested that the sequence would be critical in helping to design antibiotics and vaccines that may be used against this disease organism. Another member of the group, Rick Titball, is leading a team that has produced a vaccine that is currently in clinical trials. Titball believes that “the information available [about the genome] is of much greater advantage to people defending against biological warfare than to those intending to use it.”2 Plague as a Bioterrorist Agent

The Centers for Disease Control and Prevention (CDC) has placed plague in Category A, which is the highest-priority category for an agent that could be used for bioterrorist activity. Plague meets the criteria set up by the CDC, which are that:

• It is easily disseminated or transmitted from person to person.

• It results in high mortality rate and has the potential for major public health impact.

• It might cause panic or social disruption. • It requires special action for public health preparedness. The Working Group on Civilian Biodefense prepared a consensus statement that concluded: Given the availability of Y. pestis around the world, capacity for its mass production and aerosol dissemination, difficulty in preventing such activities, high fatality rate of pneumonic plague, and potential for secondary spread of cases during an epidemic, the potential use of plague as a biological weapon is of great concern.3

In 1970, the WHO issued a report suggesting that up to 36,000 deaths and more than 150,000 incapacitating ill-

Concerns for the Future

nesses would result from the airborne release of 50 kilograms (approximately 110 pounds) of dried powder containing Yersinia pestis in a country such as the United States. These numbers represent only direct impacts and do not take into account the number of secondary infections and deaths that would result from subsequent person-to-person contact. NIAID Research

Because bioterrorism continues to be a real concern for the United States, the National Institute for Allergy and Infectious Diseases (NIAID) and other agencies have accelerated microbial research and development of diagnostic, preventive, and treatment methods. Microbial genomics, the study of the totality of genetic information found in an organism, has become an important part of this overall approach. The complete collection of an organism’s genetic information is known as its genome. Understanding an organism’s genetic program may aid in understanding how an organism carries out its biochemical and physical processing and operates as a complex biological system. This genetic information can be used to develop genebased diagnostic and sampling tests to quickly detect dangerous germs and assess their susceptibility to different types of treatment. Genomes also provide molecular fingerprints of different strains of a given microbe, thereby enabling investigators to better track future outbreaks to their source. Current long-term research of NIAID includes:

• Identifying genes in the Y. pestis bacterium that infect the digestive tract of fleas and researching how the bacterium is transferred to humans.

• Studying the disease-causing proteins and genes of Y. pestis that allow the bacterium to grow in humans and how they function in human lungs.

NIAID is also working with the Department of Defense, the CDC, and the Department of Energy to:

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• Develop a vaccine that protects against inhalationally acquired pneumonic plague.

• Develop promising antibiotics and intervention strategies to prevent and treat plague infection.

In 2002, NIAID released its “Biodefense Research Agenda for CDC Category A Agents.” The bubonic plague organism was high on the list of the greatest bioterror threats. Hopes for developing a viable vaccine have led to a number of collaborations between groups, companies, and nations. In 2005 the United States, Canada, and the United Kingdom signed an agreement to work together to develop a vaccine for plague. The United States and the United Kingdom have each developed vaccines that were being tested in clinical trials. At the end of 2005, they selected a vaccine to continue on the testing trail.4 In 2006, the Biodesign Institute at Arizona State University, Icon Genetics (Halle, Germany), and the U.S. Army Medical Research Institute for Infectious Disease demonstrated a vaccine whose components had been grown in tobacco leaves as the biological factories. Animal testing showed that the vaccine protected the animals against an aerosol form of the plague organism.5 And in 2007, Ichor Medical Systems was awarded a twoyear contract valued at more than $2.3 million by the Defense Threat Reduction Agency (DTRA). Ichor is working with the Biological Defense Research Directorate (BDRD) of the Naval Medical Research Center (NMRC) in Rockville, Maryland, in the development of a DNA vaccine for anthrax and plague.6 Cautionary notes

Plague has already been classified as a reemerging disease. Although its impact today is less dramatic than its past history it is still a severe problem. The Wildlife Conservation Society (WCS) has spotlighted plague as one of the 12 most deadly

Concerns for the Future

diseases capable of causing pandemics because of natural infections stimulated by climate change. “Plague is spread by rodents and their fleas and alterations in temperatures and rainfall are expected to change the distribution of rodent populations,” said Steven Sanderson, WCS president.7 In a November 2008 article entitled “Study of Ancient and Modern Plagues Finds Common Features,” three prominent NIAID researchers discuss disease emergence. “There appear to be common determinants of disease emergence that transcend time, place and human progress,” says NIAID director Anthony S. Fauci, M.D., one of the study authors. International trade and troop movement during wartime, poverty, lack of political will, and changes in climate, ecosystems and land use are some of the determinants the article mentions. “A better understanding of these determinants is essential for our preparedness for the next emerging or reemerging disease that will inevitably confront us,” says Dr. Fauci. “The art of predicting disease emergence is not well developed,” says David Morens, M.D., another NIAID author. “We know, however, that the mixture of determinants is becoming ever more complex, and out of this increased complexity comes increased opportunity for diseases to reach epidemic proportions quickly.” Our ability to understand and, more importantly, to predict when, where, how and why disease-causing microbes cause their diseases and how they make the jump from animals to humans requires continuing research. Only by looking at the broad framework as well as the specific pieces can scientists effectively combat these disease organisms.”8

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9 Hopes for the Future Techniques originally used to help analyze the human genome may soon be

used as tools to decipher the DNA of Yersinia pestis, investigate how anti­ biotic resistance develops, and make earlier detection possible. Researchers are also looking at ways to investigate a number of host-pathogen interactions, including how pathogens use host cell membrane proteins to enter host cells. PCR (polymerase chain reaction)-based plasmid typing has become a standard method to screen multiple cultures to identify specific genetic sequences. PCR assays could also be used to look for antibiotic resistance plasmids in foods and within the stages of the food production process. Conceivably, such test procedures could prove useful for detecting these plasmids in biothreat organisms including Y. pestis.1 Detecting Bacterial DNA Sequences

Antibiotic resistance in bacteria is an inherited characteristic, but attempting to analyze all of the DNA of all of the organisms present in an infection is not practical. A new method is under development at the University of Rochester Medical Center in Rochester, New York, that detects the presence of specific DNA sequences. Certain portions of DNA molecules take particular shapes based on their ability to find a complementary information sequence at the end of the DNA molecule. Once the DNA sequence that represents antibiotic resistance is found in one bacterial species, that sequence can be used as a model to find the same sequence in other bacteria. A fluorescent molecule is attached to one end of the DNA strand and can be used as a molecular beacon when bacteria suspected of being antibiotic resistant are checked on 86

Hopes for the Future

Microarrays Microarrays, also called DNA chips, gene chips, DNA micro­ arrays, or gene expression microarrays, represent a blending of computer technology and DNA. Researchers cover a small glass chip with hundreds to thousands of fragments of DNA. Each of these fragments represents a different gene within the cell being studied. By adding a fluorescent molecule to the fragments, they can be made to light up when they come into contact with a complementary gene. This tells scientists about the activity level of the genes in question. Microarrays make it possible for researchers to determine which genes are being activated and under what conditions. These microarrays are being used to try to understand the genetics involved with how microorganisms respond to drugs, how they respond to different environmental conditions, and how they are able to infect different kind of host cells. This new technology gives scientists the opportunity to test many genes simultaneously, thus speeding up the process of detecting change within the cell.2

a slide. The hope is that a single microarray or DNA chip would be able to test different species as well as different strains within a species to determine if they are resistant to a specific antibiotic. Discovering How to Disable Bacterial Weapons

In May 1999, researchers announced that they had found a way to prevent a number of bacteria from causing disease. It is hoped that the findings, published in the journal Science, can be used to develop a new generation of vaccines and antibiotics. The researchers reported that they had identified a “master switch” controlling the production of many chemicals that bacteria use to cause infection. When

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scientists knock out the switch, the bacteria are no longer capable of causing disease. It seems that the master switch for pathogenic bacteria, such as Vibrio cholerae (cholera), Yersinia (plague), Salmonella (typhoid fever), and Shigella (dysentery), is the same. Treatment of these bacterial infections, as well as others, may be affected by this discovery. It is also hoped that this discovery can be used in the fight against newly emerging, drug-resistant pathogens. Michael Mahan and his colleagues used a mutated strain of Salmonella with the master switch always on. This allowed researchers to discover all the tricks the organisms use for getting past the stomach and intestines and into organs and tissues. “This [knowledge of how to stop organisms from invading] has two important consequences,” Mahan states. “The bacteria are completely disabled in their ability to cause disease, and these crippled bacteria work as a vaccine since they stimulate immune defenses to defend against subsequent infections.”3 Researchers at the University of Illinois and the University of Massachusetts at Amherst have developed a method that causes a bacterium to reprogram its genetic information and cause its own death. “The basic idea is for an antimicrobial to target something in a bacterium that, in order to gain immunity, would require the bacteria to kill itself through a suicide mutation,” said Gerard Wong, a professor of materials science and engineering, of bioengineering and physics at the University of Illinois. The researchers have been working with a class of antimicrobial molecules that causes holes to be created in the cell membrane of the bacterium. A synthetic “hole punching” antimicrobial depends on the presence, in the membrane, of a lipid molecule called phosphoethanolamine (PE). This molecule is found in high concentrations within gram-negative bacterial membranes. PE lipids are used to kill the bacteria, but without the lipids the bacteria would die. “It’s a catch-22,” Wong said.

