1. Zoonotic infections (bacterial) Definition: Infectious diseases of animals that are transmitted to humans either by contact, food sources or by a vector (insects) Many bacterial pathogens are zoonotic pathogens because they display host ranges involving multiple mammalian species and are to humans and vice versa; examples are, Lyme disease, Rocky Mountain Spotted Fever, S. aureus, Salmonella gastroenteritis, EPEC, EHEC. Other bacterial pathogens cause disease only in humans; animals, wild or domesticated, play no role in ecology and disease establishment: Neisseria gonorrhoea, Neisseria meningitidis, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus infleunzae, Mycobacterium tuberculosis, Corynebacterium diphtheriae. This lecture will discuss bacterial zoonotic pathogens causing severe and frequently lethal infectious diseases in humans: anthrax, plague, tularemia and brucellosis. The pathogens Bacillus anthracis, Yersinia pestis and Francisella tularensis are considered biological weapons of mass destruction and their possession, storage and proliferation is regulated by the US Patriot Act.

2. Biological warfare agents (CDC) Select Agents, Category A Plague (Yersinia pestis) Anthrax (B. anthracis) Tularemia (F. tularensis) Botulism (BoNTs) Smallpox Viral Hemorrhagic Fevers (Ebola, Marburg etc.) Category B (partial) Brucellosis Typhus (R. prowazekii) Q Fever (C. burnetii)

3. Plague, a recently evolved disease impacts humanity Mass graves of plague victims Lazzaretto Vecchio Island, Venice Black rat Xenopsylla cheopsis (oriental rat flea)

4. Three plague pandemics decimate the human population (50-60%) Justinian (541-740), Black Death (1346-1731), Asian (1855- 1935) Wagner et al. (2014) Lancet Infect. Dis. 14:319

5. Ancient Y. pestis genomes – SNPs, deletions and mobile elements Wagner et al. (2014) Lancet Infect. Dis. 14:319

6. Current plague epidemiology Global distribution of Y. pestis, low incidence of human disease Stenseth et al. (2008) PLoS Med.5:e3

7. Natural cycles of plague in the United States

8. Yersinia pestis Enterobacteriaceae, evolved from Y. pseudotuberculosis (1,500-20,000 years ago) Y. pestis shares 70 kb pCD1 plasmid & type III secretion pathway (LcrV needle cap protein) with Y. pseudotuberculosis and Y. enterocolitica Acquired two unique plasmids pFra (F1 capsule/pilus production) and pPCP (Pla, plasminogen activator) for virulence and plague pathogenesis Acquired factors for flea-borne transmission to mammals Infects most vertebrate animal species to cause lethal plague disease

9. Bubonic plague Transmission: Y. pestis inoculated via flea bite from insects that fed on infected human or rodent Incubation: 2-6 days Clinical: Fever, chills, headache, myalgias, prostration, occasionally nausea and diarrhaea, swollen lymph node or bubo Diagnosis: Lymph node aspirate, Wayson staining, culture; serological test (F1 specific antibody positive after 8-14 days) used for retrospective diagnosis Treatment: streptomycin or gentamycin, (ciprofloxacin, levofloxacin as alternatives) Prognosis: 40-60% fatal without treatment (14% of U.S. cases fatal, 1970-present) because Y. pestis disseminates to cause septicemic plague; bubonic plague survivors are immune to Y. pestis infection (F1-specific antibodies). Epidemiology: 4-40 cases in the US rural West

10. Pneumonic & septicemic plague Transmission: Pneumonic plague -Y. pestis inhaled via aerosol droplets typically from plague infected humans (~5-10% develop secondary pneumonia) under crowded conditions; septecimic – flea inoculation into the bloodstream, animal bite/scratch, direct inoculation Incubation: 1-3 days Clinical: acute, fulminating disease with sepsis, shock and lethal outcome within 1-3 days Diagnosis: Blood cultures (Wayson stain), NAAT (PCR); serological test (F1 specific antibody positive after 8-14 days) used for retrospective diagnosis Treatment: ICU, symptomatic, streptomycin or gentamycin, (ciprofloxacin, levofloxacin as alternatives) Prognosis: 100% fatal without treatment Epidemilogy: 1-10 cases in the US rural West Pneumonic plague Septicemic plague

