Two Med Students in Search of Answers Tam-Linh Nguyen Elizabeth Leddy The Plague Two Med Students in Search of Answers Tam-Linh Nguyen Elizabeth Leddy

Intro Patient Info large swelling of lymph nodes at arm pit, neck and groin (buboes) high fever delerium black splotches on skin as the disease progressed, the buboes burst

Culture/Isolation of Organism Organism taken from blood or buboes Media used: blood agar or other enteric media After 24 hours the colonies were pinpoint After 48 hours the colonies were 1-1.5 mm in diameter, and grey to greyish white, and slightly mucloid In broth culture (bile or bile salts), clumps adhere to side of tube

Tests performed

Yersinia Species Three species of Yersinia cause disease in humans Y. pseudotuberculosis and Y. enterocolitica are enteric food and water borne pathogens acquired by ingestion of contaminated food can cross the gastrointestinal mucosa infection is localized

Y. pestis Discovered in 1894 by French bacteriologist Alexandre Yersin Gram negative rod shaped bacilli found in low frequency in wild rodent populations, such as Rattus rattus (small and black) and Rattus norreguis (large grey sewer rat) Oriental rat flea Xenopsylla cheopis is prime transmitter of disease members.aol.com/landmate/ biological.htm

Modes of Transmission Rattus rattus Rattus norvegicus Xenopsylla cheopis

Y. pestis morphology Part of Enterobacteriaceae family Optimal growth at 28 deg. C Non-motile, slow in vitro growth Closely related to E. coli by DNA-DNA hybridization Unable to survive outside of a host organism

A little bit of history 6th Century (Justinian’s plague 541-767 AD)

A little bit of history Black Death (1346 to nearly the 19th century) this was also known as the “Great Dying” or the “Great Pestilence” Responsible for the death of 30% of the European population Third Pandemic (1894)

Transmission of Y. pestis Disease endemic to rat species Transmitted by fleas The flea bites the rat, and ingests the organism The Y. pestis replicates in the digestive tract of the flea A solid mass forms which obstructs the fleas gut

Transmission of Y. pestis www.cdc.gov/ncidod/dvbid/ plague/cheob6x4.htm

Transmission of Y. pestis Flea can’t ingest blood Gets increasingly hungry, therefore increasingly voracious The flea attacks more rats, which are then infected with the microbe When the rat population dwindles, the flea moves onto other available hosts, such as humans

Human to Human Transmission Inhalation of infected respiratory droplets When an infected human coughs, some microbe is released Airborne pathogen can be inhaled by another human in close proximity Accounts for ability of such a large outbreak in Europe

Stages of Disease Bacteria travel through the blood to the nearest lymph nodes In lymph nodes, Y. pestis is ingested by fixed macrophages Y. pestis is able to grow in inactivated macrophages and replicate Elicits an inflammatory response (the bubo)

Stages of Disease The inflammatory response - “bubo” www.exn.ca/Stories/ 2000/09/12/52.asp http://www.cdc.gov/ncidod/dvbid/images/bubo.jpg

Stages of Disease Bacteria from the bubo leak into the blood stream. (septicemic plague) Lysis of the bacteria releases LPS, which causes septic shock Eventually bacteria reach the lung, where they parasitize the lung macrophages (pneumonic plague)

Stages of Disease At the pneumonic stage, the bacteria can be spread to others via aerosols. (respiratory droplets) Direct inhalation at this point of the disease, induces more rapid development (than flea) At this stage the bacteria has well developed virulence factors needed to colonize the human body

Stages of Disease http://gsbs.utmb.edu/microbook/images/fig29_4.jpg

Virulence Mechanism of Y. pestis Many genes necessary for virulence are found on plasmids Due to similarity of Y. pestis and Y. pseudotuberculosis, it is possible to eliminate certain genes located on Y. enterocolitica as critical to causing plague

Virulence Mechanism of Y. pestis The three similar Yersinia species have 70-75 kbp plasmids These plasmids carry a number of genes related to virulence

Virulence Mechanism of Yersinia These virulence genes produce virulence factors that fall into four general categories: Adhesion and invasion (YadA) Execreted antiphagocytic proteins (Yops) Proteins involved in processing and excreting Yops (Ysc) Regulatory proteins (Lcr)

Virulence Mechanism of Y. pestis Genes represented are known to be encoded on the 75-kbp plasmid of Yersinia species

Virulence Mechanism of Y. pestis What do you think would happen if Y. pestis was cured of its virulence plasmids?

