Seminar in Nucleic Acids Dr Geoffrey Zubay March

Seminar in Nucleic Acids Dr. Geoffrey Zubay March 23, 2004

• Bubonic • Septicemic Primary Secondary • Pneumonic Primary Secondary

Bubonic q Most common form (~85% of all cases) q causes swollen lymph nodes (buboes) q can only spread from person to person via direct contact with bubo drainage q 1%-15% death rate if treated; if not treated, 40%-60% death rate q the backbone of the survival of y. pestis because it can develop into both secondary septicemic and pneumonic types Septicemic q fatality rate of 3050% in treated cases, 50 -90% in untreated cases q causes severe blood infection throughout the body and gangrene of acral regions (nose and digits) if untreated Primary: q occurs when a flea inserts y. pestis directly into the bloodstream Secondary: q occurs as a severe development from bubonic or pneumonic (when y pestis migrates to bloodstream) Pneumonic q 100% death rate if not treated within first 24 hrs q can be transmitted via direct inhalation of the germs q least common, yet most dangerous form Primary: q occurs via inhalation of pneumonic respiratory droplets Secondary: q occurs when bubonic or septicemic plagues spread to the lungs

Lungs of a pneumonic plague patient

Reservoirs Vectors • Urban and domestic rats • Ground squirrels • Rock squirrels • Prairie dogs • Deer mice • Field mice • Gerbils • Voles • Chipmunks • Marmots • Guinea pigs • Xenopsylla cheopis (the oriental rat flea; nearly worldwide in moderate climates) • Oropsylla montanus (United States) • Nosopsyllus fasciatus (nearly worldwide in temperate climates) • Xenopsylla brasiliensis (Africa, India, South America) • Xenopsylla astia (Indonesia and Southeast Asia) • Xenopsylla vexabilis (Pacific Islands) • Kangaroo rats …over 200 identified reservoirs ~30 identified flea vectors Incidental Hosts • humans • Domestic and feral cats • Dogs • Lagomorphs (rabbits and • hares) • Coyotes • Camels • Goats • Deer • Antelope *http: //www. cidrap. umn. edu/cidrap/con tent/bt/plague/biofacts/plaguefactsheet. html#_Reservoirs/Vectors/Modes_of_T ransmissio

• *BITES FROM FLEA VECTORS* • Direct contact with infectious body fluids or tissues while handling an infected animal (which can be dead or alive) • Ingestion of raw or uncooked meat from an infected animal (marmots, prairie dogs, goats, camel) • Inhalation of infectious droplets

BUBONIC 1º 2° SEPTICEMIC • Bites from flea vectors where Y pestis is inserted • Bites or scratches from directly into bloodstream infected animals, such as cats –no discernible bubo present • Direct contact with infected animal carcasses, such as rodents (especially marmots), rabbits, hares, carnivores (eg, wild cats, coyotes), and goats • Develops as a complication of bubonic or 1º pneumonic plague –When Y pestis enters the bloodstream PNEUMONIC • Inhalation of respiratory droplets from infected animals such as cats • Inhalation of respiratory droplets from a person with primary or secondary pneumonic plague • Handling Y pestis cultures in the laboratory setting • Bubonic and 1° septicemic spread plague bacilli hematogenously to the lungs

To 2° only 1° Or only Septicemic

Plague patients in the U. S.

Medieval doctor cutting off a bubo from a plague victim

*The first record of plague was an outbreak among the Philistines in 1320 B. C. when God enacted his vengeance upon them for capturing his Ark, which belonged to Israel: “He [the Lord] brought devastation upon them and afflicted them with tumors. And rats appeared in their land, and death and destruction were throughout the city…The LORD’S hand was against that city throwing it into great panic. He afflicted the people of the city, both young and old, with an outbreak of tumors. ” I Samuel 5: 6 -12 “The Philistines asked, “What guilt offering should we send to Him? They replied, “Five gold tumors and five gold rats because the same plague has struck both you and your rulers. ” I Samuel 6: 4

• Justinian’s Plague (~540 -700 AD) • The Black Death (1346 -1350) • The China Epidemic (~1855 -1908) AKA “The Modern Pandemic”

• The first recorded and confirmed pandemic • Occurred during the reign of the Roman Emperor Justinian • It spread from Egypt through the known world • Population losses of 5060% occurred in North Africa, Europe, and central and southern Asia for an approximate total of 100 million deaths Justinian the Roman Emperor

