Immunologic indicators of clinical progression during canine Leishmania infantum infection

Abstract

In both dogs and humans Leishmania infantum infection is more prevalent than disease, as infection often does not equate with clinical disease. Previous studies additively indicate that advanced clinical visceral leishmaniasis is characterized by increased production of anti-Leishmania antibodies, Leishmania-specific lymphoproliferative unresponsiveness, and decreased production of gamma interferon (IFN-gamma) with a concomitant increase of interleukin-10 (IL-10). In order to differentiate infection versus progressive disease for better disease prognostication, we temporally evaluated humoral and cellular immunologic parameters of naturally infected dogs. The work presented here describes for the first time the temporal immune response to natural autochthonous L. infantum infection in foxhounds within the United States. Several key changes in immunological parameters should be considered when differentiating infection versus clinical disease, including a dramatic rise in IgG production, progressive increases in antigen-specific peripheral blood mononuclear cell proliferation, and IFN-gamma production. Polysymptomatic disease is precluded by increased IL-10 production and consistent detection of parasite kinetoplast DNA in whole blood. This clinical presentation and the immuno-dysregulation mirror those observed in human patients, indicating that this animal model will be very useful for testing immunomodulatory anti-IL-10 and other therapies.

FIG. 1.

Anti-L. infantum IgG1 and IgG2 responses increase with CVL disease progression. L. infantum-specific IgG responses were measured via ELISA using sera from a control (one dog), noninfected (two dogs), infected resistant (four dogs), infected susceptible (two dogs), and clinical (three dogs) animals. Blood samples were collected and centrifuged to clarify serum. Results shown are OD values from the antigen-specific IgG1 (A) and IgG2 (B) ELISAs. Lines indicate mean values for each group.

FIG. 2.

Decreased lymphoproliferative response in PBMC from L. infantum-infected, clinical dogs. (A) PBMC proliferative response evaluations from a control (one dog), noninfected (two dogs), infected resistant (four dogs), infected susceptible (two dogs), and clinical (two dogs) animals were repeated monthly over 3 to 6 months. PBMC were isolated, stained with CFSE, restimulated with freeze-thawed L. infantum antigen, and incubated for 7 days at 37°C. Cells were then harvested and stained with a PE-conjugated anti-CD4 antibody. PMBC CD4⁺ T-cell proliferation was assessed via CFSE dilution using flow cytometry. Each point is indicative of a blood draw from each animal over a 3- to 6-month period and subsequent proliferation assay. At least four separate proliferation assays were carried out over time on each dog in every group. Horizontal lines indicate the mean responses for each group. *, significant difference (P < 0.05). (B) PBMC proliferative responses to L. infantum antigen (Ag), distemper vaccine (DHPP), or ConA stimulation for the control (one dog), infected susceptible (three dogs), and clinical (two dogs) animals. PBMC were isolated and processed as described for panel A and stimulated with L. infantum antigen for 7 days, with DHPP for 10 days, or with ConA for 4 days. CD4⁺ T-cell proliferation was assessed via CFSE dilution using flow cytometry. At least three separate experiments were carried out for each dog in every group. Bars indicate the average proliferation for each group (± standard errors of the means).

FIG. 3.

Disease progression correlates with decreased IFN-γ and increased IL-10 production. Shown are PBMC effector cytokine responses from the control (one dog), noninfected (two dogs), infected resistant (four dogs), infected susceptible (two dogs), and clinical (two dogs) animals. Culture supernatants were collected from PBMC cultures stimulated with L. infantum antigen for 7 days and analyzed via ELISA for IFN-γ (A) and IL-10 (B). Each point is indicative of one experiment. At least three separate experiments were carried out for each dog in every group. Lines indicate the mean responses for each group. *, significant difference (P < 0.05). ND, not detectable.