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