11. Plague pathogenesis Marketon et al. (2005) Science 309:1739

12. Yop effectors subvert innate defenses of immune cells

13. Y. pestis establishes a biofilm and blocks the flea proventriculus uninfected flea Y. pestis infected flea blood meal blocked flea proventriculus Y. pestis biofilm esophagus midgut

14. Asian plague pandemic ends in 1935 Girard and Robic (Institut Pasteur) inoculatd 500,000 Malagasies with Y. pestis EV76 (pgm-iron defective) and achieve plague protection Girard & Robic (1942) Bull. Soc. Pathol. Exotique 35:42

15. Yersiniabactin, a siderophore for iron scavenging from transferrin, is absent from the EV76 vaccine Crosa and Walsh (2002) MMBR 66:223

16. LcrV vaccine protects animals (mice, rats, guinea pigs, non-human primates) against plague currently no licensed vaccine in the US

17. Tularemia – Francisella tularensis Definition: infection with Francisella tularensis, Gram-negative facultative intracellular pathogen, whose natural reservoirs are wild-animals, small rodents, hares, rabbits; 100-200 cases/year in the US Transmission: ticks (other arthropods), deer flies, contact or aerosol; very low infectious dose; 1-14 day incubation period; untreated 7% mortality for all disease forms Clinical: six clinical entities depend on site and route of transmission: ulceroglandular (75% of tularemia), glandular, oropharyngeal, oculoglandular & pneumonic or typhoidal (highest mortality; fever, malaise, shock and death; subacute disease occurs frequently; lymphadenopathy with necrosis of immune cells. Diagnosis: lesion swab, LN biopsy, sputum, pharyngeal wash: growth on buffered-charcoal and yeast extract (BCYE); 4x increase in F.t. serology also diagnostic; NAATs (PCR), fluorescent antibody: presumptive diagnosis Treatment: 21 days streptomycin/gentamycin, doxycycline or ciprofloxacin Prevention: live-attenuated vaccine (LVS) is not licensed for general use; protective equipment and precaution for hunters; avoiding ticks

18. Tularemia disease states & transmissiom ulceroglandular: most common form, characterized by ulcer at entry site and swelling of regional lymph nodes; insect or contact transmission glandular: similar to above, but without ulcer oculoglandular: bacteria enter through the eye; contact with infected animals oropharyngeal: sore throat, mouth ulcers, tonsillitis, glandular swelling in the neck; transmission by eating infected animals pneumonic: most serious form characterized by cough, chest pain and respiratory distress; result from inhalation of the pathogen (lawn mowers aerosolize F.t. from dead animals) typhoidal: systemic infection in the RES, high mortality ticks deer fly hare lawn mower

19. US tularemia cases, 2003-2013

20. Tularemia ulceroglandular pneumonic

21. Francisella pathogenesis escape from the endosome and intracellular replication

22. Francisella survival inside macrophages requires escape from the phagosome and specific genes

23. Brucellosis Definition: Gram-negative, facultative-intracellular coccobacillus transmitted from animals; replicates in cells of the RES to cause persistent infection with granuloma formation in liver/spleen, undulating fever and secondary manifestations Epidemiology: 500,000 new cases/yr worldwide; 90-150 US cases Transmission: inhalation (aborted animal fetus), consumption of contaminated milk & milk products, or direct contact of bacteria with skin lesions (animal workers) Initial symptoms: fever, malaise, anorexia, headache, muscle pain, arthralgia, hepatomegaly, splenomegaly Recurrent symptoms: fever, arthritis, osteomyelitis, meningoencephalitis and endocarditis (1-2%) Diagnosis: Blood culture (Castaneda bottle- 6 weeks); Brucella microagglutination test (BMAT, to detect serum Abs against B. abortus, B. melitensis & B. suis: 2x for increase in Ab titer) Treatment: 6-8 week course of rifampin and doxycycline Prevention: no vaccine available for humans; milk pasteurization; protective equipment; vaccinate animals At risk: farmers, animal workers, veterinarians, laboratory scientists Sir David Bruce