Hopes for the Future

“Some mutations bacteria can tolerate, and some mutations they cannot tolerate. In this case, the bacteria would have to go through a mutation that would kill it, in order to be immune to these antimicrobials. The antimicrobial reorganizes PE lipids into holes in the membrane,” explained Wong. “The perforated membranes leak, and the bacteria die.” As the number of emerging pathogens increases, and many become more resistant to the available antibiotics, it becomes critically important that we “more fully understand how our molecular ‘hole punchers’ work, [so that] we can look for similar ways to make antimicrobials [to which] bacteria cannot evolve immunity.”4 Deciphering the DNA of Yersinia pestis

Determining the sequence of the bubonic plague organism will hopefully lead to the design and development of new antibiotics and vaccines. In this case, we will also learn a great deal about how infectious bacteria have evolved over time. According to researchers from the Yersinia gene sequencing team, the plague organism descended from an intestinal organism, Yersinia pseudotuberculosis, which two thousand years ago gave you a mild tummy ache. One reason for the rapid evolution of Y. pestis may be its ability to shuffle segments of its chromosome, an activity common among pathogens. Yersinia pestis also carries genes for insecticidal toxins that are now deactivated but may have aided its move into the fleas. The Y. pestis organism contains about 150 pseudogenes — repeating, gene-like sequences of DNA that are no longer active in the organism. These sequences may eventually be lost from the organism completely. The Yersinia pestis genome was added to the Comprehensive Microbial Resource (CMR) in December of 2002. The CMR is a free Web site used to display information on all of the publicly available, complete prokaryotic genomes.5 Since 2002 a number of additional studies have provided valuable

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information about the role of several genetic sequences within that genome.6 How Bacterial Cells Communicate

Biologists have long speculated that bacteria are able to communicate with each other and their environment by releasing and then detecting various types of chemicals. This activity is a form of molecular census-taking and is usually called quorum sensing. Evidence exists that indicates more than 70 different types of organisms, including Yersinia pestis, are engaged in this activity. According to researcher Rong-guang Zhang, the quorum-sensing process signals bacteria to create biofilms , or mats of bacterial cells over a solid surface. The researchers used the Structural Biology Laboratory at Argonne National Laboratories to determine the molecular structure of a protein called TraR. This key protein seems to act as a relay that senses chemicals called pheromones. Once the bacterial cell senses these molecules, it activates specific genes that allow it to create biofilms. Knowing the structure of this protein allows scientists to pursue two different avenues of research. They may be able to find or create drugs that would block the chemical signals and thus prevent formation of biofilms. Conversely, they could stimulate the production of biofilms for useful projects such as water filtration. New Antibacterial Drugs

Researchers at the Wistar Institute in Philadelphia have identified and isolated a group of insect peptides (short proteins) that attack specific molecules in bacteria. Insects lack an immune system and have devised strategies to eliminate bacteria and other germs. The insect host may use peptides to create holes in bacterial membranes, or the peptides may bind to target sites within the bacterial cells causing death in some poorly understood way. Working with a European

Hopes for the Future

sap-sucking insect, Pyrrhocoris apterus, scientists identified the antimicrobial peptide called pyrrohocoricin. The target of this molecule is another protein called DnaK whose job is to identify and correct the shape of proteins that have folded up incorrectly. DnaK is a “heat-shock protein” and, when it is bound by the pyrrhocori­cin, DnaK is unable to complete its work of repairing misshapen proteins. This usually leads to the death of the bacterium. The insect peptide does not bind to mouse or human equivalents of DnaK and thus is not toxic to humans. Nanoparticles Can Be Used to Kill Bacteria

Researchers at Kansas State University have developed nanoparticles made of magnesium oxide to kill various bacteria, including bioterorrism agents such as anthrax and E. coli, in about five minutes. Kenneth J. Klabunde and his team have shown that these particles have an electrical charge opposite to that of the electrical charge on bacteria. The opposite charges attract the particles to the bacteria. The sharp edges of the particles penetrate even endospores of anthrax bacteria. Because the particles are alkaline rather than acidic, they can penetrate bacterial cell walls. Nanoparticles are solid rather than gaseous or liquid, which would allow them to be placed in air filtration units or sprayed like a powder. Research continues into the use of nanoparticles for direct killing of microbes as well as use as a means of drug delivery and a means of detection of these pathogenic microbes. An article by Xuefei Huang and colleagues, entitled “Magnetic Glyco-Nanoparticles: A Unique Tool for Rapid Pathogen Detection, Decontamination, and Strain Differentiation,” was published in the Journal of the American Chemical Society. It tells of a “magnetic glyco-nanoparticle (MGNP),” a unique compound that combines magnetic nanoparticles with sugars.7

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Many scientists worry that silver nanoparticles may be potentially harmful or at least not as benign as people once believed. Silver has long been used to kill microbes, but it kills good microorganisms along with the bad. Silver nanoparticles are already present in many consumer products. Many of the silver particles are showing up in drinking water supplies with the potential to kill beneficial bacteria. “Nanoparticles are very small, and they are interacting with the bacteria and rupturing the cell wall,” says chemist George John of The City College of New York and lead author of a study published in the journal Nature Materials. This rupturing kills the bacteria, he explains.8 New Diagnostic Tests

The ability to detect the plague organism rapidly would greatly reduce the number of deaths and the likelihood of transmission among people. Scientists from the Pasteur Institute in France

Figure 9.1  The dipstick diagnostic test for plague provides reliable results in 15 minutes. (© Institut Pasteur)

Hopes for the Future

and the Ministry of Health in Madagascar developed a simple test, called a dipstick test, that provides reliable results within 15 minutes. The dipstick test recognizes a unique substance called F1 antigen, which is found in the blood and infected fluid of buboes. By rapid detection of pneumonic plague, more infected patients will be able to get rapid treatment. Since its development in 2002, the dipstick test has become the major rapid diagnostic technique used worldwide. DRUG DEVELOPMENT

The Science and Technology Review of Lawrence Livermore National Laboratory details the use of a new method called accelerator mass spectrometry. This analytical technique uses very high voltages, usually in the mega-volt range, to accelerate negative ions to identify the chemical composition of a compound or sample. Yersinia pestis is one of the four disease models the laboratory will use to find broadspectrum anti-infective drugs that can target agents, such as plague or Hantavirus, which may be modified or engineered. Scientists could identify both common and unique factors in the immune response—a critical process for fighting infection—based on research on four particular disease models including plague, Francisella tularensis (tularemia), Brucella abortus (brucellosis), and Hantavirus. The Lawrence Livermore Laboratory will be working with Trius Therapeutics a San Diego–based biotech company that recently was granted a five-year $28 million contract from the NIH to develop novel antibiotics against some possible bioterrorism microbes. This work is at a very early stage of development. The grant comes from the National Institute of Allergy and Infectious Diseases (NIAID), a unit of NIH.9 Drug development often requires 6 to 8 years of laboratory research, followed by the use of the drug by laboratory animals. The difficult part is to find a dosage that will work in humans while testing research animals. If the animal test results seem

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promising, Phase I clinical trials follow. Phase I testing typically involves 10 to 20 healthy human subjects who volunteer to participate. They are given the new drug at clinical doses to evaluate its safety, determine a safe dosage range, and help identify its possible side effects. Subsequent Phase II and III trials involve a larger number of human subjects, who are patients diagnosed with the specific illness that scientists hope the drug will help treat or cure. It is not unusual for the process to fail at the point where Phase I clinical trials begin because research has repeatedly shown that humans and animals metabolize many substances differently.10 New Vaccine Trials

Currently in clinical trials is a new type of vaccine for the plague. At this point, vaccines do not provide 100 percent protection against the inhaled forms of the plague. Nose drops and nasal sprays of biodegradable microspheres containing antigenic subunits F1 and V are being tested on mice. This approach seems to protect the animals from inhaled and injected forms of the plague organism. Testing in larger animal models and humans will follow. This type of a vaccine has the advantage of being administered easily and to large numbers of people quickly. Another vaccine possibility involves the use of monoclonal antibodies against the V and F1 antigens of Yersinia pestis. Whether given alone or in combination, mice were protected against bubonic and pneumonic plague. The antibodies worked well whether given prior to infection or within 48 hours after infection. In June 2008, DynPort Vaccine Company (DVC) was informed it would continue development of a recombinant plague vaccine candidate called rF1V. Vaccine development includes possible licensure by the U.S. Food and Drug Administration (FDA). The vaccine is currently in Phase II clinical trials.11

Hopes for the Future

Scientists in many areas of the country, working for universities and companies, are involved in vaccine development to fight the plague. In July 2008, professor Henry Daniell and his team from the University of Central Florida reported that they had developed a vaccine whose early results indicate that it will be highly effective against the plague. The vaccine can be taken orally or by injection. Having a pill form of vaccine allows for a more rapid distribution and removes the need for special skills or sterile needles. Human trials are still needed but animal studies suggest that the vaccine will work for both the bubonic and pneumonic forms of the plague.12 In August 2008, researchers from the Pasteur Institute in Paris reported in the journal Infection and Immunity that they have also developed a live oral vaccine. They used a less virulent ancestor of Yersinia pestis, which shares 95 percent of its genetic information to produce their vaccine.13 New Regional Centers for Biodefense Research

In September 2003, Tommy G. Thompson, secretary of Health and Human Services, announced that $350 million in grants was being released to establish eight Regional Centers of Excellence for Biodefense and Emerging Infectious Diseases Research (RCE). These centers will be part of the nation’s plan for research in biodefense. “We have moved with unprecedented speed and determination to prepare for a bioterror attack or any other public health crisis since the terrorist attacks of 2001,” Thompson said. “These new grants add to this effort and will not only better prepare us for a bioterrorism attack, but will also enhance our ability to deal with any public health crisis, such as SARS and West Nile virus.” The NIAID is providing the grants and will administer the RCE program. “Since the terrorist attacks on American soil in 2001, NIAID has moved rapidly to bolster basic bio-

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medical research and the development of countermeasures to defend the United States against deliberately released agents of bioterrorism as well as naturally occurring infectious diseases,” said Anthony S. Fauci, director of NIAID. “The new RCE program provides a coordinated and comprehensive mechanism to support the interdisciplinary research that will lead to new and improved therapies, vaccines, diagnostics and other tools to protect the citizens of our country and the world against the threat of bioterrorism and other emerging and re-emerging diseases.”14 All of the Regional Centers for Excellence will:

• Train researchers and other personnel for biodefense research activities.

