Using genomics to track the dissemination of Yersinia pestis strains

1. Using genomics to track the dissemination of Yersinia pestisstrains Courtesy of www.cdc.gov Deng et al. 2002 J. Bacteriol. 184:16 4601-4611

2. Transmission cycle of Plague

3. Y. pestis and plague • nearly all plague infections are zoonotic with humans being accidental hosts • two different transmission cycles of Y. pestis infection: • zoonotic • human to human • zoonotic (pertaining to animals) plague: transmission due to the bite of infected fleas or contact with infected animals • Zoonotic plague is further divided into: • urban • sylvatic (rural) plague • The second type involves the pneumonic transmission of infection occurring between humans. Courtesy of www.bt.cdc.gov Information from Neil Chamberlain “Transmission Cycles of Plague” www.microbelibrary.org

4. sylvatic plague cycle • wild mammals, usually rodents) involves the passing of plague from mammal to mammal by fleas (Xenopsylla cheopis) infected with Y. pestis. • Mammal to mammal passage of Y. pestis can occur by direct contact but is less common. • Many of these mammals are relatively resistant to the lethal effects of Y. pestis infection. • Squirrels, rabbits, and field rats can all serve as long-term reservoirs for the plague bacterium (enzoonotic plague). • Other mammals are highly susceptible to the lethal effects of Y. pestis resulting in rapid spread of plague among the animals with large numbers dying in the community (epizoonotic plague; e.g., prairie dogs). • If humans enter these areas they acquire the infection via flea bites or direct contact with infected animals. Courtesy of www.cdc.gov Infected rat lymph node (bubo) containing massive numbers of Yersinia pestis with associated necrosis, hemorrhage, and coagulation (courtesy of wwwG.niaid.nih.gov)

5. urban plague cycle • involves the passing of plague from mammal to flea to mammal. • Mammal to mammal passage of Y. pestis can occur by direct contact but is less common. • These mammals, frequently rats, are usually highly susceptible to the lethal effects of Y. pestis. • As a result, large numbers of these animals die (epizoonotic plague). • The infected fleas lacking their more common rodent hosts then bite and infect humans. • Direct contact with infected animals can also result in human infection. Courtesy of www.ssi.nrcs.usda.gov

6. Vehicle of Transmission fleas • The flea draws viable Y. pestis organisms into its intestinal tract. These organisms multiply in the flea and block the flea's proventriculus. • Some Y. pestis in the flea are then regurgitated when the flea gets its next blood meal thus transferring the infection to a new host. • While growing in the flea, Y. pestis loses its antiphagocytic capsular layer. Male Xenopsylla cheopis (oriental rat flea) engorged with blood. This flea is the primary vector of plague in most large plague epidemics in Asia, Africa, and South America. Both male and female fleas can transmit the infection. Courtesy of wwwtest.cdc.gov

7. Human infections • Following flea bites or by direct contact with infected animals usually result in bubonic plague. • Most of the unencapsulated organisms are phagocytosed (ingested) and killed by the polymorphonuclear leukocytes (PMN’s or neutrophils) in the human host. • A few bacilli are taken up by tissue macrophages. • The macrophages are unable to kill Y. pestis and provide a protected environment for the organisms to synthesize their capsular and other virulence factors. • The Y. pestis are taken to the draining lymph nodes by the macrophages. Courtesy of www.cdc.gov

8. Human infection cont. • The encapsulated intracellular organisms then kill the macrophage and are released into the extracellular environment, where they resist phagocytosis by the PMN’s. • The Y. pestis multiply rapidly and cause a hemorrhagic inflammatory response making the lymph nodes hot, swollen, tender, and hemorrhagic. • This gives rise to the characteristic black buboes responsible for the name of this disease: bubonic plague. Courtesy of www.cdc.gov Courtesy of www.cdc.gov

9. Human infection cont. • In a relatively short period of time the bacteria get out of the swollen lymph nodes and into the bloodstream. • Then they infect the liver, spleen, and lungs. Bacteremia (bloodstream infection; septicemic plague) occurs rapidly if not treated and mortality rates can be as high as 75%. • A severe bacterial pneumonia can develop: pneumonic plague. Patients expel large numbers of viable organisms during coughing fits. • Other humans inhale these viable organisms and develop pneumonia. • The spread of Y. pestis via droplets from human to human is also known as the demic plague cycle. • Spread from person to person can be very rapid and has in the past caused epidemics with mortality rates over 90% if untreated. Courtesy of www2.doh.wa.gov Courtesy of www.health.utah.gov

