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

Abstract

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.

An immunoinformatic approach driven by experimental proteomics: in silico design of a subunit candidate vaccine targeting secretory proteins of Leishmania donovani amastigotes

“…The first-generation vaccine, which includes live-attenuated, killed and fractionated parasites, is the only class of human prophylactic VL vaccine that entered phase III clinical trials so far. However, this vaccine failed to achieve satisfactory results [4]. The second-generation vaccines are produced from recombinant Leishmania antigens (single peptides/polypeptides).…”

A canine model of experimental infection with Leishmania (L.) mexicana

“…While visceral leishmaniasis has been extensively studied in dogs and several experimental models have been described [20,26,34,[41][42][43], very little is known about canine cutaneous leishmaniasis. We present here a first detailed model of experimental infection with L. (L.) mexicana in Beagle dogs.…”

“…This has important implications for the definition of epidemiological control measures. Indeed, the massive euthanasia of dogs in Brazil has had a questionable efficacy to reduce infection rate in humans [17], and as a consequence, the World Health Organization has now prioritized the development of veterinary vaccines [34,41]. Therefore, our model of canine cutaneous leishmaniasis may be very helpful for the evaluation of vaccine candidates.…”

“…Leishmania (L.) mexicana MHET/MX/97/Hd18 strain was cultured in 199 media with 15% of fetal bovine serum, 5 μM mercaptoethanol, 20 mM sodium pyruvate, 100 IU penicillin and 100 mg/mL streptomycin, and 5% of filtered human urine. Under general anesthesia (ketamine/xilazine, 8:1 mg/kg, iv), Beagle dogs received 7 × 10 7 promastigotes resuspended in 50 μl of PBS solution intradermally on the shaved back [34].…”

An immunoinformatic approach driven by experimental proteomics: in silico design of a subunit candidate vaccine targeting secretory proteins of Leishmania donovani amastigotes

“…However, this vaccine failed to achieve satisfactory results [4]. The second generation vaccines are produced from recombinant Leishmania antigens (single peptides/polypeptides).…”