• Create and maintain supporting resources, including scientific equipment and trained support personnel, for use by the RCEs and other researchers in the region.

• Emphasize research focused on development and testing of vaccine, therapeutic, and diagnostic concepts.

• Make available core facilities to approved investigators from academia, government, biotechnology companies, and the pharmaceutical industry.

• Provide facilities and scientific support to first responders in the event of a national biodefense emergency.

• Support investigator-directed research. Each center comprises a lead institution and affiliated institutions located primarily in the same geographical region. The eight institutions receiving an RCE grant are Duke University, Harvard Medical School, New York State Department of Health, University of Chicago, University of Maryland (Baltimore), University of Texas Medical Branch (Galveston), University of Washington, and Washington University in St. Louis. Research to be conducted in the RCE program includes:

Hopes for the Future

• Developing new approaches to blocking the action of anthrax, botulinum, and cholera toxins.

• Developing new vaccines against anthrax, plague, tularemia, smallpox, and Ebola.

• Developing new antibiotics and other therapeutic strategies. • Studying bacterial and viral disease processes. • Designing new advanced diagnostic approaches for biodefense and for emerging diseases.

• Conducting immunological studies of diseases caused by potential agents of bioterrorism.

• Developing computational and genomic approaches to combating disease agents.

• Creating new immunization strategies and delivery systems.

In addition to the RCE, the NIAID has funded construction of two National Biocontainment Laboratories (NBLs) and 13 Regional Biocontainment Laboratories (RBLs). The two NBLs include the National Emerging Infectious Dieases Laboratory at Boston University Medical Center and the Galveston National Laboratory at the University of Texas Medical Branch at Galveston. The 13 RBLs include: • Colorado State University (Fort Collins), Regional Biocontainment Laboratory • Duke University Medical Center (Durham), Regional Biocontainment Laboratory at Duke • George Mason University (Manassus, VA), George Mason University Biomedical Research Laboratory • Tufts University, Cummings School of Veterinary Medicine (Grafton, MA), Regional Biosafety LaboratoryNew England

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plague • Tulane National Primate Research Center (Covington, LA), Regional Biocontainment Laboratory • University of Alabama at Birmingham, School of Medicine, Southeast Biosafety Laboratory Alabama • University of Chicago, The Ricketts Laboratory • University of Hawaii at Manoa, Pacific Regional Biocontainment Laboratory • University of Louisville, Center for Predictive Medicine • University of Medicine and Dentistry of New Jersey (Newark) • New Jersey Medical School Center for Infectious Disease Research—RBL • University of Missouri–Columbia College of Veterinary Medicine, University of Missouri–Columbia Regional Biocontainment Laboratory • University of Pittsburgh, Regional Biocontainment Laboratory at the Bioscience Tower III (BST3) • University of Tennessee Health Science Center (Memphis), University of Tennessee Health Science Center Regional Biocontainment Laboratory

The terrorist attacks of September 11, 2001, placed a new emphasis on improving and increasing our national health care facilities. Plague is just one of the deadly diseases that has come to the attention of the public in the wake of September 11. As a new administration takes office in Washington, scientists hope that protection against plague continues to be a high priority. Funding efforts ranging from basic science research to final product development need to continue. Scientists hope that greater awareness and additional research will help stop the plague once and for all.

Notes Chapter 1 1.

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3.

4.

5.

6.

New Mexico Department of Health, “Plague Data in New Mexico,” http:// www.nmhealth.org/epi/plague.html (accessed April 8, 2009). Cameron Walker, “Bubonic Plague Traced to Ancient Egypt,” National Geographic News, March 10, 2004, http://news.nationalgeographic.com/ news/2004/03/0310_040310_blackdeath.html (accessed April 8, 2009). S. N. DeWitte and J. W. Wood, “Selectivity of Black Death Mortality with respect to Preexisting Health,” Proceedings of the National Academy of Sciences (US) 105: 1436-1441. World Health Organization (WHO), “Human Plague in 2002 and 2003,” Weekly Epidemiological Record; 79 (33): 301-308, http://www.who.int/wer/2004/ en/wer7933.pdf (accessed April 8, 2009). A. Matteelli and N. Saleri, “Respiratory Diseases,” in Travel Medicine, eds. J.S. Keystone, P.E. Kozarsky, D.O. Freedman, H.D. Nothdurft, and B.A. Connor (Philadelphia: Elsevier Ltd., 2004); WHO, “Human Plague in 1997,” Weekly Epidemiological Record; 74 (41): 337-348, http://www.who. int/docstore/wer/pdf/1999/wer7441.pdf (accessed April 8, 2009); WHO, “Plague in Algeria—Update 2,” WHO Disease Outbreak News, http://www.who.int/ csr/don/2003_07_10/en (accessed April 8, 2009). Edward Marriott, Plague: A Story of Science, Rivalry, and the Scourge That Won’t Go Away. New York: Henry Holt, 2003.

Chapter 2 1.

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Bob Christie, “Plague Killed Grand Canyon Biologists,” Associated Press, Discovery News: Discovery Channel, November 9, 2007, http://dsc.discovery. com/news/2007/11/09/plague-grandcanyon.html (accessed April 8, 2009). Traci Angel, “Boy Scout Contracts Plague in Teton Area,” Jackson Hole Daily, August 28, 2008, http://www. jacksonholenewsandguide.com/article

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.php?art_id=3517 (accessed April 8, 2009); Richard B. Stolley, “The Case of the Coughing Cat: The Plague and its Prevention in New Mexico,” SantaFe. com, April 1, 2008, http://santafe.com/ Pages/1021 (accessed April 8, 2009). Ronald E. Viola, Lyudmyla Yerman, Janet M. Fowler, Cindy G. Arvidson, and Robert R. Brubaker, “A Missense Mutation Causes Aspartase Deficiency in Yersinia pestis “ Microbiology 154 (2008): 154: 1271-1280. Alicia P. Gregory, “Countering Bioterror: The New Threat of Plague,” Odyssey, http://www.rgs.uky.edu/odyssey/fall02/plague.html (accessed July 9, 2009).

Chapter 3 1. 2.

Fictional account. “Climate Linked to Plague Increase,” BBC News, August 22, 2006, http://news.bbc.co.uk/2/hi/science/ nature/5271502.stm (accessed April 8, 2009).

Chapter 4 1.

2.

M. J. R. Gilchrist, “The Progress, Priorities and Concerns of Public Health Laboratories,” presented at the Forum on Infections, Biological Threats and Terrorism, Institute of Medicine, November 28, 2001, http://www.aphl. org/docs/Advocacy/Testimonies/IOMBioterrorism11-28-01.pdf D. Niemeyer, “The Laboratory Bioterrorism Response Network,” Journal of Homeland Security March 2002, http:// www.homelandsecurity.org/journal/ Articles/niemeyer.html; Centers for Disease Control and Protection, “Laboratory Network for Biological Terrorism,” http://www.bt.cdc.gov/lrn/biological. asp (updated December 6, 2006); Center for Infectious Disease Research & Policy, “Plague: Current, comprehensive information on pathogenesis, microbiology, epidemiology, diagnosis, and treatment,” http://www.cidrap.umn.edu/cidrap/content/bt/plague/biofacts/plaguefactsheet. html (updated March 24, 2009); S. A. Morse et. al, “Detecting Biothreat

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Agents: The Laboratory Response Network,” American Society of Microbiology News 69, 9 (2003), http://www.asm. org/ASM/files/CCLIBRARYFILES/FILENAME/0000000605/nw09030114p.pdf.

2.

Chapter 5 1.

“Non-Toxic Disinfectant Kills Bubonic Plague Bacteria and Anthrax,” PR Newswire, September 4, 2003.

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Chapter 6 1.

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3.

4.

