RESUMO
Visceral leishmaniasis represents an important public health issue in different parts of the world, requiring that measures be put in place to control the spread of the disease worldwide. The canine leishmaniasis diagnosis is not easy based on clinical signs, since dogs may not develop the infection with recognizable signs. Thus, the laboratorial diagnosis is essential to ascertain the incidence and prevalence of canine leishmaniasis especially in areas with major control efforts. Although, the diagnosis can be performed by the use of different approaches, the molecular methods such as PCR have become an indispensable tool for leishmaniases diagnosis. A TaqMan assay for real-time PCR (Linj31-qPCR) was developed to determine the parasite occurrence in clinical cases of leishmaniasis. The assay targets an L. (L.) infantum hypothetical protein region. The specificity of the assay was verified by using Leishmania World Health Organization reference strains including parasites belonging to subgenus L. (Leishmania), subgenus L. (Viannia), other Leishmania species and Trypanosoma cruzi. The sensitivity was verified by using isolates of L. (L.) amazonensis and L. (L.) infantum. The usefulness of the assay for diagnosis was ascertained by testing 277 samples from dogs in regions endemic for visceral and/or cutaneous leishmaniasis and from regions in which leishmaniasis was not endemic in São Paulo State, Brazil. Diagnosis of canine visceral leishmaniasis (CVL) was determined on these animals by conventional PCR and three serological tests. The dog samples were divided into four groups. I, dogs with CVL (n = 101); II, dogs with other diseases and without CVL (n = 97); III, dogs with American cutaneous leishmaniasis (n = 7), and, IV, dogs without CVL (n = 72) from areas where leishmaniasis was not endemic as control group. Results indicated that Linj31-qPCR was able to identify parasites belonging to subgenus L. (Leishmania) with no cross-amplification with other parasite subgenera. The Linj31-qPCR detected Leishmania parasites DNA in 98% of samples from Group I. In conclusion this methodology can be used as routine diagnostic tools to detect parasites from subgenus Leishmania.
Assuntos
Animais , Padrões de Referência , Proteínas de Protozoários/genética , DNA de Protozoário/química , Sensibilidade e Especificidade , Leishmania infantum/genética , Leishmania infantum/química , Doenças do Cão/diagnóstico , Doenças do Cão/parasitologia , Cães , Leishmania/classificação , Leishmania/genética , Leishmania/química , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/veterinária , AnimaisRESUMO
Leishmune is the industrialized version of the FML-saponin vaccine which has been shown to develop 92-95% protection in vaccinated dogs and 76-80% vaccine efficacy against field canine visceral leishmaniasis (CVL) in Brazil. Leishmune has been proven to be safe and tolerable and a transmission-blocking vaccine which renders vaccinated dogs non-infectious to sand fly vectors. In the present investigation, 550 healthy seronegative dogs of endemic and epidemic areas of Brazil were monitored for Leishmune-induced immunogenicity during a 2-year trial. Another group of 588 untreated exposed dogs was also studied in parallel. Both groups were seronegative on day 0. The strong immunogenicity induced by Leishmune vaccine was demonstrated by the 98% of FML-seroconversion, increase in absorbencies, the 82.7% DTH positive reactions and increase in skin test size diameters, the average increase in CD8+ total lymphocytes population in blood (27.1%), expected for QS21 saponin-containing vaccine, the sustained proportions of CD4+ T cells, and the average increased proportions of CD21+ B lymphocytes (42.3%). The Leishmune-induced protection against CVL is demonstrated by the results: 98.8% asymptomatic dogs (at the end of first year) and 99% healthy survivors (at the end of the second year) among vaccinated dogs, compared to the 79.4% asymptomatic and 61% survivor dogs (p<0.001) monitored in the untreated exposed cohort. In spite of the low vaccine coverage, it was possible to detect a 66.1% (p<0.005) reduction in Belo Horizonte and an 80.2% (p<0.005) reduction in Araçatuba of the incidence of CVL among vaccinated dogs, when compared to the global incidence of CVL of each town, respectively. Our preliminary results support the potential use of Leishmune to prevent CVL epidemics.
