RESUMO
The spleen plays a central role in human and canine visceral leishmaniasis, where the activation of the immune response occurs in one of the tissues where Leishmania infantum reproduces. Therefore, this organ is both a target to understand the mechanisms involved in the parasite control and a parameter for assessing the therapeutic response. In this sense, this study aimed to evaluate the main histological, immunological and parasitological aspects in the spleen of symptomatic dogs naturally infected by L. infantum treated with the therapeutic vaccine LBMPL. For this, dogs were divided into four groups: dogs uninfected and untreated (NI group); L. infantum-infected dogs that were not treated (INT group); L. infantum-infected dogs that received treatment only with monophosphoryl lipid A adjuvant (MPL group); and L. infantum-infected dogs that received treatment with the vaccine composed by L. braziliensis promastigote proteins associated with MPL adjuvant (LBMPL group). Ninety days after the therapeutics protocol, the dogs were euthanized and the spleen was collected for the proposed evaluations. Our results demonstrated a reduction of hyperplasia of red pulp and follicular area of white pulp, increased mRNA expression of IFN-γ, TNF-α, IL-12 and iNOS, and decreased IL-10 and TGF-ß1, and intense reduction of splenic parasitism in dogs treated with the LBMPL vaccine. These results possibly suggest that the pro-inflammatory environment promoted the progressive organization of the splenic architecture favoring the cellular activation, with consequent parasite control. Along with previously obtained data, our results propose the LBMPL vaccine as a possible treatment strategy for canine visceral leishmaniasis (CVL).
RESUMO
Diagnosing canine visceral leishmaniasis (CVL) is a critical challenge since conventional immunoserological tests still present some deficiencies. The current study evaluated a prototype flow cytometry serology test, using antigens and fluorescent antibodies that had been stored for 1 year at 4°C, on a broad range of serum samples. Noninfected control dogs and Leishmania infantum-infected dogs were tested, and the prototype test showed excellent performance in differentiating these groups with high sensitivity, specificity, positive and negative predictive values, and accuracy (100% in all analyses). When the CVL group was evaluated according to the dogs' clinical status, the prototype test showed outstanding accuracy in all groups with positive serology (asymptomatic II, oligosymptomatic, and symptomatic). However, in dogs which had positive results by PCR-restriction fragment length polymorphism (RFLP) but negative results by conventional serology (asymptomatic I), serological reactivity was not observed. Additionally, sera from 40 dogs immunized with different vaccines (Leishmune, Leish-Tec, or LBSap) did not present serological reactivity in the prototype test. Eighty-eight dogs infected with other pathogens (Trypanosoma cruzi, Leishmania braziliensis, Ehrlichia canis, and Babesia canis) were used to determine cross-reactivity and specificity, and the prototype test performed well, particularly in dogs infected with B. canis and E. canis (100% and 93.3% specificities, respectively). In conclusion, our data reinforce the potential of the prototype test for use as a commercial kit and highlight its outstanding performance even after storage for 1 year at 4°C. Moreover, the prototype test efficiently provided accurate CVL serodiagnosis with an absence of false-positive results in vaccinated dogs and minor cross-reactivity against other canine pathogens.
Assuntos
Anticorpos Antiprotozoários/sangue , Doenças do Cão/diagnóstico , Citometria de Fluxo/veterinária , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/veterinária , Testes Sorológicos/veterinária , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Reações Cruzadas/imunologia , Doenças do Cão/parasitologia , Cães , Feminino , Citometria de Fluxo/métodos , Leishmania infantum/imunologia , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/imunologia , Masculino , Sensibilidade e EspecificidadeRESUMO
The control of canine visceral leishmaniasis (CVL) is imperative, but euthanasia of seropositive dogs has been highly criticized. Commonly used, immunodiagnostic tests, including Dual-Path Platform®, enzyme-linked immunosorbent assay, and immunofluorescent antibody test, have failed at detecting asymptomatic dogs in endemic areas. In this context, new serological methods are needed. Flow cytometry serology has demonstrated potential as a test with excellent performance for CVL. In this study, we proposed to establish the best conditions for preserving Leishmania infantum promastigote antigens employed in this serology test. During 12 months of follow-up, promastigotes were maintained in different preservatives (phosphate-buffered saline with 3% fetal bovine serum, phenol 0.35%, thimerosal 0.01%, and formaldehyde 0.5%) and stored at 3 distinct temperatures (25 °C, 4 °C, and -20 °C). During the study period, the morphological characteristics of the promastigotes were assessed by flow cytometry according to the forward and side scatter parameters and also under optical microscopic analysis. Reactivity performance was evaluated as the percentage of positive fluorescent parasites in the sera of naturally infected and noninfected dogs. Microbiological analysis was performed at 2 time points, the first and sixth months, to rule out contamination of stored promastigotes. Taken together, our results indicated that the best conditions to preserve fixed L. infantum antigens were storage in formaldehyde at 4 °C. Promastigotes presented the best morphological profile, with appropriate antigenic stability even at 4 °C, in an inexpensive preservative for a long period of conservation.
Assuntos
Antígenos de Protozoários/análise , Doenças do Cão/diagnóstico , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/veterinária , Animais , Anticorpos Antiprotozoários/química , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/química , Antígenos de Protozoários/imunologia , Doenças do Cão/sangue , Doenças do Cão/imunologia , Doenças do Cão/parasitologia , Cães , Citometria de Fluxo/veterinária , Técnica Indireta de Fluorescência para Anticorpo , Formaldeído/química , Imunoglobulina G/química , Imunoglobulina G/imunologia , Leishmania infantum/imunologia , Leishmaniose Visceral/sangue , Leishmaniose Visceral/imunologia , Estágios do Ciclo de Vida , Conservantes Farmacêuticos/química , Estabilidade Proteica , TemperaturaRESUMO
Cellular and humoral immune responses of dogs to a candidate vaccine, composed of Leishmania braziliensis promastigote protein plus saponin as adjuvant, have been investigated as a pre-requisite to understanding the mechanisms of immunogenicity against canine visceral leishmaniasis (CVL). The candidate vaccine elicited strong antigenicity related to the increases of anti-Leishmania IgG isotypes, together with higher levels of lymphocytes, particularly of circulating CD8(+) T-lymphocytes and Leishmania chagasi antigen-specific CD8(+) T-lymphocytes. As indicated by the intense cell proliferation and increased nitric oxide production during in vitro stimulation by L. chagasi soluble antigens, the candidate vaccine elicited an immune activation status potentially compatible with effective control of the etiological agent of CVL.