U.S. Geological Survey, “Ouch! Taking a Shot at Plague: Vaccine Offers Hope for Endangered Ferrets in Plague Outbreak,” news release, July 16, 2008, http://www.usgs.gov/newsroom/article. asp?ID=1976 (accessed April 8, 2009). National Park Service, “Wind Cave National Park Dusts to Prevent Spread of Plague,” news release, November 13, 2008, https://www.nps.gov/wica/parknews/ pr11132008.htm (accessed April 8, 2009). “Bubonic Plague Hits San Francisco,” http://www.pbs.org/wgbh/aso/databank/entries/dm00bu.html (accessed May 6, 2009). “China Intensifies Rat Plague Prevention,” China Daily, September 5, 2007, http://www.chinadaily.com. cn/china/2007-09/05/content_6083876. htm (accessed April 8, 2009).

Chapter 7 1.

2.

Infectious Diseases Society of America, “NIAID Resistance Research Agenda, Shortcomings Highlighted in JID,” IDSA News 18, 3 (March 2008). T. J. Welch, W. F. Fricke, P. F. McDermott, D. G. White, M. L. Rosso, et al., “Multiple Antimicrobial Resistance in Plague: An Emerging Public Health Risk,” PLoS ONE 2, 3 (March 21, 2007): e309, http://www.plosone.org/ article/info:doi%2F10.1371%2Fjournal. pone.0000309 (accessed April 8, 2009).

Chapter 8 1.

G. Prehna, M. I. Ivanov, J. B. Bliska, and C. E. Stebbins, “Yersinia Virulence Depends on Mimicry of Host Rho-

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Family Nucleotide Dissociation Inhibitors,” Cell 126, no. 5 (2006): 869-880. John Whitfield, “Black Death’s DNA: The Plague Bacterium Joins the Ranks of Completed Genomes,” Nature, October 4, 2001, http://www.nature.com/ news/2001/011004/full/news01100412.html. Working Group on Civilian Biodefense, “Plague as a Biological Weapon,” Journal of the American Medical Association 283, no 17 (May 3, 2000), http://jama.amaassn.org/cgi/content/full/283/17/2281. Center for Infectious Disease Research and Policy (CIDRAP), “US, UK, Canada to Jointly Test Plague Vaccines,” CIDRAP News, April 21, 2005, http://www.cidrap. umn.edu/cidrap/content/bt/plague/news/ april2105plague2.html (April 8, 2009). L. Santi and H. Mason, “Protective Plague Vaccine Produced in Tobacco Leaves,” Information Systems for Biotechnology News Report, April 2006, http://www. isb.vt.edu/news/2006/news06.apr. htm#apr0602 (accessed April 8, 2009). “Navy Partners with Ichor Medical Systems to Develop a Vaccine for Anthrax and Plague,” NewsRx: TB & Outbreaks Week, August 21, 2007, http://www.newsrx.com/newsletters/ TB-and-Outbreaks-Week/2007-0821/40082120075TW.html (accessed April 8, 2009). Kathryn Senior, “Yersinia pestis: A force to be reckoned with,” MD Consult, http://www.mdconsult.com/das/article/ body/114185429-2/jorg=journal&sour ce=&sp=21505360&sid=0/N/670326/1. html?issn=1473-3099 (accessed April 8, 2009). D. M. Morens, G. K. Folkers, and A. S. Fauci, “Emerging Infections: A Perpetual Challenge,” The Lancet Infectious Diseases 8, 11 (2008): 710-719, http:// www3.niaid.nih.gov/about/directors/ pdf/EmergingInfectionsLancetID.pdf (accessed April 8, 2009).

Chapter 9 1.

T. J. Welch, W. F. Fricke, M. Eppinger, M. J. Rosovitz, P. Mcdermott, D. White,

2.

3.

4.

5.

6.

7.

G. D. Wiens, J. Ravel, “Distribution of Yersinia pestis pIP1202-like Multidrug Resistance Plasmids Among Foodborne Pathogens,” May 21, 2007, http://www.ars.usda.gov/research/publications/Publications.htm?seq_no_ 115=207556 (accessed April 8, 2009). U.S. Department of Health and Human Services, National Institutes of Health, National Institute of Allergy and Infecitous Diseases, Division of Microbiology and Infectious Diseases, “Deciphering Pathogens: Blueprints for New Medical Tools,” (September 2002), http://cmgm. Stanford.edu/biochem/gfx/images/ brown_arrayimage.jpg. University of California at Santa Barbara, “Breakthrough on Microbial Disease Published in Science,” press release, May 6, 1999, http://www.ia.ucsb.edu/ pa/display.aspx?pkey=302 (accessed July 9, 2009). James E. Kloeppel, “Scientists Fool Bacteria into Killing Themselves to Survive,” news release, News Bureau: University of Illinois at Urbana-Chambaign, December 15, 2008, http://news. illinois.edu/news/08/1215bacteria.html (accessed April 8, 2009). Comprehensive Microbial Resource, “Yersinia Pestis KIM Genome Page,” http://cmr.jcvi.org/tigr-scripts/CMR/ GenomePage.cgi?database=ntyp02 (accessed April 8, 2009). X. Wang et al., “Yersinia Genome Diversity Disclosed by Yersinia pestis Genome-wide DNA Microarray,” Canadian Journal of Microbiology 53, 11 (2007): 1211-1221; Z. Dongsheng et al., “DNA Microarray Analysis of Genome Dynamics in Yersinia pestis: Insights into Bacterial Genome Microevolution and Niche Adaptation,” Journal of Bacteriology 186, no.15 (2004): 5138-5146.  ScienceDaily, “Magnetic Nanoparticles Detect And Remove Harmful Bacteria,” news release, November 20, 2007, http://www.sciencedaily.com/releases/ 2007/11/071119110910.htm (accessed April 8, 2009).

8.

9.

10.

11.

12.

13.

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M. Heger, “A Silver Coating in the Fight Against Microbes,” Scientific American, May 2, 2008, http://www. sciam.com/article.cfm?id=silvercoating-fights-microbes (accessed April 8, 2009); News-Medical.net, “Silver Nanoparticles May Kill Beneficial Bacteria,” April 29, 2008, http://www. news-medical.net/?id=37887 (accessed April 8, 2009). L. Timmerman, “Trius Therapeutics Wins $28M Contract From NIH For Bioterror Antibiotics,” Xconomy, October 22, 2008, http://www.xconomy. com/san-diego/2008/10/22/triustherapeutics-wins-28m-contractfrom-nih-for-bioterror-antibiotics (accessed April 8, 2009). K. Walter, “Developing New Durgs and Personalized Medical Treatment,” Science and Technology Review, September/October 2008, https://www.llnl.gov/ str/SepOct08/turteltaub.html (accessed April 8, 2009). Huliq News, “CSC’s Dynport Vaccine Company To Continue Plague Vaccine Development,” http://www.huliq. com/61412/csc039s-dynport-vaccinecompany-continue-plague-vaccinedevelopment (accessed April 8, 2009). ScienceDaily, “Vaccine To Protect Against Black Plague Bioterror Attack Being Developed,” July 31, 2008, http:// www.sciencedaily.com/releases/2008/0 7/080730140829.htm (accessed April 8, 2009). T. Blisnick, P. Ave, M. Huerre, E. Carniel, C. E. Demeure, “Oral Vaccination against Bubonic Plague Using a Live Avirulent Yersinia pseudotuberculosis Strain,” Infection and Immunity 76, 8 (2008): 3808, http://www3.niaid. nih.gov/topics/plague/research.htm (accessed April 8, 2009). U.S. Department of Health and Human Services, “HHS Announces New Regional Centers for Biodefense Research,” news release, September 3, 2003, http://www3.niaid.nih.gov/ news/newsreleases/2003/hhs_rce.htm (accessed July 10, 2009).

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Glossary anaerobic—Not requiring oxygen. antibiotic—A chemical produced either by a microorganism such as a bacterium

or a fungus such as a mold used to treat bacterial infections. Antibiotics are not useful in fighting viruses. Some antibiotics are currently produced synthetically either completely or in part (e.g., semisynthetic penicillins such as ampicillin). antibiotic resistance—Also called drug resistance; the condition when organ-

isms such as bacteria are no longer killed or inhibited by an antibiotic; the opposite of antibiotic sensitivity. antibody—An immune protein or immunoglobulin. These proteins are

produced by white blood cells called lymphocytes. When stimulated by a foreign protein, chemical, or cell part, lymphocytes are converted into protein- (antibody-) producing factories. antigen—A molecule, group of molecules, or part of a cell that is recognized

by the host immune cells as being nonself or foreign. Antigens stimulate production of antibodies (antibody generating). antiphagocytic—Unable to perform phagocytosis, or able to prevent phago-

cytosis. attenuated—Weakened. bacillus—When the term is used with a capital “B,” it refers to a specific genus

of bacteria that have a rod-like shape; when used with a lowercase “b,” the term refers to the a rod-like shape (a rod is longer than it is wide). bipolar staining—Staining technique in which dye molecules concentrate at

the ends or poles of the cells, giving them the appearance of a safety pin characteristic of Yersinia and Franciscella species. bubo—A swelling of the lymph nodes. Adjective form is bubonic. cholecystitis—Inflammation of the gallbladder that may be the result of infec-

tion in the gallbladder; gallstones may be present. clinical trials—Rigorous scientific evaluation of a procedure, device, or

drug(s) used for prevention, diagnosis, or treatment of a disease. Usually three phases (Phases I, II, III) are required for approval by the Food and Drug Administration (FDA): Phase I: Evaluation of clinical pharmacology, involves volunteers; testing for safety.

