Pneumonic plague pathogenesis and immunity in Brown Norway rats

Abstract

The Brown Norway rat was recently described as a bubonic plague model that closely mimics human disease. We therefore evaluated the Brown Norway rat as an alternative small animal model for pneumonic plague and characterized both the efficacy and potency of vaccine candidates. When infected by intranasal instillation, these rats rapidly developed fatal pneumonic plague within 2 to 4 days of infection. Plague disease was characterized by severe alveolar edema and vascular hemorrhage in the lung in addition to fulminant necrotizing pneumonia caused by massive bacterial replication and inflammation. Twenty-four hours before death, animals developed systemic disease with an apparent delayed inflammatory response. We evaluated the ability of the protective antigen, LcrV, and a mutant derivative, V10, to protect these rats from pneumonic plague. Both were highly effective vaccines because complete protection was observed at challenge doses of 7500 LD(50). Antibody analyses suggested stronger potency of V10 immune sera compared with LcrV in the passive transfer of immunity to bubonic plague, with multiple neutralizing epitopes in LcrV. Taken together, these data demonstrate the effectiveness of inhibiting type III secretion in the prevention of pneumonic plague in rats and reveal critical contributions from both the cellular and humoral immune systems. Thus, the Brown Norway rat is an appealing alternative small animal model for the study of pneumonic plague pathogenesis and immunity.

Figure 1

Brown Norway rats are highly susceptible to pneumonic plague but only sometimes generate immunity after sublethal challenge. Groups of four to six Brown Norway rats were challenged with increasing doses of Y. pestis CO92 delivered by intranasal instillation. A: Animals succumbed to disease between 60 and 108 hours after infection, with a mean 50% lethal dose (LD50) of 171 cfu. Data shown are representative of three independent experiments. B: Rats that survived sublethal infection (R1 to R5) did not consistently develop antibodies to dominant plague antigens, CaF1 and LcrV, because only three of five survivors (R1, R2, and R4) from the experiment in A had seroconverted by day 14 after challenge.

Figure 2

Time course of bacterial dissemination in rats infected with 600 LD50 Y. pestis CO92 showed systemic spread by 48 hours after infection. Viable bacteria recovered from the lung, liver, and spleen after 24 hours (A), 36 hours (B), 48 hours (C), or 60 hours (D). Three animals were analyzed per time point; two of the three animals in group 4 succumbed to the infection before 60 hours and are indicated as × on the graph. Y. pestis was identified by colony morphology and pigmentation on heart infusion agar with Congo Red; each sample was plated in triplicate; data shown are average cfu/ml of homogenized tissue. This experiment was repeated with similar results; a representative experiment is shown.

Figure 3

Lungs from rats infected with Y. pestis CO92 were harvested and perfused with 10% formalin and stained with H&E after 60 minutes (control, A and B), 24 hours (C and D), or 48 hours (E–H) after infection. Boxes in lower magnification images indicate location where the higher magnification images were taken. Original magnifications: ×100 (A, C, E, G); ×400 (B, D, F, H).

Figure 4

H&E-stained spleens show development of severe pathological lesions affecting primarily red pulp by 48 hours after infection. Spleens were harvested and fixed in 10% formalin after 1 hour (A and B), 24 hours (C and D), or 48 hours (E and F) after infection by Y. pestis CO92. Original magnifications: ×100 (A, C, E); ×400 (B, D, F).

Figure 5

Lung macrophages are recruited to the infection and rapidly killed by Y. pestis. Left lower lobe sections of lungs harvested 1 hour (A and B), 24 hours (C and D), or 48 hours (E and F) after infection were perfused in 10% formalin followed by immunohistochemistry with antibodies to CD68. Images are representative of lesions found in the tissues. Original magnifications: ×100 (A, C, E); ×400 (B, D, F).

Figure 6

Cleaved caspase-3 is found in infected areas of the lungs suggesting host cells are undergoing apoptosis. Left lower lobe sections of lungs harvested 1 hour (A and E), 24 hours (B and F), or 48 hours (C, D, G, and H) after infection were perfused in 10% formalin followed by immunohistochemistry with antibodies to cleaved caspase-3. C and G: In moderate lesions, caspase-3 staining appears limited to the periphery of the lesion. D and H: More intense staining and higher background were found in congested areas. Images are representative of lesions found in the tissues. Original magnifications: ×100 (A–D); ×400 (E–H).

Figure 7

Humoral and cell-mediated immune responses to V and V10 in Brown Norway rat reveals complexity in antibody-mediated neutralization of plague. A: LcrV peptide epitope mapping of V and V10 immune sera shows different epitope binding profiles elicited by the two antigens. B: Competition for binding LcrV between neutralizing LcrV monoclonal antibody BA-5 and V or V10 rat immune sera. C: Memory T-cell assay in splenocytes purified from rats immunized with V or V10 shows greater IFN-γ production in V-immunized rats compared with V10-immunized rats.