• • • The 2 nd pandemic is thought to have originated in the Gobi desert in the 1330’s where the bacillus was active and abundant China was a major player in trade, thus it took less than a decade for the plague to spread across western Asia and into Europe In Oct 1347, Italian merchant ships returned to Sicily from a trip to the Black Sea, and by the time they docked, many of the Lists of the dead were merchants were already dying from the plague It is believed to have been spread via fleas embedded in the fur they traded published regularly By the following August (1348), it spread all the way north to England The disease lay dormant in winter (due to flea inactivity), but within 5 years of its onset, 25 million people were killed in Europe (1347 -1352) 25 million = 1/3 of the population; however, these numbers could be askew due to the presence of other diseases that may have assisted in depopulation of Europe After the first 5 yr cycle, the death toll lessened; however, for the next 130 years, outbreaks occurred in 2 -5 year cycles A period of rest was seen from 1480 -17 th C. , then the Great Plague struck killing ~100, 000 more people in London The plague caused major political, cultural, and religious ramifications It is also responsible for the introduction of hospitals as care centers rather than quarantine locations as it catalyzed the movement toward more effective health care and a cleaner, healthier style of living 17 th Century London (plague locations + death toll in London)

• • Beginning in the Yunnan province of China in 1885, the 3 rd pandemic spread to all inhabited continents excluding Australia It spread to Canton and Hong Kong in 1894 and Bombay in 1898 By 1900, it had spread via steamship to the rest of the world By 1903, India was losing an average of 1 million people per year Ultimately, it killed more than 12 million people in India and China alone in the period from 1898 -1918 Small outbreaks of plague continue to occur as a result of the stable enzootic foci found round the world from the 3 rd pandemic (except, of course, in Australia) This pandemic was the least severe of the three due to understanding its nature and the advent of effective public health measures (100→ 25→ 12 million) Most importantly, antibiotics were discovered during this outbreak, and some patients were actually cured, which is why it is sometimes referred to as the modern pandemic

• Ø Ø § § June 1894: Alexandre Yersin successfully isolates the plague organism which he calls Bacterium Pestis. Kitasoto makes the same discovery independent of Yersin; however, his data shows discrepancies, thus Yersin is credited. He develops a treatment (an antiserum) to combat the disease and cures a plague patient in 1896 He is the first to suggest that it may be caused by the rodent/flea pathway, and he also identifies the black rat as the reservoir for the Manchurian outbreak 1897: Masanori Ogata and Paul-Louis Simond independently discover the role of the flea in plague transmission 1910: W. M. Haffkine demonstrates the efficacy of his vaccine Post-1911: L-T. Wu recognizes the Manchurian outbreak as the pneumonic form and establishes measures to prevent the spread of disease via aerosolization 1970: After 3 name changes, plague bacterium is renamed Yesinia Pestis in honor of Alexandre Yersin Costume worn by plague doctor to protect against 'miasmas' of poisonous air

• World Health Organization reports ~ 1000 -3000 cases/year worldwide w/ an average of ~1700/yr • For the period of 1954 -1997, a total of 80, 613 cases were reported with 6, 587 deaths • The maximum number of reported plague cases (6004) occurred in 1967 and the minimum (200) occurred in 1981 • This is vastly underreported due to lack of proper surveillance and laboratory capabilities in many countries • There are only 38 countries that report plague activity • Generally occurs in rural areas where enzootic foci and rodent populations abound (L. A. outbreak in 1924 was last urban outbreak)

• There are 7 countries that have been affected by plague every year: Brazil, Democratic Republic of the Congo, USA (with the exception of 1955, 64, 68), Madagascar, Myanmar, Peru, and Vietnam • There have been three periods of increased plague activity from 1954 -97: 1) During the mid-60’s 2) Between 1973 -1978 3) mid-80’s-present • The rise of reported plague morbidity has increased worldwide in the 90’s, especially in Africa GEOGRAPHICAL SHIFTS: ASIA→AMERICAS→AFRICA • In the 1950’s, plague was a problem primarily in Asia, with little activity in the Americas • By the early 60’s, plague activity in the Americas increased, and in Africa, it began to play a role • The mid-60’s-early 70’s show a magnificent increase in plague activity in Asia which is primarily due to the Vietnam War • For the past 15 -20 years (since~1982), a dramatic rise in activity can be seen for Africa

Number of cases of plague reported to World Health Organization, 1954 -1997

• A plague epidemic in Vietnam from 1966 to 1972 was largely responsible for the increased plague activity during the mid-sixties • This epidemic is largely a result of the defoliation of vast areas (which contained the enzootic foci for plague) during military operations, as well as the disruption of the economy, ecosystem and infrastructure as a result of prolonged armed conflict The number of reported cases of plague with data from Viet Nam excluded: Asia is primary location for plague activity in the 50’s The trend shifts in the 60’s and the Americas become more dominant Africa shows a drastic increase in the 80’spresent

