Apoptosis in T lymphocytes from spleen tissue and peripheral blood of L. (L.) chagasi naturally infected dogs.

Dogs are the main domestic reservoirs of L. (L.) chagasi. Once in the vertebrate host, the parasite may cause visceral leishmaniasis, which can also be transmitted to humans. Infected symptomatic dogs show disorganization in the white pulp in spleen tissue and a reduction in T lymphocytes in peripheral blood. To investigate whether apoptosis is involved in white pulp disorganization and diminished T cell counts in peripheral blood, apoptotic T cells from the spleen and peripheral blood of dogs naturally infected with L. (L.) chagasi and presenting clinical manifestations were quantified and compared with healthy dogs. Thirteen symptomatic adult dogs infected by L. (L.) chagasi and six healthy dogs from a nonendemic area (controls) were included in the study. Samples from spleen and peripheral blood were used to quantify apoptosis in CD3 lymphocytes by flow cytometry using Anexin V and Multicaspase kits; the results were compared using the Mann Whitney test. The percentage of total T cells was lower in Leishmania infected dogs compared to healthy controls (P<0.05). Apoptosis levels in T cells from PBMC and spleen were higher in infected dogs than in controls (P<0.05). The least squares method test was used to determine the effect between the degree of structural organization of spleen white pulp and the percentage of apoptosis in the spleen. A significant effect on the level of white pulp morphological disorganization and percentage of apoptosis in spleen T cells was observed (F=20.45; P=0.0014). These data suggest that apoptosis is an important for the immunopathogenesis of canine visceral leishmaniasis.

Comparison between ELISA using total antigen and immunochromatography with antigen rK39 in the diagnosis of canine visceral leishmaniasis.

In this study, an enzyme-linked immunosorbent assay using crude total antigen (CTA-ELISA) and immunochromatography with antigen rK39 were compared in the diagnosis of canine visceral leishmaniasis (CVL). Fifty-two total blood samples from symptomatic dogs obtained from a location endemic for leishmaniasis and 52 blood samples from healthy dogs from a nonendemic region were tested. Polymerase chain reaction (PCR) was used to detect DNA from Leishmania spp. in both groups. Symptomatic dogs with positive PCR were considered infected by Leishmania spp. and the PCR technique was chosen as a gold standard test. The sensitivity determined for CTA-ELISA was 100%, with specificity of 91.2%, while the immunochromatographic assay with the antigen rK39 showed sensitivity of 91.5%, with specificity of 94.7%. A strong correlation was verified between CTA-ELISA and immunochromatography with antigen rK39, with a kappa coefficient of agreement of 0.88. Analysis of the results suggested that both assays presented good sensitivity and specificity for diagnosing CVL; however, immunochromatography with the antigen rK39 may be more advantageous when a fast field test is required.

Potential role for dog fleas in the cycle of Leishmania spp.

Several species of Leishmania spp. cause diseases in humans that range from self-healing cutaneous lesions to fatal visceral leishmaniosis. It has been observed that besides being transmitted by sand flies, Leishmania spp. may also be transmitted by arthropods such as ticks and fleas. To investigate the possible role of dog fleas in the transmission of Leishmania spp., Ctenocefalides felis were removed from 22 dogs which were positive according to ELISA and rK-39 tests. A C. felis sample from each of the 22 dogs was used to infect a hamster. The 22 hamsters were euthanized 4 months after infection with the fleas and the blood was subjected to ELISA to detect antibody anti-Leishmania spp., and the spleen samples were submitted to PCR for detection of Leishmania spp. DNA. PCR and ELISA were both positive in 18.1% (4/22), with PCR alone being positive in 45% (10/22) and ELISA alone in only 9% (2/22). These results suggest the participation of dog fleas in the Leishmania spp. cycle. Confirmation that C. felis indeed transmit leishmaniosis to dogs requires new strategies against leishmaniosis to be enforced by public health authorities and which focus on better ways to keep dogs free of fleas.

Evidence of Leishmania spp. antibodies and DNA in bush dogs (Speothos venaticus) in Brazil.

The municipality of Ilha Solteira, São Paulo, Brazil is an endemic area of leishmaniasis. At the Companhia Energética de São Paulo (CESP) Wild Animal Center of Ilha Solteira, two bush dogs (Speothos venaticus) showed clinical signs of this disease. The amastigote form of Leishmania was detected in lymph-node smears taken by fine-needle biopsy. In addition, serum samples from both animals, screened with an enzyme-linked immunosorbent assay (ELISA), were positive for anti-Leishmania antibodies. Moreover, tissue samples from one of the bush dogs were evaluated for the presence of Leishmania DNA by means of a polymerase chain reaction (PCR). DNA of the parasite was indeed detected in the tissue samples of the liver and the lymph nodes; however, no DNA from the parasite was detected in samples of the skin and spleen. These findings confirm a Leishmania infection in bush dogs (S. venaticus).

An investigation of Leishmania spp. in Didelphis spp. from urban and peri-urban areas in Bauru (São Paulo, Brazil).

Blood and bone marrow samples were taken from 112 Didelphis spp., collected between March 2005 and February 2006, from urban and peri-urban areas of Bauru, São Paulo State, Brazil, to evaluate the hypothesis that these animals might constitute a reservoir of Leishmania spp. Anti-Leishmania ssp. antibodies were screened in the serum samples using an enzyme-linked immuno-sorbent assay (ELISA) and the polymerase chain reaction (PCR). PCR was performed on fragments of DNA samples from Leishmania spp. using primers 13A and 13B, and showed a positive outcome in 91.6% of the 112 samples tested. Of the 107 samples analyzed by ELISA, 71% were positive. Evidence of epidemiological risk factors such as a circulating parasite and freely moving vectors suggests that Didelphis spp. may participate in the transmission cycle of Leishmania spp. in Bauru.