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Phase II: Performed in a small group of patients; testing for dosage and overall desired clinical effect. Phase III: Large, comparative study using patients to establish a clear clinical benefit; control groups using placebos or comparisons to established or current procedures. coccus—Refers to shape of a bacterial cell that resembles a sphere or circle

(plural is cocci). Staphylococcus (a cluster of cocci) and streptococcus (a chain of cocci) are common arrangements of specific groups of cocci well known for causing diseases. cytoskeleton—An internal protein framework inside eukaryotic cells. endemic—Refers to frequency of disease cases in a well-defined geographic

region; frequency is low but cases are constantly present. endotoxin—A metabolic product released from the cell walls of gram-nega-

tive bacteria when they die. Composed of lipids, polysaccharides, and peptides, endotoxins are often toxic to the cells and may cause an inflammatory reaction. enzootic reservoirs—Populations of organisms that maintain a pathogenic

organism, such as Yersinia pestis, within their population in low levels, resulting in low mortality. enzyme—A protein that serves as an organic catalyst, speeding up the rate of

a biochemical reaction, but not consumed or used up in that reaction. All biochemical reactions within living systems are controlled or regulated by enzymes. epidemic—A dramatic increase in the number of individuals showing the

symptoms of a disease within a specified area and during a specified time period. In the United States, statistics to determine a true epidemic are collected and maintained by the Centers for Disease Control and Prevention (CDC). epizootic reservoir—Population of organisms that are highly susceptible to a

pathogen such as Yersinia pestis, with resulting high mortality. eukaryote, eukaryotic—A type of cellular organization in which an organism

contains a nucleus and one or more cells with a nucleus and other welldeveloped compartments known as organelles. These organelles consist of membranes or are bounded by membranes.

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facultative anaerobe—An organism that can grow and reproduce in the pres-

ence or absence of oxygen. flagellin—A protein that is used to construct the flagella of various bacteria. flagellum—A whip-like tail structure that helps a bacterium to move. Plural

is flagella. free radical oxygen—Reactive by-products of metabolism; free radicals have

only single electrons in their outer energy level and remove electrons from other molecules to complete their outer energy level; this electron stealing sets off a chain of biochemical events in the cells, creating instability that could destroy the cell. GAP proteins—Proteins that activate an enzyme (GTPase) that breaks down

an energy storage molecule known as GTP (guanine triphosphate), thus releasing energy to be used in various cellular activities. gastroenteritis—Inflammation of the stomach and the intestines; may result in

nausea and vomiting and/or diarrhea. genetic or gene mutation—Change in the genetic information of a cell or

virus (either DNA or RNA in some viruses); changes in genetic information usually lead to new proteins, altered proteins or loss of proteins. genome—The sum total of all the genetic information in a cell or virus. genomics—The study of the totality of genetic information found in an

organism. glycoprotein—A molecule made of simple sugars such as glucose (glyco-) and

a protein; commonly found on the outside of cell membranes and involved in cellular recognition and rejection. gram-negative—Refers to the designation given to bacteria that have been

stained by the Gram stain method. Gram-negative bacteria lose the color of the first or primary stain (crystal violet) and take the color of the second or counterstain (safranin—a red color). Thus, gram-negative cells are red to pink in color. The organisms responsible for typhoid fever, cholera, and bubonic plague are gram-negative. gram-positive—Refers to the designation given to bacteria that have been

stained by the a Gram stain method. Gram-positive bacteria retain the color of the crystal violet stain due to the composition of the cell wall and thus are colored purple to violet. 104

inflammation—A series of responses consisting of redness, increased heat in

the area, swelling, and pain; this is followed by repair of the inflamed area and is part of the nonspecific defenses of the body. invasin—A protein found on the outer membrane of certain bacteria, such as

Yersinia species, which allows the bacteria to attach to the cell membrane of human cells. kinase—An enzyme involved in the transfer of phosphate groups on

molecules. lipopolysaccharide (LPS)—A molecule composed of fatty acids and sugars, it

makes up a portion of the cell wall of gram-negative bacteria such as Yersinia. lymphatic system—Includes the lymph nodes, spleen, tonsils, adenoids, and

thymus as well as scattered patches of tissue in various area of the body, such as the intestinal mesentery. The lymphatic tissue system is part of the body’s immune system, producing cells that help protect the body from bacteria and other microbes. lymph nodes—Bean-shaped organs of the lymphatic system that contain

various types of white blood cells and become swollen when infected. lymphocytes—One of the five types of white blood cells produced by

humans; divided into B and T lymphocytes, both of which are essential to proper immune function. macrophage—A modified version of the monocyte, one of the five types of

white blood cells in humans. A large cell that seeks out and engulfs foreign particles and cells through phagocytosis; literally, a “large eater.” mutation—A change in the genetic information of a cell or virus (either DNA

or RNA in some viruses). Changes in genetic information usually lead to new proteins, altered proteins, or loss of proteins. nonencapsulated—Lacking a capsule or envelope. organelles—“Miniature organs;” compartments within eukaryotic cells that

are limited or bounded by membranes; they represent the sites of various metabolic actions and functions. pandemic—A worldwide epidemic or widespread disease in humans; it results

when person-to-person contact occurs between individuals who have the virus but no current immune protection against it (these types of individuals are sometimes called “immunologically naïve”). 105

pathogenic—Disease-causing. plasma—The liquid portion of the blood. plasmid—A small circle of DNA outside of the bacterial chromosome that is

capable of self-replication. These miniature chromosomes carry a limited set of genes that can be copied and sent by means of protein tube from one bacterium to another, increasing the genetic variability of the recipient bacterium. pneumonic—Refers to the lungs; a form of plague that invades the lung tis-

sue and is highly contagious. preclinical—Studies of drugs or vaccines that are carried out in tissue or

cell cultures or animals; this phase occurs before clinical trials involving humans. prokaryote, prokaryotic—A type of cellular organization in which cells lack

membrane-enclosed organelles such as a nucleus; bacteria and archaea are prokaryotic cells. protease—An enzyme that can break down proteins. septicemic, septicemia—Referring to large numbers of bacteria in the bacte-

rial infection of the bloodstream; sometimes known as blood poisoning. serology—The study of the serum. serum—The portion of the blood remaining after the blood cells, platelets, and

proteins (the formed elements) have been removed. spirillum—A bacterium that is shaped like a spiral or curved like the letter “c.” surveillance—A continuous and organized collection and analysis of data

regarding all aspects of a disease. This information is sent to national and regional public health professionals, who use it to provide an up-to-date prevention and control program. sylvatic plague—Plague caused by organisms such as prairie dogs and

squirrels, found in natural areas such as woodlands, grasslands, or forests, that carry the fleas that carry the plague organisms. transduction—A means by which bacteria acquire fragments of DNA and

viruses transport DNA fragments from a donor cell to a recipient cell.

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transformation—Alteration of the normal genetic information of a cell such as

a bacterium by the inclusion of additional DNA from an outside source. urban plague—Plague caused by organisms such as rats, found in cities, that

carry the fleas that carry the plague organisms. vaccine—A substance, organism, viral particle, or group of molecules that,

when injected or put into the body by other means, causes the immune system to mount an immune response to that specific agent. vectors—Organisms such as flies, fleas, and ticks that carry pathogenic organ-

isms and transmit them to other organisms. virulence—Ability to cause disease.

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Further Resources Books and Articles Adjemian, J.C., P. Foley, K.L. Gage, and J.E. Foley. “Initiation and Spread of Traveling Waves of Plague, Yersinia pestis, in the Western United States.” American Journal of Tropical Medicine and Hygiene 76 (2007): 365–375. Bahmanyar, M., and D.C. Cavanaugh. Plague Manual. Geneva: World Health Organization, 1976. Barras, Colin. “Black Death Casts a Genetic Shadow over England.” New Scientist August 2007, http://www.newscientist.com/article.ns?id=dn12393&feed Id=online-news_rss20 (accessed May 7, 2009). BBC News. “Black Death and Plague ‘Not Linked.’” (April 12, 2002) http:// news.bbc.co.uk/2/hi/health/1925513.stm (accessed May 7, 2009). Canizares, M.C., L. Nicholson, and G.P. Lomonossoff. “Use of Viral Vectors for Vaccine Production in Plants.” Immunology and Cell Biology 83, no. 3 (2005): 263–270. Centers for Disease Control and Prevention. “Human Plague—Four States, 2006.” Morbidity and Mortality Weekly Report 55, no. 34 (September 1, 2006): 940–943. Centers for Disease Control and Prevention. “Laboratory Test Criteria for Diagnosis of Plague.” http://www.cdc.gov/ncidod/dvbid/plague/lab-testcriteria.htm (accessed May 7, 2009) Centers for Disease Control and Prevention. “Plague.” http://www.cdc.gov/ ncidod/dvbid/plague/index.htm (accessed May 7, 2009). Centers for Disease Control and Prevention. “Prevention of Plague. Recommendations of the Advisory Committee on Immunization Practices (ACIP).” Morbidity and Mortality Weekly 45 (RR-14) (1996): 1–15. Chanteau, S., et al. “Development and Testing of a Rapid Diagnostic Test for Bubonic and Pneumonic Plague.” The Lancet 361, no. 9353 (January 18, 2003): 211–216. Cobbs, C.G., and D.H. Chansolme. “Plague.” American Journal of Clinical Dermatology 22, no. 3 (July 2004): 303–312. Collinge, S.K., et al. “Testing the Generality of a Trophic-cascade Model for Plague.” Ecohealth 2 (2005): 1–11. Craven, R.B., and D.T. Dennis. “Plague,” in Maxcy-Rosenau-Last Public Health and Preventive Medicine, 14th ed., R.B. Wallace, ed. Stamford, CT: Appleton & Lange, 1998.