24. Brucella species and their potential to cause human disease SpeciesHost preferenceZoonosis a Brucella melitensisSheep, goatHigh Brucella abortusCattleModerate Brucella suisPigModerate Brucella canisDogMild Brucella cetiDolphin, porpoise, whaleMild Brucella pinnipedialisSealMild Brucella inopinataUnknownMild Brucella ovisSheepNo Brucella neotomaeDesert woodratNo Brucella microtiCommon voleNo Brucella sp. (baboon)BaboonNo

25. Ecology of zoonotic Brucella infections

26. Brucella replicate in ER vesicles within macrophages

27. Subversion of innate immune responses by Brucella melitensis Btp1/TcpB

28. Non-opsonic entry and subsequent trafficking of Brucella spp. in macrophages

29. Microgranuloma formation by Brucella spp. in the liver

30. Undulant fever during brucellosis Blood culture Castaneda bottle

31. U.S. bovine brucellosis eradication program RB51 vaccination and culling Affected Herds –in 1957 : 124,000 –in 2000 : 6 Financial impact – in 1957 : > $400 million – in 2000 : < $1 million Feb, 1957 Brucellosis infected herds

32. Surveillance, vaccination, eradication

33. Anthrax Definition: Gram-positive spores of Bacillus anthracis transmitted from animals or the environment germinate and replicate in host tissues and cause fatal disseminated disease; not transmitted between humans Epidemiology: worldwide incidence unknown; 1-2 cases in the US/yr Transmission: contact with spores (cutaneous A.), ingestion (gastro- intestinal A.), injection by IVDAs (i.A.), inhalation (respiratory A.; wool sorter’s disease) Symptoms: eschar, lymphadenopathy (c.A.); fever, sore throat, nausea, vomiting, diarrhea (g.-i.A.); fever, chest pain, cough, respiratory distress (r.A.); eschar, local necrosis, meningitis (i.A.); anthrax is rapidly fatal Diagnosis: Culture B. anthracis from lesion (swab), blood, CSF, sputum; retrospective diagnosis (8-14 days) anti-PA antibody titer Treatment: 8 week course of antibiotics (ciprofloxacin and others); spores do not germinate in the presence of antibiotics; anti-toxin (PA) Prevention: AVA vaccine (FDA approved); anti-PA injection, ciprofloxacin for 60 days. At risk: farmers, hunters, veterinarians, laboratory scientists

34. B. anthracis life cycle B. anthracis spores contaminate soil, are taken up by herbivores and invade GI tract. Animals develop gastrointestinal anthrax and die. Sores contaminate the environment Spore vegetative forms

35. Clinical appearance of anthrax CDC cutaneous anthrax respiratory anthrax cutaneous anthrax

36. Anthrax in intravenous drug (heroin) users

37. B. anthracis virulence plasmids, pXO1 & pXO2 attenuated strains and whole cell live-attenuated vaccines The Sterne vaccine strain 4F32 is used world-wide to prevent anthrax in domesticated farm animals; the Soviet Union and other countries used a Sterne type strain to prevent cutaneous anthrax in humans, vaccinating 30 million people Max Sterne: B. anthracis pXO1 +, pXO2 - Louis Pasteur: B. anthracis pXO1 -, pXO2 +

38. pXO1-encoded protective antigen (PA), lethal factor (LF) and edema factor (EF) are secreted to form the lethal (PA+LF) and edema (PA+EF) toxins

39. pXO2-encoded genes synthesize the poly-D-γ- glutamic acid capsule of B. anthracis

40. Sverdlovsk (Ukraine), 1979 anthrax outbreak and anthrax weapon facility

41. B. anthracis as a weapon for biological warfare Definition: Preparation and dissemination of infectious spores, e.g. with explosives, to harm large segments of the population Target: inhalational anthrax causes lethal infections within 24-48 hours Generation of Bacillus anthracis strains resistant to antibiotics Generation of recombinant Bacillus strains evading the protective immunity of vaccinated individuals Prevention: AVA vaccination Treatment: Monoclonal antibody against PA