Africa • Beginning in the 1980 s, there has been a steep upward trend in the number of plague cases in Africa • A total of 19, 349 cases and 1, 781 deaths in Africa from 1980 to 1997, comprising 66. 8% and 75. 8% of the world's total with an average case fatality rate of 9. 2% • From 1980 -1997, human plague was reported from 13 countries in Africa (Angola, Botswana, Democratic Republic of the Congo, Kenya, Libya, Madagascar, Malawi, Mozambique, South Africa, Uganda, United Republic of Tanzania, Zambia, Zimbabwe) • Madagascar and the United Republic of Tanzania accounted for 62. 5% of the total plague cases reported in Africa during 1982 -1997

Asia • Most cases of plague worldwide were reported from Asia from 1954 -80’s • There were outbreaks in Tanzania and Madagascar in the 1990 s • Outbreaks occurred in India in 1954, 1963, and then again 30 years later in 1994 • The plague outbreak in India in 1994 is a result of the earthquake in September 1993 that disturbed the equilibrium density of domestic rodents and their fleas • A holiday that brought crowds together is also thought to have facilitated the spread of human plague

The Americas • Human plague was reported from five countries (Bolivia, Brazil, Ecuador, Peru and the United States of America) • Three of these countries have notified some cases of human plague every year (Brazil, Peru, and the United States of America) • Brazil and Peru accounted for 82% of the total cases reported in the Americas during the last 15 years • Totals for the period from 1980 -1997 were 3, 137 cases with 194 deaths • The mean case fatality rate was 6. 2% during the period

q Human plague has been reported most often from the four western states of New Mexico, Arizona, Colorado and California q 341 cases of human plague were reported during 19701995 q The overwhelming majority of cases were bubonic plague

• For the last 45 years, the mean perennial plague case fatality for the world (i. e. the average over the past 45 years of the annual reported number of plague deaths divided by the annual reported number of plague cases) has been 11. 8%. • There is wide variation in reported case fatality rates by continent and by year • There is also considerable variation from country to country and from epidemic to epidemic • Despite the availability of a number of highly effective therapeutic agents, mortality due to plague in many countries was high during the period 1954 -1997 which can most likely be attributed to the fact that it often went unrecognized until too late • also, the majority of the countries don’t have the capital to afford the proper health care required

Outbreaks from the 20 th Centurypresent • • 1900 San Francisco >Arrival of plague in the United States when Chinese laborer is found dead in a hotel basement 1924 Los Angeles >the last US urban outbreak >Mexican male is misdiagnosed with STD >31 of the 33 total cases die before proper health measures are taken 1967 -72 Outbreaks in Vietnam 1992 Arizona >case results in death due to misdiagnosis of pneumonia > Source believed to have been from household cat

• • • 1994 India >induces widespread panic >causes more than 600, 000 people to flee Surat >110 of these people are plague victims > Source is rats found in grain stockpiles >Begins in bubonic form and develops into pneumonic >~5150 cases suspected from 26 states; 53 confirmed fatalities with 300 more suspected January 1997 Zambia >90 cases with 22 fatalities >Outbreak possibly linked to heavy rains and flooding which force rodents into populated areas August 1997 Mozambique >115 cases reported between June 7 th and July 4 th ; No fatalities reported October 1997 Malawi >43 cases reported between September 29 th and October 23 rd >582 total cases reported >Over 60% of cases are children under 5 years >No fatalities reported November 1997 Mozambique >update-335 total cases since outbreak began in June >No fatalities reported Plague outbreak in India reported in Newsweek, 1996

• • 1998 Uganda >49 cases reported, no fatalities recorded May 1999 Namibia >39 confirmed cases >8 recorded fatalities July 1999 Malawi >74 suspected cases, no confirmed deaths March 2001 Zambia >436 cases, 14 deaths February 2002 India >16 cases reported, 4 deaths May 2002 Malawi >71 cases of bubonic reported, no deaths Depiction of death carts in London June-July 2003 carrying victims of plague Algeria >10 cases reported; 8 bubonic, 2 septicemic >1 fatality reported Nov 2003 New York >2 cases; couple contracted plague in Santa Fe >No deaths, but the male had to get his foot amputated

Bubonic 1° Septicemic 1° Pneumonic Flea Skin Blood vessels Inhaled infected respiratory droplets (from either cat or human) Lymphatic vessels Organs Forms buboes Blood Organs Lungs 2°septicemic 2°pneumonic Lungs

Molecular Biology of Yersinia Pestis Characteristics Biovars of Y. pestis Evolution of Y. pestis Pathogeneisis of plague

Yersinia Pestis Gram negative coccobacillus n Non-motile n Enterobacteriaceae family n Non-spore forming (unlike Anthrax) n Facultative anaerobe n Obligate parasite n

Yersinia Pestis n 0. 5 -0. 8 μm in diameter n 1 -3 μm long Grows optimally at 28ºC and a p. H of 7. 2 -7. 6 n n Bacterial cell wall n F 1 Protein Envelope http: //www. nature. com/genomics/papers/y_pestis. html

Biovars of Y. Pestis n n n There are 3 biovars of Y. pestis, each named for the pandemic that it is thought to have caused They are named based on their ability to convert nitrate to nitrite and ferment glycerol Glycerol Nitrite n Antiqua (1 st pandemic) + + n Medievalis (2 nd pandemic) + n Orientalis (3 rd pandemic) + The 3 biovars exhibit no difference in their virulence or pathology in animals or humans.