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Eisen, R.J., et al. “Early-phase Transmission of Yersinia pestis by Unblocked Fleas as a Mechanism Explaining Rapidly Spreading Plague Epizootics. Proceedings of the National Academy of the Sciences 103, no. 42 (2006): 15380–15385. Eisen, R.J., A.P. Wilder, S.W. Bearden, J.A. Montenieri, and K.L. Gage. “Earlyphase Transmission of Yersinia pestis by Unblocked Xenopsylla cheopis (Siphonaptera: Pulicidae) Is as Efficient as Transmission by Blocked Fleas.” Journal of Medical Entomology 44 (2007): 678–682. Eisen, R.J., J.L. Lowell, J.A. Montenieri, S.W. Bearden, and K.L. Gage. “Temporal Dynamics of Early-phase Transmission of Yersinia pestis by Unblocked Fleas: Secondary Infectious Feeds Prolong Efficient Transmission by Oropsylla montana (Siphonaptera: Ceratophyllidae).” Journal of Medical Entomology 44 (2007): 672–677. Eisen, R.J., P.J. Reynolds, P. Ettestad, et al. “Residence-linked Human Plague in New Mexico: A Habitat-suitability Model.” American Journal of Tropical Medical Hygiene 77 (2007): 121–125. Eisen, R.J., R.E. Enscore, B.J. Biggerstaff, et al. “Human Plague in the Southwestern United States, 1957-2004: Spatial Models of Elevated Risk of Human Exposure to Yersinia pestis.” Journal of Medical Entomology 44 (2007): 530–537. Gage, K.L. “Yersinia,” in Cecil’s Textbook of Medicine, 23d ed. L. Goldman and D. Ausiello, eds. Philadelphia: Elsevier, 2007. Gage, K.L., and M.Y. Kosoy. “The Natural History of Plague: Perspectives from More Than a Century of Research.” Annual Review of Entomology 50 (2005): 505–528. Galimand, Marc, et al. “Minireview: Resistance of Yersinia pestis to Antimicrobial Agents.” Antimicrobial Agents and Chemotherapy (October 2006). Gleba, Y., V. Klimyuk, and S. Marillonnet. “Magnifection—A New Platform for Expressing Recombinant Vaccines in Plants.” Vaccine 23, no. 17 (2005): 2042–2048. Guarner, J., et al. “Persistent Yersinia Pestis Antigens in Ischemic Tissues of a Patient with Septicemic Plague.” Human Pathology 36 (2005): 850–853. Huang, Xiao-Zhe, et al. “Current Trends in Plague Research: From Genomics to Reed, Kurt D. “Dissecting Plague.” Clinical Medicine & Research 4, no. 3 (2006). Inglesby, T.V., et al. “Plague as a Biological Weapon.” Journal of the American Medical Association (May 3, 2000), http://jama.ama-assn.org/cgi/content/ full/283/17/2281 (accessed May 7, 2009).

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Josko, D. “Yersinia Pestis: Still a Plague in the 21st Century.” American Soceity for Clinical Laboratory Science 17, no. 1 (Winter 2004): 25–29. Lazarus, A.A., and C.F. Decker. “Plague.” Respiratory Care Clinics of North America 10, no. 1 (March 2004): 83–98. Lowell, J.L., et al. “Identifying Sources of Human Plague Exposure.” Journal of Clinical Microbiology 43 (2005): 650–656. Lowell, J.L., et al. “Phenotypic and Molecular Characterizations of Yersinia pestis Isolates from Kazakhstan and Adjacent Regions.” Microbiology 153, pt. 1 (2007):169–177. Koirala, J. “Plague: Disease, Management, and Recognition of Act of Terrorism.” Infectious Disease Clinics of North America 20, no. 2 (June 2006): 273–287. Kool, J.L. “Risk of Person-to-Person Transmission of Pneumonic Plague. Clinical Infectious Diseases 40, no. 8 (April 15, 2005): 1166–1172. MacKenzie, Deborah. “Case Reopens on Black Death Cause.” New Scientist (September 11, 2003), http://www.newscientist.com/article.ns?id=dn4149 (accessed May 7, 2009). Mason, H.S., R. Chikwamba, L. Santi, et al. “Transgenic Plants for Mucosal Vaccines,” in Mucosal Immunity 3d. ed., J. Mestecky et al. (eds.). San Diego, Calif.: Elsevier, 2004, 1053–1060. Mwengee, W., et al. “Treatment of Plague with Gentamicin or Doxycycline in a Randomized Clinical Trial in Tanzania.” Clinical Infectious Diseases 42, no. 5 (March 1, 2006): 614–21. Orent, Wendy. Plague: The Mysterious Past and Terrifying Future of the World’s Most Dangerous Disease. New York: Free Press, 2004. Santi, L., et al. “Protection Conferred by Recombinant Yersinia pestis Antigens Produced by a Rapid and Highly Scalable Plant Expression System.” Proceedings of the National Academy of the Sciences 103, no. 4 (2006): 861–866. Sebbane, F., et al. “Adaptive Response of Yersinia pestis to Extracellular Effectors of Innate Immunity During Bubonic Plague.” Proceedings of the National Academy of the Sciences 103, no. 31 (August 1, 2006): 11766–11771. Stenseth, N.C., N.I. Samia, H. Viljugrein, K.L. Kausrud, M. Begon, and S. Davis. “Plague Dynamics Are Driven by Climate Variation.” Proceedings of the National Academy of the Sciences 103, no. 35 (August 29, 2006): 13110–13115.

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University of Maryland Medical Center. “Plague: Overview.” http://www.umm. edu/ency/article/000596.htm (accessed July 9, 2009.) Webb, C.T., C.P. Brooks, K.L. Gage, and M.F. Antolin. “Classic Fleaborne Transmission Does Not Drive Plague Epizootics in Prairie Dogs.” Proceedings of the National Academy of the Sciences 103, no. 16 (2006): 6236–6241. Weir, E. “Plague: A Continuing Threat.” Canadian Medical Association Journal 172, no. 12 (June 7, 2005): 1555. World Health Organization. “Plague Manual: Epidemiology, Distribution, Surveillance and Control.” http://www.who.int/csr/resources/publications/ plague/WHO_CDS_CSR_EDC_99_2_EN/en/index.html (accessed May 7, 2009). World Health Organization. “Plague.” http://www.who.int/mediacentre/factsheets/fs267/en/ (accessed May 7, 2009). World Health Organization. “Outbreak News: Plague, Democratic Republic of the Congo.” Weekly Epidemiological Record 81, no. 42 (October 20, 2006): 397–398. Web Sites Center for Infectious Disease Research and Policy http://www.cidrap.umn.edu

Centers for Disease Control and Prevention http://www.cdc.gov

National Institute of Allergy and Infectious Diseases http://www3.niaid.nih.gov

National Institutes of Health http://www.nih.gov

World Health Organization http://www.who.int/en

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Index accelerator mass spectrometry, 93 Advice Against the Plague (Harvey), 52, 54 Algeria, 13 American Biotech Labs, 58 amino acids, 22 anaerobic environment, 24, 102 animal feed, 70 animals plague vaccine for, 66–67 as primary hosts, 28, 35–36, 39, 61–63 anthrax, 91 anthrax vaccine, 66 antibacterial drugs, 90–91 antibiotic(s) and antibiotic resistance, 70 and bacterial structure, 23 bioterrorism treatments, 57 defined, 102 introduction of, 51 for pneumonic plague, 56 as preferred treatment, 54 as prevention strategy, 60, 63 antibiotic resistance, 69–78 bacterial transfer of, 73 and biological weapons, 81 defined, 102 DNA sequencing to detect, 86–87 evolution of resistant strains, 79–81 and “hole punching” antimicrobials, 89 “master switch” research, 88 in Yersinia pestis, 76 antibodies defined, 102 and serologic testing, 46 in serum, 25 stimulation by vaccine, 66 tests for, 11 and V antigens, 26 Yersin’s experiments, 15 antigen, 66, 102 antiphagocytic capsule, 24, 102 archaebacteria (archaea), 22 Arizona, 19 ASAP-AGX-32 disinfectant, 58 aspartase, 26 aspartic acid, 26 attenuated strains, 66, 102

