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

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. Enzootic (maintenance) cycle Model assumes that fleas remain infected after feeding on immune hosts Susceptible Hosts Resistant/ Immune Hosts Resistant/ Immune Hosts X 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 of other 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 Infect fleas with Y. pestis Allow fleas to feed on immunized or naïve mice Freeze live fleas, screen for infection 3 days Immunize mice 2 days 7-8 weeks

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: LD 50 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 Expresses F1 and 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. Plasmid profile CO96-3188 (pgm-) Control CO96-3188 9.5 kb (pla) 100-110 kb (F1) 70-75 kb (lcrV) 19 kb dimer (pla)

15. Inducing immunity in mice Week0 7 5 Inoculate (CO96-3188(pgm - )) Boost Fleas feed Draw blood to determine feeding-day titer 3 Draw blood, test serum to verify seroconversion (titer ≥ 1:128) 68 Infect fleas

16. Using an artificial feeding system to infect fleas Fleas feed for 1 hour Circulating 37ºC water keeps blood warm Fresh rat blood spiked with ~10 9 cfu/ml CO96-3188 in glass reservoir fleas Mouse skin membrane

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 χ2 = 5.32 DF = 6 P = 0.50 Infection Prevalence

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 Fisher’s Exact χ2 = 3.93 DF = 1 P = 0.14 Infection Prevalence

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 Fisher’s Exact χ2 = 0.10 DF = 1 P = 0.43 Infection Prevalence

28. X. cheopis infection prevalence by mouse titer Likelihood Ratio Χ 2 = 6.35 DF = 4 P = 0.17 Infection Prevalence

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 10 6 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 Flea-Borne Disease Activity Becky Eisen Ken Gage Sara Vetter Mike Woods Jenn Holmes John Montenieri Anna Schottoefer Scott Bearden Diagnostic and Reference Activity Martin Schriefer Jeannine Petersen Chris Sexton John Young Ryan Pappert Animal Care John Liddell Erin Molloy Andrea Peterson Lisa Massoudi

32. Thank You

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

34. Titer effect? X. cheopis Infection Prevalence by Mouse Titer TiterInfection PrevalenceNo. of fleas 1:12898%40 1:25688%26 1:51287%54 1:102487%152 1:204896%26

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 of other 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