The Omptins of Yersinia pestis and Salmonella enterica Cleave the ...
The Role of Host Immunity in Interepizootic Maintenance of Yersinia Pestis
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Transcript of The Role of Host Immunity in Interepizootic Maintenance of Yersinia Pestis
The Role of Host Immunity in Interepizootic Maintenance
of Yersinia Pestis
Christine Graham
APHL/CDC EID Training Fellow
Bacterial Diseases Branch, DVBID, CDC
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)
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)
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
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
Enzootic (maintenance) cycle
• Y. pestis persists in infected fleas, susceptible hosts• Fleas survive by feeding on resistant/immune hosts
SusceptibleHosts
Resistant/Immune
Hosts
Resistant/Immune
Hosts
X
X
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)
Enzootic (maintenance) cycle
• Model assumes that fleas remain infected after feeding on immune hosts
SusceptibleHosts
Resistant/Immune
Hosts
Resistant/Immune
Hosts
X
X
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)
Study design
Infect fleas with
Y. pestis
Allow fleas to feed on
immunized or naïve
mice
Freeze live fleas, screenfor infection3
days
Immunize mice
2 days
7-8 weeks
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
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
Expresses F1 and lcrV, encoding proteins known to elicit immune response
Expresses pla, insures dissemination
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
Plasmid profile
CO
96-3188
(pgm
-)
Control
CO
96-3188
9.5 kb (pla)
100-110 kb (F1)
70-75 kb (lcrV)
19 kb dimer (pla)
Inducing immunity in mice
Week 0 75
Inoculate (CO96-3188(pgm-))
Boost Boost
Fleas feed
Draw blood to determine feeding-day
titer
3
Draw blood, test serum to verify seroconversion
(titer ≥ 1:128)
6 8
Infect fleas
Using an artificial feeding system to infect fleas
Fleas feed for 1 hour
Circulating 37ºC water
keeps blood warm
Fresh rat blood spiked with ~109 cfu/ml CO96-3188 in
glass reservoir
fleas
Mouse skin membrane
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
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
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
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
Infection prevalence in immune-fed O. montana by mouse group
χ2 = 5.32DF = 6P = 0.50
0%
20%
40%
60%
80%
100%
Immune Mouse Group
not infected
infected
Infe
ctio
n P
reva
len
ce
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
Results: Infection prevalence in O. montana
0%
20%
40%
60%
80%
100%
Immune Naïve
Host Immune Status
Not Infected
Infected
Fisher’s Exact χ2 = 3.93DF = 1P = 0.14
Infe
ctio
n P
reva
len
ce
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
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
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
Results: Infection prevalence in X. cheopis
Fisher’s Exact χ2 = 0.10DF = 1P = 0.43
0%
20%
40%
60%
80%
100%
Immune Naïve
Host Immune Status
Not Infected
Infected
Infe
ctio
n P
reva
len
ce
X. cheopis infection prevalence by mouse titer
0%
20%
40%
60%
80%
100%
128 256 512 1024 2048
Host Titer
not infected
infected
Likelihood RatioΧ2 = 6.35DF = 4P = 0.17
Infe
ctio
n P
reva
len
ce
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.
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?
Acknowledgements
Flea-Borne DiseaseActivity• 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
Thank You
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
Titer effect?
X. cheopis Infection Prevalence by Mouse Titer
Titer Infection Prevalence No. of fleas
1:128 98% 40
1:256 88% 26
1:512 87% 54
1:1024 87% 152
1:2048 96% 26
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)
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)
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