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Australia, 68 Avant Immunotherapeutics, 66 bacillus, 24, 75, 102 Bacillus anthracis, 66 bacteria. See also specific bacteria, e.g.: Yersinia pestis and antibiotic resistance, 70–72 cell communication among, 90 cell organization, 21 and climate change, 40 defined, 20–21 disabling of disease-causing mechanism in, 87–89 DNA sequencing, 86–87 important structures of, 21–24 motility of, 23 transfer of antibiotic resistance in, 73 bacterial cultures, 45 Barts and London Hospital, 18 BDRD (Biological Defense Research Directorate), 84 Beth Israel Medical Center, 10 biodefense, 54, 57, 95–98 “Biodefense Research Agenda for CDC Category A Agents” (NIAID report), 84 Biodesign Institute (Arizona State University), 84 biofilms, 90 Biological Defense Research Directorate (BDRD), 84 biological weapon, plague as, 81–82 Biology of Plagues, The (Scott and Duncan), 16 biosafety level(s) (BSL), 49 Biosafety Level 2 (BSL-2), 46, 47 Biosafety Level 3 (BSL-3), 47, 58 Biosafety Level 4 (BSL-4), 47, 50 bioterrorism, 48, 57, 82–83 bipolar staining, 24, 102 Black Death, 11–13, 16–18, 43 bloodstream, 29–31 bodily fluids, 56 Bradley, Richard, 54 Brubaker, Robert, 26 Brucella, 50 BSL. See biosafety levels

buboes, 12, 30, 34, 43, 102. See also lymph nodes, swollen bubonic plague case study, 8–11 characteristics, 41, 43 diagnosis of, 44–46 as endemic disease, 13 and inflammation, 30 Buffalo Gap National Grasslands, South Dakota, 62–63 calcium, 26 California, 19 capsules, 30, 74 carbohydrates, 22, 46 caspases, 29 Category A bioterror agent, 82 cats, 19, 20 causative agent, 13–16 causes of plague, 19–31 CDC. See Centers for Disease Control and Prevention cell(s), organization of, 21 cell membrane, 29, 72, 88 cell walls, 21–22, 25 Centers for Disease Control and Prevention (CDC), 11, 48, 50, 82 charge, electrical, 91 China, 12–13, 63, 66 Chinatown, San Francisco, 64 chipmunks, 19 chloramphenicol, 54, 71 cholecystitis, 102 chromosomes, 89 Cipolla, Carlo, 51 climate change, 39–40, 85 Clinical Laboratory Improvement Act (CLIA), 48 clinical trials, 82, 84, 94–95, 102–103 clotting, 26 CMR (Comprehensive Microbial Resource), 89 coccobacillary cells, 45 coccus, 24, 103 coffee, 52, 54 Colorado, 19 communication, among bacterial cells, 90 competition, among microorganisms, 69

Comprehensive Microbial Resource (CMR), 89 conjugation, 72, 73, 76 Connecticut, 19–20 contagion, early theories of, 51–52 cultures, 46 cytoskeleton, 28, 103 Daniell, Henry, 95 DEET (N,N-diethyl-m-toluamide), 61–62 Defense, U.S. Department of, 66 Defense Threat Reduction Agency (DTRA), 84 definitive diagnosis, 45–46 Dennis, David, 75–77 deoxyribonucleic acid (DNA). See DNA diagnosis, 41–50, 92–93 dipstick diagnostic test, 92, 93 disinfectants, 58 DNA, 18, 29, 71–73 DNA chips, 87 DnaK protein, 91 DNA sequencing, 82, 86–87, 89–90 DNA vaccine, 84 Dongting Lake (China), 63, 66 Donner Memorial State Park (California), 19 doxycycline, 54, 63 droplet transmission, 56 DTRA (Defense Threat Reduction Agency), 84 Duncan, Christopher, 16 DynPort Vaccine Company (DVC), 94 Ebers Papyrus, 12 Ebola virus, 17 efflux systems, 74 Egypt, 11, 12 emeralds, powdered, 52 Emergency Preparedness and Response, 50 endemic disease, 8, 13, 103 endotoxin, 25, 103 England, 12, 16 Enterobacteriaceae, 25 Environmental Protection Agency (EPA), 58 environmental surfaces, treatment of, 56, 58–59

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enzootic reservoirs, 36, 103 enzyme, 26, 72, 79, 103 EPA (Environmental Protection Agency), 58 epidemics, 14, 103. See also pandemics epizootic reservoirs, 37, 103 Escherichia coli, 76, 78, 91 ethidium bromide, 74 eukaryotes, 21, 22, 103 eukaryotic cells, 21, 103 European plague pandemic (1300s), 12 extremophiles, 22 F1 antigen, 46, 93, 94 facultative anaerobe, 24, 104 Fauci, Anthony S., 85, 96 FDA. See Food and Drug Administration, U.S. ferrets, 62–63 fibrin, 26 flagella, 23, 104 flagellin, 23, 104 fleas, 37 and antibiotic-resistant plague strain, 76 and climate change, 40 eradication as prevention strategy, 60, 61 in Nile Valley, 12 Simond’s research, 17 as vector of plague, 11, 12 Yersinia pestis in, 28 and Yersin’s theories on plague transmission, 14 flowers, 60 fluorescent molecules, 86, 87 folk medicine, 51 Food and Drug Administration, U.S. (FDA), 54, 94 Francisella, 50 free radical oxygen, 104 Frieden, Thomas, 11 fumigation, 61 future issues antibacterial drugs, 90–91 cell communication, 90 concerns for, 79–85 diagnostic tests, 92–93

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disabling bacterial weapons, 87 DNA sequencing, 86–87, 89–90 drug development, 93–94 evolution of resistant strains, 79–81 hopes for, 86–98 nanoparticles, 91–92 vaccine trials, 94–95 gangrene, 43, 44 GAP proteins, 27, 104 garlic, 51, 52 Garlimand, Marc, 77 gastroenteritis, 20, 77, 104 gene expression microarrays, 87 gene sequencing, 75 genetic exchange, 76, 77 genetic information, 72 genetic (gene) mutation, 71, 104. See also mutation genome, 82, 83, 104 genomics, 83, 86, 104 gentamicin, 54 Global Outbreak and Response Network, 80 global warming, 63, 85 glucose, 22, 45 glycoprotein, 24, 104 gold, molten, 52 gonorrhea, 72, 74 gram-negative bacteria and ASAP-AGX-32, 58 and bubonic plague, 44–45 defined, 104 efflux systems in, 74 Gram’s stain to identify, 22 and PE, 88 Yersinia as, 25 gram-positive bacteria, 22, 104 Gram’s stain, 22, 41, 44 grave robbers, 52 Great Pestilence. See Black Death groin, 44 Guiding Principles for International Outbreak Alert and Response (WHO document), 80 Hantavirus, 93 Harvey, Gideon, 52, 54

Heska Corporation, 66 history of plague, 8–18 early means of prevention, 60 early treatments, 51–54 use as biological weapon, 81–82 “hole punching” antimicrobials, 88–89 Hong Kong epidemic (1894), 14 horizontal transfer, 76 horses, 15 host animal. See animals, as primary hosts host cell, invasion by Yersinia pestis, 26–29 Huang, Xuefei, 91 Hughes, James, 75–77 Ichor Medical Systems, 84 Icon Genetics (Halle, Germany), 84 immune system evasion by Yersinia, 79 and flagellin, 23 and inflammation, 30 macrophages as part of, 28–30 and O antigen, 25 and peptidoglycan, 22 immunofluorescent staining of capsule, 44, 45 inactivated proteins, 15 incubation period, 44 India, 13, 15–16 Indonesia, 13 inflammation, 20, 30, 105 inoculation, 15. See also vaccination insecticides, 61 insect repellents, 61 insects, peptides in, 90–91 intestinal bacteria, 70 invasin, 26, 105 in vitro dosage, 58 ions, 93 iron, 24 isolation, 55, 56. See also quarantine John, George, 92 Kaffa, Ukraine, 81 Kansas State University, 91 Kazakhstan, 39 kinase, 27, 105 kingdoms (categories), 20–21

Klabunde, Kenneth K., 91 Kramer, Vicki, 19 laboratory diagnosis techniques, 44–48, 50 Laboratory Response Network (LRN), 47, 48, 50, 59 Lawrence Livermore Laboratory, 93 leeches, 52 lesions, 44, 56 lipopolysaccharide (LPS), 25, 105 live oral vaccine, 95 liver, 30, 44 Liverpool University School of Biological Sciences, 16 livestock, 70, 77–78 London, England, 53 LPS. See lipopolysaccharide LRN. See Laboratory Response Network lucky charms, 60 lungs, 30, 31, 42 lymphatic system, 30, 105 lymph nodes, 30, 42, 105 lymph nodes, swollen and Black Death, 12 and bubonic plague, 16, 41, 43, 44 and immune system, 30 and pneumonic plague, 31 lymphocytes, 105 macrophage, 28–30, 105 Madagascar, 76 magnesium oxide, 91 magnetic glyco-nanoparticle (MGNP), 91 “Magnetic Glyco-Nanoparticles: A Unique Tool for Rapid Pathogen Detection, Decontamination, and Strain Differentiation” (Huang et al), 91 Mahan, Michael, 88 mammals, 37, 39 mannitol, 46 Marker, Lucinda, 8–11 marmots, 20 “master switch,” 87, 88 MDR (multiple drug-resistant) pathogens, 74 membrane, 29, 72

