The Role of Host Immunity in Interepizootic Maintenance of Yersinia Pestis

1. The Role of Host Immunity in Interepizootic Maintenance of Yersinia Pestis Christine Graham APHL/CDC EID Training Fellow Bacterial Diseases Branch, DVBID, CDC

2. Plague • Caused by Gram-negative coccobacillus,Yersinia pestis • Rare, highly-virulent zoonotic disease • Can infect virtually all mammals • Principal hosts: rodents,lagamorphs, musk shrew in Southeast Asia, Madagascar • Transmission • Flea bite • Direct contact • Exposure to airborne bacteria (pneumonic plague)

3. Plague is characterized by epizootic and quiescent periods • Long silences may be followed by sudden explosions of rodent plague • Human exposure is most likely during an epizootic • Some recent human outbreaks followed decades of quiescence • South Africa (1982) – 10 years • Botswana (1989-1990) – 38 years • India (1994) – 30 years • Mozambique (1994) – 16 years Reference: WHO Plague Manual: Epidemiology, Distribution, Surveillance and Control (1999)

4. How does Yersinia pestis persist between epizootics? • Implications for public health • Enzootic (maintenance) cycle hypothesis • Investigating an assumption underlying this hypothesis • Methods • Results • Conclusions • Next steps

5. Interepizootic maintenance of Y. pestis: implications for public health • Human exposure is most likely during an epizootic • Human plague is rare, often lethal, treatable anticipate, prepare • Understanding interepizootic maintenance of Y. pestis • Improve surveillance • Implement control measures

6. X Resistant/ Immune Hosts Resistant/ Immune Hosts Susceptible Hosts X Enzootic (maintenance) cycle • Y. pestis persists in infected fleas, susceptible hosts • Fleas survive by feeding on resistant/immune hosts

7. Potential enzootic hosts • Deer mice (Peromyscus maniculatus) • California voles (Microtus californicus) • Northern grasshopper mice (Onychomys leucogaster) • Kangaroo rats (Dipodomys spp.) • Rock squirrels (Spermophilus variegatus) • California ground squirrels (Spermophilus beecheyi) • Commensal rats (Rattus rattus, Rattus norvegicus)

8. X Resistant/ Immune Hosts Resistant/ Immune Hosts Susceptible Hosts Enzootic (maintenance) cycle • Model assumes that fleas remain infected after feeding on immune hosts X

9. Investigating an assumption • Our hypothesis: Feeding on an immune host will clear Y. pestis infection from a flea. • Bell (1945): fleas lose infection more quickly after feeding on an immune host • Host antibodies suppress growth or transmission ofother pathogens in arthropod vectors • Plasmodium vivax - mosquitos (Mendis et al. 1987) • Borrelia burgdorferi - ticks (Fikrig et al. 1992; Gomes-Soleki et al. 2006) • Rickettsia typhi – fleas (Azad & Emala 1987)

10. Study design Immunize mice 7-8 weeks Allow fleas to feed on immunized or naïve mice Infect fleas with Y. pestis Freeze live fleas, screen for infection 2 days 3 days

11. Colony-reared adult female fleas • Xenopsylla cheopis • Commonly infest commensal rats • Primary plague vector in most large epidemics in Asia, Africa, South America • Y. pestis colonizes proventriculus and midgut, can form proventricular block • Oropsylla montana • Commonly infest California ground squirrels and rock squirrels • Primary vector of Y. pestis to humans in North America • Y. pestis colonizes midgut, does not block readily

12. Determining which Y. pestis strains to use • Infecting strain: CO96-3188 • Virulent: LD50 of 10-100 cfu in lab mice • biovar: Orientalis • Immunizing strain: CO96-3188(pgm-) • Avirulent, spontaneously-occurring mutant (10-5) • Corresponds to infecting strain • Contains all 3 plasmids • ExpressesF1and lcrV, encoding proteins known to elicit immune response • Expresses pla, insures dissemination

13. Verifying plasmid and chromosomal content of each strain • Plate on Congo Red • Red colonies: pgm+ • White colonies: pgm- • Plasmid profile analysis • Isolate DNA from overnight cultures by rapid lysis (50 mM Tris, 50 mM EDTA, 4% SDS, pH 12.45-12.6) • Visualize plasmids by gel electrophoresis

14. CO96-3188 (pgm-) CO96-3188 Control 100-110 kb (F1) 70-75 kb (lcrV) 19 kb dimer (pla) 9.5 kb (pla) Plasmid profile

15. Inducing immunity in mice Inoculate (CO96-3188(pgm-)) Infect fleas Boost Boost Week 0 3 5 6 7 8 Fleas feed Draw blood, test serum to verify seroconversion (titer ≥ 1:128) Draw blood to determine feeding-day titer

16. Using an artificial feeding system to infect fleas Circulating 37ºC water keeps blood warm Fresh rat blood spiked with ~109 cfu/ml CO96-3188 in glass reservoir fleas Mouse skin membrane Fleas feed for 1 hour

17. Identifying fed fleas • Identify and separate fleas with red blood meal in proventriculus and/or midgut • Fed fleas presumed infected, held for 2 days Image source: http://www.upmc-biosecurity.org/bin/d/i/rat_flea.jpg

18. Allowing infected fleas to feed on immune or naïve mice • Capsule feeding system • Surviving infected fleas split among naïve and immunized mice • 1 hour feed • Identify, separate fed fleas • Hold fed fleas 3 days

