Use of the intradermal leishmanin test (Montenegro skin test) for feline visceral leishmaniosis: Detection of cellular immunity.

This study evaluated the humoral and cellular response in 100 cats living in an endemic area of visceral leishmaniosis (VL) using the Montenegro Skin Test (MST) and serological diagnosis and compared the MST with other diagnostic techniques. Sixty 60%, (60/100) cats were positive for MST and the diameter of positive skin reactions ranged from 5 to 9 mm. By serological methods, 74% (74/100) and 34% (34/100) had antibodies against Leishmania spp. by Immunofluorescence Antibody Test (IFAT) and Indirect Enzyme-Linked Immunosorbent Assay (ELISA), respectively. Comparing tests, the observed profiles were (1) IFAT (+)/MST (-) = 27 cats, (2) IFAT(-)/MST(+) = 13 cats, (3) IFAT(+)/MST(+) = 47 cats, (4) ELISA(+)/MST(-) = 12 cats, (5) ELISA(-)/MST(+) = 38 cats and (6) ELISA(+)/MST(+) = 22 cats. Through the combination of serological diagnosis and MST, a positivity frequency of 87% (87/100) by IFAT + MST and 72% (72/100) by ELISA + MST was identified in this cat population. Five cats (5%) were positive for Leishmania donovani complex DNA by molecular analysis, and two cats (2%) had Leishmania spp. amastigotes in lymph node smears. Therefore, the agreement between tests was classified as poor for all tests by Kappa index. The IFAT (+)/MST (+) response was the most frequent considering all cats (47%; 47/100); nonetheless, the most frequent immune expression in Polymerase Chain Reaction (PCR)-positive cats was the IFAT (+)/MST (-) profile (80%; 4/5). Five sick and PCR-positive cats, negative for Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV), that PCR sequencing matched 100% with L. donovani complex, all but one were MST negative. These results suggest that cats develop a significant cellular response against infection by parasites of the L. donovani complex, and most PCR and parasitological positive cats may be unable to develop a significant cellular response.

DNA extraction from individual Phlebotomine sand flies (Diptera: Psychodidae: Phlebotominae) specimens: Which is the method with better results?

Phlebotomine sand flies (Diptera: Psychodidae: Phlebotominae) are a group of small insects of great concern for Public Health. These dipterous are intensely studied worldwide due to their involvement in the transmission of several pathogens, mainly Leishmania spp. parasites. Nowadays, the molecular tools have been included in Phlebotomine sand flies studies and has shown to be powerful tools in bioecology studies of these dipterous. Thereby, when molecular approaches are employed, there is a great concern regarding the amount and quality of the DNA obtained for analysis. Here, seven methods of DNA extraction, between commercial kits and in house extraction protocols were evaluated. We considered measure of DNA concentration and purity ratios using a spectrophotometer to check the performance of each protocol. In addition, the quality evaluation of the DNA extracted was performed by endogenous gene PCR on samples. The results of the seven evaluated DNA extraction protocols and their implications are discussed.

Feline leishmaniosis: hematological and biochemical analysis.

One hundred and sixty-six cats from two animal shelters were subjected to enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence antibody test (IFAT), conventional polymerase chain reaction (cPCR), quantitative PCR (qPCR) and parasitological tests (PA) for the diagnosis of Leishmania spp. Among them, 15% (25/166), 53.6% (89/166), 3.6% (06/166) and 1.8% (03/166) were positive by ELISA, IFAT, both PCRs and PA, respectively. The sequencing of ITS-1 PCR amplicons revealed a 100% match with Leishmania infantum. After the Leishmania spp. survey, 12 cats were selected and divided into two groups for clinical, hematological, and biochemical analysis: six L. infantum positive cats (G1) and six Leishmania spp. negative cats (G2). All the cats were negative for feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV). A statistical analysis indicated significantly low platelet counts and significant hyperproteinemia associated with hypoalbuminemia in positive cats (p<0.05). Our results suggest that in endemic areas, cats with clinical signs of feline leishmaniosis (such as skin lesions, weight loss and/or enlarged lymph nodes) and that exhibit hematological and biochemical changes, such as low platelet counts and hyperproteinemia with hypoalbuminemia, should be tested for Leishmania spp. infection.

Xenodiagnosis in four domestic cats naturally infected by Leishmania infantum.