Genome n 1 chromosome – 4. 65 Mb n Orientalis and Medievalis strains have been sequenced n 3 plasmids n p. MT 1 – 96. 3 kb n p. YV – 70. 3 kb n p. Pla – 9. 6 kb n The plasmids are crucial to the virulence of Y. pestis

Evolution of Y. pestis n There are 11 species of Yersinia n 3 pathogenic species of Yersinia n Yersinia enterocolitis – enteropathogen n Yersinia pseudotuberculosis – enteropathogen n Yersinia pestis – systemic pathogen n Y. pestis evolved from Y. pseudotuberculosis 1500 -15, 000 years ago

Evolution of Y. pestis

Y. pseudotuberculosis vs. Y. pestis n Disease: Enteric infection n Disease: Bubonic Plague n Transmission: enters mammals through food and water n Transmission: rodent to humans through flea vector n Chromosomal DNA – 4. 74 Mb n n Extrachromosomal DNA n p. YV n Chromosomal DNA – 4. 65 Mb n 90% chromosomal DNA relatedness with Y. pseudotuberculosis Extrachromosomal DNA n p. YV n p. Pla n p. MT 1 n “Two thousand years ago, it only gave you a tummy ache” - Brendan Wren, geneticist n “Within a few hundred years - an evolutionary eye blink- Y. pestis learned to leap between fleas and mammals, to live in the blood instead of the intestine, and to cause the swelling, coughing and hemorrhaging of medieval nightmares. ” http: //www. nature. com/nsu/011004 -12. html

Plasmids crucial to virulence of Y. pestis Plasmid Name Size (kb) Virulence determinants Role in disease p. MT 1* 96. 2 F 1 capsule antigen Bacterial transmission by Fleas p. YV 70. 3 Several Yops, Type III secretion system Toxicity. Avoidance of immune system p. Pla* 9. 6 Plasminogen activator Dissemination from intra-dermal site of infection *unique to Y. pestis only

Pathogenesis of plague (focus on Bubonic plague)

Manner in which fleas transmit plague Flea feeds on Y. pestis-infected blood Y. Pestis enters flea’s midgut & multiplies logarithmically Clump of Y. pestis forms in the midgut, blocking fleas foregut During next meal, blood cannot enter the midgut & flea gets very hungry Flea bites vigorously & regurgitates the contents of its midgut into the next wound

Importance of flea blockage http: //www. asm. org/ASM/files/CCLIBRARYFILES/FILENAME/0000000467/nw 20030086 p. pdf • Unblocked, uninfected flea on the left (A) and blocked, infected flea on the right (B). • After flea feeds on Y. pestis infected blood, the bacteria enter the midgut of the flea, where it will grow and multiply, eventually forming a large mass that can lodge in the flea’s foregut. During next meal, blood cannot enter midgut. • The ensuing blockage causes the starving flea to go into a “blood-feeding frenzy, ” in which it regurgitates the mass of Y. pestis and transmits it to a mammalian host. • Experiments indicate that only blocked fleas effectively transmit plague to mammals.

Y. pestis mechanisms that contribute to flea blockage n Hemin storage proteins (Hms) n Genes located on chromosome n Necessary for flea blockage, which is essential for efficient transmission of plague from flea to mammals n Hms play a very important role in the transmission of plague, changing the Y. pestis from a harmless inhabitant in the flea vector’s midgut to one that amasses in the foregut, causing the blockage. n In the flea, the Hms proteins alter the hydrophobicity of the bacterial cell, thereby promoting aggregation and clumping of bacteria within the blood meal. This is one of the main mechanisms by which blocking of fleas occur.

Hms is temperature dependent n Experiments indicate that fleas do not become blocked at higher temperatures (above 28ºC = 82. 4ºF) n It is not known however whether it is the expression of the Hms gene that is affected by temperature, or rather its protein product is affected by temperature. n For instance, if held at 30ºC, fleas survive Y. pestis infections in an unblocked state, perhaps explaining why human bubonic plague epidemics often end after the onset of warmer temperatures. n In addition, if you refer to the World Distribution of Plague Map, you will notice that plague does not occur in the equatorial regions, evidence which further supports this theory.