10. Worldwide distribution of Y. pestis and plague Courtesy of www.cdc.gov

11. Historic 3 pandemics of plague -pandemic: is defined as an epidemic that spreads throughout the human population across a large region such as a continent or worldwide -1st pandemic ~550 A.D. confined to mainly Africa and some parts of the middle ease -2nd pandemic originated in Central Asia and spread via trading routes into Europe (Killed ~30% of Europe population) -3rd pandemic started in 1850’s in China’s Yunnan providence century confined mainly to Asia Courtesy of edsitement.neh.gov

12. 2nd pandemic The black death is first reported in Messina, Italy in 1347 Courtesy of edsitement.neh.gov

13. Identifying the causative agent of the plague -French researcher Paul-Louis Simond postulated a connection between human and rodent plague and identified the flea as a possible vector -In 1894, in Hong Kong, bacteriologist Alexandre Yersin isolated the responsible bacterium (Yersinia pestis) and determined the common mode of transmission -A short time later, Japanese physician and researcher Shibasaburo Kitasato independently identified the plague bacillus (after mis-identifying the bacterium at an earlier point). Courtesy of www.cdc.gov Wayson stain of Y. pestis courtesy of www.cdc.gov

14. The first two genomes of Yersinia pestis CO92 & KIM Parkhill et al. 2001 Nature 413, 523-527 Deng et al. 2002 J. Bacteriol. 184:16 4601-4611 FIG. 2. Comparison of KIM and CO92 at the DNA level. The outer circles show the CO92 C-G skew. The second circle shows CO92 IS elements: IS100 (red), IS1541A (blue), IS285 (green), and IS1661 (yellow); short ticks represent partial IS elements. The third circle shows CO92 rRNA operons. The fourth circle shows the CO92 genome in 27 blocks (numbered according to KIM genome order), regions that are conserved by both locations and orientations (red), a single intrareplichore inversion region (yellow), multiple-inversion regions (various blues), and genome-specific sequences (green). The inner four circles show KIM rRNA operons, the KIM genome in blocks, KIM IS elements, and KIM C-G skew. Colors are coded as for CO92. (Deng et al. 2002)

15. As of 04/2008 there are 7 complete and 14 Y. pestis draft genomes Traditionally the strains are classified as serovars (Antiqua, Mediaevalis, Orientalis, and other) based on the following phenotypic characteristics: -Antiqua = East Africa: (glycerol positive, arabinose positive, and nitrate positive) -Mediaevalis = Central Asia: (glycerol positive, arabinose positive, and nitrate negative) -Orientalis Central Asia (glycerol negative, arabinose positive, and nitrate positive) -other (ie Microtus, Pestoides) not consistent for these phenotypes

16. Paleomicrobiology Partial view of the grave in Dreux investigated in this work, which illustrates anthropologic features of a mass grave suitable for paleomicrobiology research. (courtesy of www.cdc.gov) -the prefix paleo comes from the Greek work palaios meaning “ancient” -bacterial colonization of dental pulp can occur during bacteremia -Bacteremia (also known as plague septicaemia with Y. pestis) is the presence of bacteria in the blood Courtesy of www.nidcr.nih.gov

17. Extraction of bacterial DNA from Dental pulp -Some historians believed that a flu-like virus and not Y. pestis was responsible for the 1st and 2nd pandemics -DNA detected in dental pulp confirm that Y. pestis was the cause -Which serovar(s) are most similar to the Y. pestis strain(s) from the dental pulp from the corpses? Figure 1The original protocol developed in our study allows recovering the dental pulp and minimizes the risk of laboratory-acquired contamination of the specimen. The tooth was encasted into sterile resin (1a) ; the apex was sterily sectioned (1b) to give access to the canal system (1c) ; solutions were injected (1d) ; after incubation, the tooth was put upside down into sterile tube (1e) and centrifuged (1f). Tran-Hung et al. PLoS ONE v.2(10); 2007

18. Use of genomic tools to study Y. pestis Concepts in this module that you will address: #1) mutations that affect the production of a full functional gene product that has phenotypic consequences (insertions, deletions, single nucleotide polymorphisms [SNP’s]) to study the genes glpD, napA, and araC #2) Paleomicrobiology investigation, determine which serovar(s) have the most similar matching genes compared to the amplified sequence from the dental pulp of 3 corpses. #3) use of genome alignments; determine a island that is unique to the 4 genomes that infect humans and is absent in Y. pestis strain 91001 #4) determine the conservation of a virulence factor in the 5 strains in the genome alignment. Determine if it is a full functional product in strain 91001.