Assuntos
Doenças do Cão/imunologia , Leishmania donovani/imunologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/veterinária , Adjuvantes Imunológicos/farmacologia , Animais , Antígenos de Protozoários/imunologia , Brasil , Doenças do Cão/parasitologia , Doenças do Cão/prevenção & controle , Cães , Citometria de Fluxo , Hipersensibilidade Tardia/imunologia , Hipersensibilidade Tardia/parasitologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/prevenção & controle , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/parasitologia , Saponinas/imunologia , Saponinas/farmacologiaRESUMO
In order to assess the immunotherapeutic potential on canine visceral leishmaniasis of the Leishmune vaccine, formulated with an increased adjuvant concentration (1mg of saponin rather than 0.5mg), 24 mongrel dogs were infected with Leishmania (L.) chagasi. The enriched-Leishmune vaccine was injected on month 6, 7 and 8 after infection, when animals were seropositive and symptomatic. The control group were injected with a saline solution. Leishmune-treated dogs showed significantly higher levels of anti-FML IgG antibodies (ANOVA; p<0.0001), a higher and stable IgG2 and a decreasing IgG1 response, pointing to a TH1 T cell mediated response. The vaccine had the following effects: it led to more positive delayed type hypersensitivity reactions against Leishmania lysate in vaccinated dogs (75%) than in controls (50%), to a decreased average of CD4+ Leishmania-specific lymphocytes in saline controls (32.13%) that fell outside the 95% confidence interval of the vaccinees (41.62%, CI95% 43.93-49.80) and an increased average of the clinical scores from the saline controls (17.83) that falls outside the 95% confidence interval for the Leishmune immunotherapy-treated dogs (15.75, CI95% 13.97-17.53). All dogs that received the vaccine were clustered, and showed lower clinical scores and normal CD4+ counts, whereas 42% of the untreated dogs showed very diminished CD4+ and higher clinical score. The increase in clinical signs of the saline treated group was correlated with an increase in anti-FML antibodies (p<0.0001), the parasitological evidence (p=0.038) and a decrease in Leishmania-specific CD4+ lymphocyte proportions (p=0.035). These results confirm the immunotherapeutic potential of the enriched-Leishmune vaccine. The vaccine reduced the clinical symptoms and evidence of parasite, modulating the outcome of the infection and the dog's potential infectiosity to phlebotomines. The enriched-Leishmune vaccine was subjected to a safety analysis and found to be well tolerated and safe.
Assuntos
Leishmaniose Visceral/imunologia , Leishmaniose Visceral/terapia , Vacinas Protozoárias/imunologia , Saponinas/química , Animais , DNA de Protozoário , Cães , Imunoglobulina G/sangue , Imunoterapia , Fatores de TempoRESUMO
A group of 600 healthy and asymptomatic dogs from Brazilian canine visceral leishmaniasis endemic areas was vaccinated with three sc doses of Leishmune which is the industrialized formulation of the FML-saponin, recently licensed for commercialization in Brazil, which previously showed 76-80% vaccine efficacy against canine visceral leishmaniasis. Safety evaluation was performed for 14 days after each vaccine injection and disclosed transient reactions of local pain (40.87%), anorexia (20.48%), apathy (24.17%), local swelling reactions (15.90%), vomit (2.4%) and diarrhoea (1.5%). All effects showed significantly correlating declines, from the first to the third dose (p<0.0001). Most of the noticed reactions of pain (73%), anorexia (79%) and local swelling (84.7%) were mild. No significant differences between puppies and adults dogs were found in the number of adverse reactions. Adult dogs developed however, 94.5% of the small swelling reactions (<3 cm), and indicating that they are more resistant to the inflammatory response promoted by the saponins. No dead by anaphylaxis occurred, and only two dogs (0.1%) showed allergic reactions (facial oedema and itching) after the third dose. Transient alopecia on injection site occurred in only five poodles (0.28%) with total recovery and no need of treatment. All the mild adverse events in response to Leishmune injection were transient and disappeared before the injection of the following vaccine dose, confirming the tolerability of the vaccine. The Leishmune preparation was less haemolytic (HD(50)=180 microg/ml) than expected for a QS21 saponin-containing vaccine, indicating that its formulation with the FML antigen diminished the potential in vitro toxicity.
Assuntos
Doenças do Cão/imunologia , Leishmania donovani/imunologia , Leishmaniose Visceral/veterinária , Vacinas Protozoárias/imunologia , Animais , Brasil , Doenças do Cão/parasitologia , Doenças do Cão/prevenção & controle , Cães , Edema/induzido quimicamente , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/prevenção & controle , Vacinas Protozoárias/administração & dosagem , Vacinas Protozoárias/efeitos adversos , Prurido/induzido quimicamente , Fatores de Tempo , Resultado do TratamentoRESUMO
Leishmune vaccine is the first licensed vaccine against canine visceral leishmaniasis. It contains the Fucose-Mannose-ligand (FML) antigen of Leishmania donovani. The potential Leishmune vaccine effect on the interruption of the transmission of the disease, was assayed by monitoring, in untreated (n=40) and vaccinated dogs (n=32) of a Brazilian epidemic area: the kala-azar clinical signs, the FML-seropositivity and the Leishmania parasite evidence by immunohistochemistry of skin and PCR for Leishmanial DNA of lymph node and blood samples. On month 11 after vaccination, untreated controls showed: 25% of symptomatic cases, 50% of FML-seropositivity, 56.7% of lymph node PCR, 15.7% of blood PCR and 25% of immunohistochemical positive reactions. The Leishmune-vaccinated dogs showed 100% of seropositivity to FML and a complete absence of clinical signs and of parasites (0%) in skin, lymph node and blood PCR samples (p<0.01). The positivity in FML-ELISA in untreated dogs significantly correlates with the PCR in lymph node samples (p<0.001) and with the increase in number of symptoms (p=0.006) being strong markers of infectiousness. The absence of symptoms and of evidence of Leishmania DNA and parasites in Leishmune-vaccinated animals indicates the non-infectious condition of the Leishmune-vaccinated dogs.