Pathogenesis of plague (focus on Bubonic plague) • Colonization of Y. pestis in the flea • Transmission of the Y. pestis from flea to mammalian host. A flea bite transfers 25, 000 – 100, 000 organisms to host. • While growing in the flea, Y. pestis loses its antiphagocytic F 1 capsular layer (inactivated at lower temperatures), and so many of the pathogenic organisms are phagocytosed and killed by mammalian leukocytes. • However, not all engulfed Y. pestis are killed. Bacteria that are ingested by neutrophils appear to be readily killed, but bacteria within macrophages are able to survive. • The macrophages provide a protected environment for Y. pestis to resynthesize their F 1 capsular layer and other virulence antigens (activated by the warm 37ºC body temperature). The ability of Y. pestis to survive and grow in macrophages is critical to the early pathogenesis of plague.

Pathogenesis of plague n Y. pestis within the macrophages are then trafficked to the local draining lymph node. The massive infiltration of phagocytic cells within the lymph nodes cause them to become hot, swollen and hemorrhagic. n This gives rise to the characteristic black buboes responsible for the name of this disease.

Pathogenesis of plague n n Within the bubo, by an unknown mechanism, the bacteria then escapes from the infected macrophages to adopt an extracellular lifestyle, where they further grow and replicate. The organisms, with their newly formed antiphagocytic F 1 envelope, can now resist phagocytosis by the leukocytes. In addition, Y. pestis can actually kill macrophages with an apparatus called the Type III secretion system. n Eventually, the infection can spill out into the bloodstream, leading to involvement of the liver, spleen, and lungs (which leads to 2° septicemic and 2° pneumonic development). n 1° Septicemic n Flea inserts directly into the bloodstream causing migration of y. pestis to organs n 1° Pneumonic n Inhaled Y. pestis bacilli would enter into lungs

Bubonic Plague vs. Pneumonic Plague

Mechanisms that allow spreading of Y. pestis in mammalian host n Plasminogen activator protease (Pla) n Genes located on smallest plasmid p. Pla n Pla is required for the migration of Y. pestis from the subcutaneous infection site into the circulation n Pla derives its name from the fact that it can activate the mammalian plasma enzyme plasminogen to plasmin. Plasmin is responsible for the breakdown of fibrin n Main virulence role of Pla: Cleaves fibrin deposits that trap Y. pestis, thereby promoting plague infection http: //horizon. unc. edu/projects/monograph/CD/Professional_Schools/Mo. By/10 hr m. doc

Mechanisms that allow intracellular lifestyle in the mammalian host n The determinants which allow survival and growth of in the macrophage are not known n However, Y. pestis has been shown to possess a two-component regulatory system called Pho/Pho. Q which is associated with protection from macrophage killing mechanisms. n The ability of Y. pestis to survive in macrophages is critical to the early pathogenesis of the disease.

Mechanisms that allow extracellular lifestyle in the mammalian host n F 1 antigen n Genes located on largest plasmid p. MT 1 n Exposure to temperatures of around 37ºC in mammalian host results in production of large amounts of F 1 antigen, which is exported to Y. pestis surface to assemble into an antiphagocytic envelope. n Yersiniabactin (Ybt) – siderophore n Genes located on chromosome n Used to obtain nutritional iron necessary for bacterial growth from eukaryotic proteins transferrin and lactoferrin n Bacteria requires iron in order to cause infection n Type III secretion system n Genes located on the middle-sized p. YV plasmid n In extracellular environment, this is the weapon used by Y. pestis to kill macrophages n It is the key virulence mechanism that allows Y. pestis to protect itself from phagocytosis.

Importance of siderophores http: //gsbs. utmb. edu/microbook/ch 007. htm

Type III secretion system n Type III secretion system is upregulated at 37ºC, i. e. within the mammalian host. n This system allows Y. pestis that are in contact with a macrophage to inject a range of effector proteins called Yersinia Outer Proteins (Yops) into the macrophage through a “syringe-like” apparatus. The Yops essentially function as a poison that destroys a macrophage’s phagocytic and signalling capabilities, ultimately inducing its apoptosis. http: //www. rkm. com. au/imagelibrary/index. html

n n n Yops When placed in environment that is around 37ºC and with a low Calcium concentration, Y. pestis ceases to grow and expression of Yops is induced. Altogether, there are 29 Yops but not all play a role in Type III secretion system. There at least 6 Yops which directly contribute to the killing of a macrophage: n Yops E, H, J, O, M n Yops B and D n Required for pore formation in the macrophage n Low Calcium Response V antigen (Lcr. V) n Important for the activation of the Type III secretion system