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MGNP (magnetic glyco-nanoparticle), 91 miasma, 51 miasmatic paradigm, 51–52 mice, 19, 36 microarrays, 87 microbial genomics, 83 Microtus, 36 Middle Ages, 52, 53 midgut, 76, 79 Ministry of Health (China), 63 mitochondria, 29 Moeller, Keith, 58 monkeys, 19 monoclonal antibodies, 94 Morens, David, 85 “Multidrug Resistance in Yersinia pestis Mediated by a Transferable Plasmid” (Garlimand et al), 77 multiple drug-resistant (MDR) pathogens, 74, 77–78 mutation, 26, 71, 89, 105 N,N-diethyl-m-toluamide (DEET), 61–62 nanoparticles, 91–92 National Biocontainment Laboratories (NBLs), 97 National Institute of Allergy and Infectious Diseases (NIAID), 75, 83–85, 93, 95–96 national LRN laboratories, 47, 59 National Medical Society, 50 Natural Bridges National Monument (Utah), 19 NBLs (National Biocontainment Laboratories), 97 New Mexico, 8, 11, 13, 20, 32–35 New York City, 8–11 New York City Department of Health, 10 NIAID. See National Institute of Allergy and Infectious Diseases Nile rat, 12 Nile Valley, Egypt, 12 nonencapsulated bacteria, 24, 105 North Africa, 11 Norway rat, 36 nucleus, 21 O antigen, 25 odor, 60

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Old Dominion University (Norfolk, Virginia), 74 online diagnosis, 50 oral vaccine, 66, 95 organelles, 21, 26–27, 105 oriental rat flea, 37 outer membrane, 25 Oxford University, 18 Panagiotakopulu, Eva, 12 pandemics, 11–13, 105 parasites, 70 passive hemagglutination test, 45 Pasteurella pestis, 24 Pasteur Institute, 14, 16, 92–93, 95 pathogenic species, 20, 106 PCR-based plasmid typing, 86 PE (phosphoethanolamine), 88–89 penicillinase, 74 penicillinase-producing Neisseria gonorrhea (PPNG), 74 peptides, 90–91 peptidoglycan, 22, 23, 25 perfumes, 60 permethrin, 61 Peromyscus, 36 pesthouses, 60 phagocytic white blood cells, 28 phagocytosis, 79–80 pheromones, 90 phosphoethanolamine (PE), 88–89 phospholipase D (PLD), 79 phospholipids, 25 pilus, 72 plasma, 15, 25, 106 plasma membrane, 29 plasmids, 24, 71, 72, 76, 106 plasminogen activator, 26 Plasmodium, 75 PLD (phospholipase D), 79 pneumonia, 41 pneumonic plague, 20, 43, 56, 93, 106 polymorphonucleated neutrophils (PMNs), 28, 30 PPNG (penicillinase-producing Neisseria gonorrhea), 74 prairie dogs, 62–63 preclinical (term), 106

presumptive diagnosis, 44–45 prevention, 60–68 priests, 52 Primas, Ronald, 9, 10 prokaryotic cells, 21, 106 protease, 27, 106 proteins, 15, 22 proventricular plague mass, 37 pseudogenes, 89 Pyrrhocoris apterus, 91 quarantine, 16, 52, 60, 64 quorum sensing, 90 rats, 14, 17, 38, 61, 63, 65 RBLs (Regional Biocontainment Laboratories), 97–98 RCE (Regional Centers of Excellence for Biodefense and Emerging Infectious Diseases Research), 95–98 recombinant vaccine, 94 reemerging disease, 77, 84–85 reference LRN laboratories, 47, 48, 50, 59 Regional Biocontainment Laboratories (RBLs), 97–98 Regional Centers of Excellence for Biodefense and Emerging Infectious Diseases Research (RCE), 95–98 repellents, 61 reproduction of microorganisms, 71 rF1V vaccine, 94 Rockefeller University (New York), 79 rock squirrels, 34, 36 rodents, 37, 38, 60, 61. See also rats rosemary, 51, 52 Salmonella, 78, 88 Sanderson, Steven, 85 San Francisco plague cases (1900-1909), 64–65 sanitation, 52 Santa Fe County, New Mexico, 11, 20 Scott, Susan, 16 sea trade, 12 select agent, 46 sentinel LRN laboratories, 47, 48 September 11, 2001, terrorism attacks, 8, 9, 56, 98

septicemia, 106 septicemic (term), 106 septicemic plague, 31, 44 serologic testing, 45, 46 serology, 46, 106 serum, 15, 16, 25, 106 17/95 biotype orientalis, 76 Seville, Spain, 52 sheep blood agar, 46 side-chains, 25 silver, 51 silver nanoparticles, 92 Simond, Paul-Louis, 17 skin lesions, 44, 56 smoke, 60 Society of General Microbiology, 18 Soviet Union, 81 Spain, 52 spirillum, 24, 106 spleen, 30, 44 sputum, 43 squirrels, 19, 34, 36 Stebbins, C. Erec, 81 Straley, Susan, 27, 30, 31 streptomycin, 54, 55 Structural Biology Laboratory (Argonne National Laboratories), 90 “Study of Ancient and Modern Plagues Finds Common Features” (NIAID report), 85 sulfonamides, 63 Suntsov, Victor, 20 surgical masks, 56 surveillance, 76–77, 106 sylvatic plague, 38, 106 symptoms, 16 Tatars, 81 temperature, 28, 40 tetracycline, 54, 63 Thompson, Tommy G., 95 thyme, 51, 52 Titball, Rick, 82 tobacco leaves, 84 toxins, 15 transduction, 72, 73, 106 transformation, 71–73, 107 transmission, 17, 79

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TraR protein, 90 treatment, 51–59 tree squirrels, 35 trimethoprim-sulfamethoxazole, 54 Tull, John, 8–11 type III secretion system, 26–27 ulcerations, 12 United States cold war biological weapons programs, 81 plague in, 10m, 11, 13 University of Central Florida, 95 University of Illinois, 88 University of Massachusetts, 88 University of Rochester Medical Center, 86 urban plague, 38, 107 U.S. Army Medical Research Institute for Infectious Disease, 84 Utah, 19 vaccination, 60, 63, 66–68 vaccines and bacterial structure, 23 clinical trials, 94–95 defined, 107 and microbe genome, 82 NIAID research, 84 Yersin’s experiments, 15 V (virulence) antigens, 25, 26, 94 vectors, 11, 107 Vietnam War, 66 viral infections, 70 Virtue and Use of Coffee with Regard to the Plague and Other Infectious Distempers (Bradley), 54 virulence, 107 virulence factors, 24–26, 81 viruses, 72, 73 W antigens, 25 Wayson stain, 41, 44 weapon, plague as, 81–82 western United States, 13 white blood cells, 25 WHO. See World Health Organization

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Wildlife Conservation Society, 84–85 Wind Cave National Park, 63 Wistar Institute, 90 Wong, Gerard, 88 Wood, James, 17 Working Group on Civilian Biodefense, 54, 57, 82 World Health Organization (WHO), 80, 82–83 World War II, 81 Wright’s stain, 23 Wyoming, 19–20 Xenopsylla cheopis, 37 X-rays, 41 Xu, X. Nancy, 74 Yersin, Alexander, 13–16, 24 Yersinia spp. , 20 Yersinia enterocolitica, 20 Yersinia pestis, 23 and alternate Black Death theories, 18 antibiotic-resistant strain, 69, 76 and ASAP-AGX-32, 58 as biological weapon, 81 as cause of plague, 8 characteristics of, 24 discovery/naming of, 14 DNA sequencing of, 89–90 evolution of resistant strains, 79–81 in fleas, 28 invasion of host cell by, 26–29 invasion of host’s bloodstream, 29–31 “master switch” research, 88 and MDR genetic information, 77 and pneumonic plague, 56 primary hosts for, 35–36 and reference laboratories, 50 vaccine against, 66 viability on external surfaces, 59 virulence factors, 24–26 Yersinia pseudotuberculosis, 20, 89 Yops toxins, 27–28 York, Eric, 19 Zhang, Rong-guang, 90

About the Author Dr. Donald Emmeluth spent most of his teaching career in upstate New York. In 1999, Dr. Emmeluth retired from the State University of New York system and moved to Savannah, Georgia. He became a member of the Biology Department of Armstrong Atlantic State University in Savannah, where he teaches a number of courses. Dr. Emmeluth has published several journal articles and is the co-author of a high school biology textbook. His most recent article appeared in the February 2002 issue of The American Biology Teacher. He has also authored three other books in the Deadly Diseases and Epidemics series: Typhoid Fever, Plague, and Botulism. Dr. Emmeluth has served as President of the National Association of Biology Teachers. During his career, Dr. Emmeluth has received a number of honors and awards including the Chancellor’s Award for Excellence in Teaching from the State University of New York system and the Two-Year College Biology Teaching Award from NABT. In 2003, Dr. Emmeluth was awarded NABT’s Honorary Membership Award for outstanding contributions to Biology and Life Science Education. This award is the association’s highest honor.

About the Consulting Editor Hilary Babcock, M.D., M.P.H., is an assistant professor of medicine at Washington University School of Medicine at Washington University School of Medicine and the Medical Director of Occupational Health for Barnes-Jewish Hospital and St. Louis Children’s Hospital. She received her undergraduate degree from Brown University and her M.D. from the University of Texas Southwestern Medical Center at Dallas. After completing her residency, chief residency, and Infectious Disease fellowship at Barnes-Jewish Hospital, she joined the faculty of the Infectious Disease division. She completed an M.P.H. in Public Health from St. Louis University School of Public Health in 2006. She has lectured, taught, and written extensively about infectious diseases, their treatment, and their prevention. She is a member of numerous medical associations and is board certified in infectious disease. She lives in St. Louis, Missouri.

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