Virulence Mechanism of Y. pestis What do you think would happen if Y. pestis was cured of its virulence plasmids? A decrease in ability to colonize in the human body

Virulence Mechanism of Y. pestis What do you think would happen if Y. pestis was cured of its virulence plasmids? A decrease in ability to colonize in the human body Disease not able to spread as easily

Virulence Mechanism of Y. pestis Y. pestis has two more plasmids than Y. pseudotuberculosis and Y. enterocolitica One 110 kbp plasmid not much is known about the genes on this plasmid one gene encodes a protein component of an antiphagocytic protein capsule (F1) another encodes a toxin that kills mice, but has not been shown to be toxic in humans

Virulence Mechanism of Y. pestis Fraction 1 (F1) is a capsule like antigen expressed at 37 deg. C located on the large plasmid found only in Y. pestis Involved in the ability of Y. pestis to prevent uptake by macrophages Does not influence the general ability of phagocytic cells Expression of F1 reduced the number of bacteria that interacted with macrophages

Virulence Mechanism of Y. pestis The second plasmid (pPCP1) is a 9.5 kbp plasmid that encodes 3 proteins: pesticin, pesticin immunity protein, and plaminogen activator

Virulence Mechanism of Y. pestis Plasminogen Activator (Pla) multifunction surface protein this aids in the spread of the disease throughout the human body may also have a role in the insect mediated transmission of the bacteria

Chromosomally encoded virulence factors Three chromosomally encoded virulence genes (yst, psa, inv) the first encodes a heat stable enterotoxin eventually leads to fluid build up in the intestine the second is the main structural component of the Yersinia fimbriae the third helps to colonize deep tissues (lymph nodes)

Yersinia Pestis Genome magpie.genome.wisc.edu/browser/ Yersinia_pestis_circle.html

Special Traits of Y. pestis Two extra plasmids account for higher virulence Plasminogen Hemin storage iron reserve also protect the bacteria from host’s defenses by making it seem more like a host component

Treatment and Prevention Antibiotic Therapy streptomycin, tetracycline, and chloramphenicol all inhibit protein synthesis essential to help the immune system clear the infection the earlier the treatment is initiated, the better the outcome

Treatment and Prevention Sanitation keep rodent population down by proper disposal of garbage eliminate crowded living conditions of substandard housing

Treatment and Prevention Vaccines effective but protects for less than a year vaccine consists of whole killed Y. pestis cells requires a series of injections over a 6 month period live attenuated vaccines injection of non-pathogenic mutant, derived from a fully virulent strain safety questionable

Plague and relation to AIDS CCR5 gene http://www.cdc.gov/ncidod/eid/vol3no3/mcnicf4.gif

“Ring Around The Rosy…” http://www.thelewisfamilymusic.com/010829kidsring.jpg

References Achtman, Mark, et al. “Yesinia pestis, the cause of plague…”. Proceedings of the National Academy of Sciences. Nov. 23, 1999 Balows, Albert. Ed. In chief. 1991. Manual of Clinical Microbiology. 5th Edition. American Society for Microbiology. Washington D.C. Du, Yidong, et al. “Role of fraction 1 antigen of Yersina pestis in inhibtion of phagocytosis.” Infection and Immunity. V70, no3. March 2002 Gerhardt, Philipp, ed in chief. 1994 Methods for General and Molecular Bacteriology. American Society for Microbiology. Washington D.C. Lederberg, Joshua, ed in chief. Encylcopedia of Microbiology. 2nd Edition, v3. Assoc. Press, New York McEvedy, Colin. “The Bubonic Plague.” Scientific American. Feb. 1998 v. 258 n2 p118

References Revel, Paula A. “Yersinia virulence: more than a plasmid. FEMS Microbiology Letters. V206 no 2. Dec. 2001” Salyers, Abigail A., and Dixie E. Whitt. 1994. Bacterial Pathenogeneis, a Molecular Approach. American Society for Microbiology. Washington D.C. Titball, Richard W, and E. Diane Williamson. “Vaccination against bubonic and pneumonic plague.” Elsevier. April 2001 Wieland, Felix. ed. “The Pla surface protease adhesin of Y. pestis mediates bacterial invasion into human endothelial cells”. FEBS Letters. V504, no.1-2. Aug. 2001 http://www.rkm.com.au/PLAGUE/ http://www.cdc.gov/ncidod/dvbid/plague/ http://www.sanger.ac.uk/Projects/Y_pestis/