Machinery of this “Biological Syringe” n The Type III secretion system consists of : n The core apparatus for secretion through two bacterial membranes n The “needle” n Yop. B, Yop. D, Yop. K, Lcr. V n Control elements n n Yop. N, Tye. A, Lcr. G The “poison” (Anti-host effector proteins) n Yop. E, Yop. H, Yop. M, Ypk. A and Yop. J

Yersinia’s Deadly Kiss

Plasmids crucial to virulence of Y. pestis Plasmid Name Size (kb) Virulence determinants Role in disease p. MT 1* 96. 2 F 1 capsule antigen Bacterial transmission by Fleas p. YV 70. 3 Several Yops, Type III secretion system Toxicity. Avoidance of immune system p. Pla* 9. 6 Plasminogen activator Dissemination from intra-dermal site of infection *unique to Y. pestis only

Clinical Aspects Signs and Symptoms of Plague n Differential Diagnosis n Laboratory Diagnosis n Treatment n

Initial Signs and Symptoms Incubation Period: 2 – 6 days Bubonic Plague Fever 100% Headache 85% Severe exhaustion 75% Vomiting Septicemic Plague Fever 100% 25 -49% Nausea & Vomiting 50% Altered mental status 38% Altered mental status common Abdominal pain 18% Abdominal pain 39% Cough 25% Skin rash 23% 2º septicemic plague 23% Diarrhea 39% 2º pneumonic plague 5 -15% Chest x-ray Patchy bilateral infiltrates

Signs and Symptoms (Bubonic Plague) • Pain/tenderness at regional lymph nodes enlarge to become “buboes” • Extremely painful • occur in groin , axilla or cervical areas • Ulcer or skin lesions at site of flea bite in <10% of cases

Septicemic plague n 1º septicemic plague is due to spreading of Y. pestis by way of the bloodstream from the site of inoculation without bubo formation n Septicemic plague may also follow an initial presentation of bubonic plague, thereby becoming 2º septicemic plague. n Spreading of Y. pestis to all organs including liver, spleen, heart, kidneys and CNS occurs, leading to septic shock and death.

Signs and Symptoms (Septicemic Plague) • Complications • Hemorrhagic changes in skin called “purpuric lesions” • Disseminated intravascular coagulation (DIC) • Extremity gangrene • It is the blackened gangrene characteristic of advanced septicemic plague that gave the pandemic of Medieval Europe the name “Black Death. ”

Signs and Symptoms Pneumonic Plague • Incubation period of 1 -3 days • Productive cough • Hemoptysis • Rapid, shallow breathing • Cyanosis • Nausea and vomiting • Abdominal pain • Chest x-ray with alveolar infiltrates

Differential diagnosis of plague Bubonic • Tularemia • Cat Scratch Disease • Chancroid • Lymphogranuloma venereum • Bacterial adenitis • Tuberculosis • Scrub Typhus Septicemic • Septicemia caused by other Gram Negative bacteria • Meningcoccemia • Rocky Mountain Spotted Fever Pneumonic • Inhalational anthrax • Tularemia • Viral Pneumonia (Influenza, Hantavirus, CMV) • Q fever

Differential Diagnosis Pneumonic Plague vs. Inhalational Anthrax • bilateral pulmonary infection, with greater infection in the left lung. • widened mediastinum, resulting in less available space for lungs

Diagnosis Conditions for Suspected Plague: 1. Clinical symptom of Plague, such as fever and buboes, in the person 2. 2. Person resides in or has recently traveled to a plague-endemic region. Exposure to rodents or fleas in the western U. S. 3. Samples taken from affected tissues that are Giemsa stained show the bacillus to have a bipolar or “safety” pin appearance. Samples are taken from bubo (bubonic plague), blood (septicemic plague), or tracheal/lung aspirate (pneumonic plague). http: //www. cdc. gov/ncidod/dvbid/plague/p 1. htm

Diagnosis Conditions for Presumptive Plague: 1. Immunofluorescence stain of sample is positive for the presence of Y. pestis F 1 antigen. Pro: This test can be done quickly (less than 2 hours) Con: Since F 1 antigen is expressed at > 33ºC, samples that have been refrigerated or are from culture that have been incubated at lower temperatures would test negative http: //www. cdc. gov/ncidod/dvbid/plague/p 4. htm

Diagnosis Conditions for Confirmed Plague: 1. Isolate Y. pestis from the specimen OR 2. Observe at least a 4 fold elevation in serum antibody titer to the F 1 antigen (smaller elevations are considered a presumptive diagnosis). Con: Neither of these 2 techniques is fast – Y. pestis grows very slowly in culture, and antibodies can take a significant amount of time after disease onset to develop – so they are usually useful only as a retrospective confirmation of plague.