19. Determining infection prevalence • Harvest and freeze live fleas (-80ºC) • Homogenize • 100 μl 10% glycerol in heart infusion broth • Plate and score • 10 μl on sheep blood agar, incubate 36-56 hours at R.T. • Y. pestis growth infected • No Y. pestis growth not infected • Proteus contamination in some X. cheopis samples  plated on selective media

20. Results: O. montana • 316 O. montana fleas, • 165 immune-fed • 151 naïve-fed • 7 immunized, 7 naïve mice • Immunized mouse titers (flea feeding day) • 1:128, n=1 mouse, 19 fleas • 1:512, n=4 mice, 94 fleas • 1:1024, n=2 mice, 52 fleas

21. Infection prevalence in immune-fed O. montana by mouse group Infection Prevalence χ2 =5.32 DF = 6 P = 0.50

22. Infection prevalence in O. montana by mouse group • Naïve-fed fleas: 100% infected; no difference between mouse groups • No mouse effect  pooled naïve-fed and immune-fed flea data

23. Results: Infection prevalence in O. montana Infection Prevalence Fisher’s Exact χ2 = 3.93 DF = 1 P = 0.14

24. Results: X. cheopis • 609 X. cheopis fleas • 298 immune-fed • 311 naïve-fed • Does not include 15 (8 immune-fed, 7 naïve-fed) with unknown infection status

25. Results: X. cheopis • 11 immunized, 12 naïve mice • Immunized mouse titers (flea feeding day) • 1:128, n=1 mouse, 40 fleas • 1:256, n=1 mouse, 26 fleas • 1:512, n=3 mice, 54 fleas • 1:1024, n=5 mice, 152 fleas • 1:2048, n=1 mouse, 26 fleas

26. Results: Infection prevalence in X. cheopis across mouse groups • Immune-fed fleas: 73%-100% infected; no significant difference between mouse groups (χ2 = 16.14, DF = 10, P =0.10) • Naïve-fed fleas: 83%-100% infected; no significant difference between mouse groups (χ2 = 19.27, DF = 11, P =0.06) • Pooled naïve-fed and immune-fed flea data • Analyzed pooled data both with and without fleas with unknown infection status, did not change results

27. Results: Infection prevalence in X. cheopis Infection Prevalence Fisher’s Exact χ2 =0.10 DF = 1 P = 0.43

28. X. cheopis infection prevalence by mouse titer Infection Prevalence Likelihood Ratio Χ2 = 6.35 DF = 4 P = 0.17

29. Conclusions • Feeding on an immune host does not appear to clear Y. pestis infection from fleas. • Longer time period and/or multiple feedings required to clear infection? • Rickettsia typhi-infected fleas exposed to immune rats antibody bound to bacterium at 3 hr; maintained on immune rats  stop transmitting after 19 days (Azad & Emala 1987) • Infected ≠ infectious • Fleas may play a role in interepizootic maintenance of Y. pestis.

30. Next Steps • Bacteria load • 3 days not long enough to clear infection  decrease in bacteria load in immune-fed vs. naïve-fed fleas? • Difference in number of immune-fed vs. naïve-fed fleas above 106 cfu threshold? (Engelthaler 2000) • Preliminary data suggest that bacteria loads are similar between naïve- and immune-fed fleas • Do results differ when fleas infected with a biofilm mutant?

31. Acknowledgements • Diagnostic and Reference Activity • Martin Schriefer • Jeannine Petersen • Chris Sexton • John Young • Ryan Pappert Animal Care • John Liddell • Erin Molloy • Andrea Peterson • Lisa Massoudi Flea-Borne Disease Activity • Becky Eisen • Ken Gage • Sara Vetter • Mike Woods • Jenn Holmes • John Montenieri • Anna Schottoefer • Scott Bearden

32. Thank You

33. Determining infection prevalence in trial with Proteus contamination Plate 10 μl on CIN agar base + 1 μg/ml Irgasan Incubate 36-56 h at R.T. Y. pestis growth? no Dilute 10 μl 1:10 in sterile saline, plate on sheep blood agar yes Incubate 36-56 h at R.T. Visible Y. pestis growth? Contamination? no yes yes no infected not infected unknown

34. Titer effect? X. cheopis Infection Prevalence by Mouse Titer

35. Potential enzootic hosts • Deer mice (Peromyscus maniculatus) • California voles (Microtus californicus) • Northern grasshopper mice (Onychomys leucogaster) • Kangaroo rats (Dipodomys spp.) • Rock squirrels (Spermophilus variegatus) • California ground squirrels (Spermophilus beecheyi) • Commensal rats (Rattus rattus, Rattus norvegicus)

36. Investigating an assumption • Our hypothesis: Feeding on an immune host will clear Y. pestis infection from a flea. • Bell (1945): fleas lose infection more quickly after feeding on an immune host • Host antibodies suppress growth or transmission ofother pathogens in arthropod vectors • Plasmodium vivax - mosquitos (Mendis et al. 1987) • Borrelia burgdorferi - ticks (Fikrig et al. 1992; Gomes-Soleki et al. 2006) • Rickettsia typhi – fleas (Azad & Emala 1987)

37. Misc. notes • “Stable colonization of the flea gut depends on the ability of the bacteria to produce aggregates that are too large to be excreted” (Hinnebusch 2005) Image source: http://www.upmc-biosecurity.org/bin/d/i/rat_flea.jpg