An experimental protocol for the establishment of dogs with long-term cellular immune reactions to Leishmania antigens.

Domestic dogs are considered to be the main reservoirs of zoonotic visceral leishmaniasis. In this work, we evaluated a protocol to induce Leishmania infantum/Leishmania chagasi-specific cellular and humoral immune responses in dogs, which consisted of two injections of Leishmania promastigote lysate followed by a subcutaneous inoculation of viable promastigotes. The primary objective was to establish a canine experimental model to provide positive controls for testing immune responses to Leishmania in laboratory conditions. After inoculation of viable promastigotes, specific proliferative responses of peripheral blood mononuclear cells (PBMCs) to either Leishmania lysate or recombinant proteins, the in vitro production of interferon-γ by antigen-stimulated PBMCs and a significant increase in circulating levels of anti-Leishmania antibodies were observed. The immunized dogs also displayed positive delayed-type hypersensitivity reactions to Leishmania crude antigens and to purified recombinant proteins. An important finding that supports the suitability of the dogs as positive controls is that they remained healthy for the entire observation period, i.e., more than seven years after infection. Following the Leishmania antigen lysate injections, the infection of dogs by the subcutaneous route appears to induce a sustained cellular immune response, leading to an asymptomatic infection. This provides a useful model for both the selection of immunogenic Leishmania antigens and for immunobiological studies on their possible immunoprotective activities.

Domestic swine in a visceral leishmaniasis endemic area produce antibodies against multiple Leishmania infantum antigens but apparently resist to L. infantum infection.

In order to investigate whether pigs can be infected by Leishmania infantum, a serological and parasitological study was carried out on swine in the Jequié municipality, Northeast of Brazil. Anti-Leishmania infantum antibodies were detected in 37 out of 92 swine (40.2%), by two different assays: an anti-L. infantum lysate and an anti-K39 recombinant protein ELISA. An experimental study was also carried out to verify the susceptibility of domestic pigs to L. infantum infection. Three sows inoculated with 10(8) stationary-phase infective L. infantum promastigotes (26% metacyclic promastigotes) per kilogram of body weight produced anti-Leishmania antibodies until the end of the experiment, 11 months later. No parasites, however, could be visualized through optical microscopy of spleen, liver and bone marrow or by in vitro culture of these organs. Homogenates of these organs were also inoculated in hamsters, without producing infection. No Leishmania DNA was detected by polymerase chain reaction (PCR) in sand flies fed on these animals. The results indicate that domestic pigs bitten by L. infantum-infected vectors in the endemic area do not display a full infection pattern, and the positive association in endemic areas between the presence of swine and infection in canines may not be ascribable to the former acting as a parasite reservoir.

An experimental protocol for the establishment of dogs with long-term cellular immune reactions to Leishmania antigens

Domestic dogs are considered to be the main reservoirs of zoonotic visceral leishmaniasis. In this work, we evaluated a protocol to induce Leishmania infantum/Leishmania chagasi-specific cellular and humoral immune responses in dogs, which consisted of two injections of Leishmania promastigote lysate followed by a subcutaneous inoculation of viable promastigotes. The primary objective was to establish a canine experimental model to provide positive controls for testing immune responses to Leishmania in laboratory conditions. After inoculation of viable promastigotes, specific proliferative responses of peripheral blood mononuclear cells (PBMCs) to either Leishmania lysate or recombinant proteins, the in vitro production of interferon-γ by antigen-stimulated PBMCs and a significant increase in circulating levels of anti-Leishmania antibodies were observed. The immunized dogs also displayed positive delayed-type hypersensitivity reactions to Leishmania crude antigens and to purified recombinant proteins. An important finding that supports the suitability of the dogs as positive controls is that they remained healthy for the entire observation period, i.e., more than seven years after infection. Following the Leishmania antigen lysate injections, the infection of dogs by the subcutaneous route appears to induce a sustained cellular immune response, leading to an asymptomatic infection. This provides a useful model for both the selection of immunogenic Leishmania antigens and for immunobiological studies on their possible immunoprotective activities.