Treatment

Precautions for Dealing with Plague Victims n n Since the only form of human to human spread occurs by respiratory droplet from a patient with pneumonic or secondary pneumonic plague, surgical masks, gloves, gowns, and goggles should be worn at all times All patients with pneumonic plague should be strictly isolated (as required by law) until they have received 48 hours of antibiotic treatment and show signs of improvement Labs should operate at biosafety level 2, unless they are performing tests that may aerosol or produce droplets in which case biosafety level 3 should be observed No environmental decontamination is necessary as the bacteria is quite fragile outside of the host environment

Treatment n n n In a contained casualty setting, parenteral antibiotic therapy, especially streptomycin or gentamycin, is suggested. In a mass casualty setting, intravenous or intramuscular therapy may not be possible, so oral therapy, preferably with doxycycline (or tetracycline) or ciprofloxacin, should be administered. Patients with pneumonic plague will suffer from complications and therefore require substantial advanced medical supportive care.

Treatment in a Contained Casualty Setting Patient Category Recommended Therapy Contained Casualty Setting Adults Preferred choices: Streptomycin, 1 g IM twice daily Gentamicin, 5 mg/kg IM or IV once daily or 2 mg/kg loading dose followed by 1. 7 mg/kg IM or IV three times daily † Alternative choices: Doxycycline, 100 mg IV twice daily or 200 mg IV once daily Ciprofloxacin, 400 mg IV twice daily ‡ Chloramphenicol, 25 mg/kg IV 4 times daily § Children|| Preferred choices: Streptomycin, 15 mg/kg IM twice daily (maximum daily dose 2 g) Gentamicin, 2. 5 mg/kg IM or IV 3 times daily † Alternative choices: Doxycycline, If >= 45 kg, give adult dosage If < 45 kg, give 2. 2 mg/kg IV twice daily (maximum 200 mg/dl) Ciprofloxacin, 15 mg/kg IV twice daily ‡ Chloramphenicol, 25 mg/kg IV 4 times daily § Pregnant Women¶ Preferred choice: Gentamicin, 5 mg/kg IM or IV once daily or 2 mg/kg loading dose followed by 1. 7 mg/kg IM or IV three times daily † Alternative choices: Doxycycline, 100 mg IV twice daily or 200 mg IV once daily Ciprofloxacin, 400 mg IV twice daily ‡

Treatment in a Mass Casualty Setting and Postexposure Prophylaxis# Adults Preferred choices: Doxycycline, 100 mg orally twice daily ** Ciprofloxacin, 500 mg orally twice daily ‡ Alternative choices: Chloramphenicol, 25 mg/kg orally 4 times daily §, †† Children|| Preferred choices: Doxycycline, ** If >=45 kg give adult dosage If <45 kg then give 2. 2 mg/kg orally twice daily Ciprofloxacin, 20 mg/kg orally twice daily Alternative choices: Chloramphenicol, 25 mg/kg orally 4 times daily §, †† Pregnant Women¶ Preferred choices: Doxycycline, 100 mg orally twice daily and Ciprofloxacin, 500 mg orally twice daily Alternative choices: Chloramphenicol, 25 mg/kg orally 4 times daily §, ††

Benefits of Treatment Type of Plague Untreated Fatality Rate with Treatment Bubonic 50 -90% 5 -20% Septicemic 50 -100% 30 -50% Pneumonic 100% and death occurs rapidly usually within 48 hours of onset Unknown (too few cases for accurate results)

How Antibiotics Work n Antibiotics inhibit prokaryote protein synthesis by preventing the transition from initiation complex to chain-elongating ribosome and causes miscoding.

Weaponization n The CDC ranks the plague as a Category A disease n “Agents in Category A have the greatest potential for adverse public health impact with mass casualties, and most require broad -based public health preparedness efforts (e. g. , improved surveillance and laboratory diagnosis and stockpiling of specific medications). Category A agents also have a moderate to high potential for large-scale dissemination or a heightened general public awareness that could cause mass public fear and civil disruption. ”

The Oldest Bioweapon n The plague has a long history as an agent of biological warfare

Yersinia Pestis as a Weapon n Pros n Cons n It is relatively easy to obtain and mass produce. n n It can be delivered in aerosol form n n Pneumonic plague causes a rapid onset of illness with a high fatality rate Plague is fragile and dies after about 1 hr Manufacturing an effective weapon using Y. pestis would require advanced knowledge and n Pneumonic plague has a high potential for secondary spread of cases during an epidemic n 100 -500 bacteria are enough to cause pneumonic plague technology

Additional Dangers of Yersinia Pestis as a Weapon n n There is no currently available pre-exposure prophylaxis or vaccine for plague Biological attack with plague might employ antimicrobialresistant strains that circumvent clinical efforts to deal with the disease n In 1995 a patient in Madagascar was found who had a Y. Pestis with a transferable multidrug resistance plasmid (natural) n Additionally, there are reports that the bioweapons operations of the former Soviet Union engineered multidrug resistant and fluoroquinolone resistant Y. Pestis

Effectiveness of Y. Pestis as a Weapon n n While antibiotic treatment of bubonic plague is usually effective, pneumonic plague is difficult to treat and often results in death regardless of treatment Most experts agree that “intentional dissemination of plague would most probably occur via an aerosol of Y pestis, a mechanism that has been shown to produce [pneumonic] disease in nonhuman primates…The size of the outbreak would depend on the quantity of biological agent used, characteristics of the strain, environmental conditions, and methods of aerosilization…people would die quickly following the onset of symptoms. ” - JAMA May 3, 2000 Vol 283, No. 17 In 1970, the WHO estimated that “if 50 kg of Y pestis were released as an aerosol over a city of 5 million, plague could occur in as many as 150, 000 persons, 36, 000 of whom would be expected to die. ” And this does not take into account the people who would die from secondary contraction of the disease. According to the CDC, “The fatality rate of patients with pneumonic plague when treatment is delayed more than 24 hours after symptom onset is extremely high. ”

Means of Detection n n With its relatively short onset time, the best defense against the plague is early notification There are currently two means of detection n Since the disease is passed directly or via a flea vector from animal reservoirs (such as rats, ground squirrels, etc) to humans, one mode of detection is observation of these reservoirs (the bacterium survives by causing chronic disease in animal reservoirs, so a sudden increase in the death of rats, will cause the flea vectors to find another source of food) n The employment of Y. Pestis as a weapon would likely involve aerosilization of the bacteria and direct release upon humans. Thus new detection methods are required

Autonomous Pathogen Detection Systems (APDS) n The LLL has just finished developing a prototype for a system capable of detecting aerosolized bacteria including Bacillus Anthracis and Yersinia Pestis.

APDS: How It Works n n The sample is collected through an aerosol collector Next the sample is delivered to a fluidics module which reproduces functions routinely performed by laboratory personnel on the bench, including various mixings, filterings, and incubations to prepare the sample for detection Next, the sample is delivered to the immunoassay detector n The detector consists of a column of polystyrene microbeads, which have antigen-specific capture antibodies attached to them n There a hundred different bead classes (various beads have different antibodies resulting in an “antibody cocktail”) n When the antigen (bacteria) binds to the appropriate antibody, secondary antibodies labeled with the fluorescent reporter phycoerythrin bind to the now immobilized antigen from the other side n The fluorescent secondary antibodies can then be detected with classification lasers and fluorescence detectors. Most of the sample is discarded as waste, but a small amount is archived, with the appropriate data

APDS: How it Detects n n n Using the previously described fluorescent “antibody sandwich” detection system, the system is able to detect the median fluorescent intensity (MFI) The detection threshold is the background MFI value plus three standard deviations n For Yersinia Pestis, the background MFI was 155 with a standard deviation of 25, resulting in a threshold detection limit at an MFI of 230 n Establishing this criteria for a detection threshold helps to eliminate false positives Because the fluorescence is detected at specific beads, which have specific antibodies, the system is able to detect the type of antigen in addition to the concentration of the antigen

APDS: Primary Testing Results and Intended Improvements n Primary testing of the APDS has given excellent results, and proven the APDS capable of continuous and unattended operation, and the platform is sensitive and specific, detecting releases with no false positives. In addition, the system is able to detect the simultaneous release of multiple pathogens. n n Scientists are currently developing a confirmatory nucleic acid-based test to augment the detection capabilities of the system The final prototype is scheduled for later this year

APDS: Where and How Can We Use It n The key functional features of the APDS are that it is autonomous and can run unattended for 8 day stretches, can detect specific pathogens in a relatively short period of time (about 1 minute) without false positives n n The system is intended for use in office complexes, transportation terminals, or convention centers where the public is at high risk of the release of bioagents. In particular, each system can be part of an integrated network of biosensors for wide-area monitoring of urban areas and major gatherings The system would need only a source of electricity and a network connection, to allow it to perform continuous sample collection, immunoassays every 30 -60 minutes, sample archiving, data reporting, and alarming

Summary The plague has a long history as a killer, and due to its notoriety can be used to inspire fear. n Although the plague is a very real threat, the CDC is taking it seriously, and steps are being taken to increase our defensive preparedness for such an attack. Perhaps the best thing that can be done is to have an early detection system, and scientists are rapidly making efforts to do just that. n