Leishmaniasis is a neglected tropical disease that continues to pose a serious public health problem. Albeit dogs have long been held as the major reservoirs of Leishmania infantum, the involvement of domestic cats in the zoonotic cycle of visceral leishmaniasis has gained prominence. Here, 240 cats were evaluated by clinical signs and haematological/biochemical changes compatible with leishmaniasis and were diagnosed by serological, molecular, and parasitological techniques. Thus, four cats naturally infected by L. infantum were submitted to xenodiagnosis. A total of 203 females of Lutzomyia longipalpis were subjected to feeding on four cats, with all females completing the blood meal. Parasitological and molecular assays were carried out to evaluate the presence of L. infantum in the sand flies' midgut. Promastigotes were observed in 10 females (6.5%) that fed on one cat, and L. infantum DNA was detected in 17 (8.4%) females that fed on two cats. Our results strengthen the evidence that naturally infected cats are capable of transmitting L. infantum to sand flies.

Leishmaniasis in cat shelters: A serological, molecular and entomological study.

An epidemiological Leishmania spp. and entomological Phlebotomine sandflies survey was performed in cat shelters at leishmaniasis endemic area of Brazil. Blood and conjunctival swab (CS) samples were collected from 94 cats in two animal protection shelters. These samples were subjected to serological tests using the indirect immunofluorescence antibody test (IFAT) and indirect enzyme-linked immunosorbent assay (ELISA) and to molecular test by polymerase chain reaction (PCR). In addition, a Phlebotomine sandflies survey was performed in the same shelters. The analyses revealed a positivity of 31.91% (30/94) through ELISA and 29.79% (28/94) through IFAT. The two serological tests showed a positive association with perfect agreement (k = 0.925). None of the cats were positive by Leishmania spp. DNA. One Lutzomyia (Lutzomyia) longipalpis male was found in one of the cat shelters. The results and the implications of our findings are discussed below.

Risk factors associated with Leishmania exposure among dogs in a rural area of Ilha Solteira, SP, Brazil.

INTRODUCTION: We sought to determine risk factors (RFs) associated with the presence of antibodies against Leishmania in dogs from a rural area of Ilha Solteira, SP, Brazil. METHODS: Serum samples were collected from 250 dogs and tested using indirect enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence antibody tests (IFATs). Data concerning dogs, their environment, and their owners' knowledge of leishmaniasis were collected using a questionnaire. To determine RFs for contact with the parasite, univariate statistical analysis based on chi-squared and Fisher's exact tests, followed by logistic regression, was used. RESULTS: It was found that 79/250 (31.6%) of the dogs were positive by IFAT, and 72/250 (28.8%) by ELISA. A total of 82/250 dogs (32.8%) were positive in at least one test. The RFs associated with occurrences of Leishmania exposure were large body size (OR = 2.25; 95% CI = 1.26-4.04; p = 0.003), presence of chickens (OR = 1.94; 95% CI = 1.05-3.65; p = 0.023), and lack of knowledge about Leishmania among dog owners (OR = 1.74; 95% CI = 0.96-3.21; p = 0.049). After multivariate analysis, the RFs for occurrence of Leishmania exposure in dogs that remained significantly associated were the dog's size (large dogs) (OR = 1.2; 95% CI = 1.06-1.35; p = 0.003) and presence of chickens on the properties (small farms) (OR = 1.15; 95% CI = 1.02-1.30; p = 0.023). CONCLUSIONS: These results may be useful for improving preventive practices to reduce the incidence of Leishmania exposure among dogs in rural areas.

Neutrophils, eosinophils, and mast cells in the intestinal wall of dogs naturally infected with Leishmania infantum.

Visceral leishmaniasis (VL) is a disease caused by the protozoa Leishmania infantum and can cause an inflammatory reaction in the gastrointestinal tract, however the role of granulocytic cells (neutrophils, eosinophils, and mast cells) in the intestine of dogs infected is not fully understood. We performed a quantitative analysis these cells in the intestinal wall of dogs with canine visceral leishmaniasis (CVL). Twenty dogs were assigned to one of three groups: group 1 (G1, n=8), dogs with CVL and L. infantum amastigotes in the intestine; group 2 (G2, n=9), dogs with CVL but without intestinal amastigotes; and group 3 (G3, n=3), uninfected dogs (control group). Granulocytic cells were counted in the crypt-villus unit (mucosa), submucosa, and muscle layer of the intestinal mucosa. Cell counts were higher in the intestinal wall of dogs from G2 followed by G1 and G3 (p≤0.05). In G1, there was a low inverse correlation between parasite burden of the small intestine and granulocyte counts (r= -0.1, p≤0.01). However, in G2 dogs, mast cell and eosinophil numbers showed positive correlation (r=0.85, p≤0.01). The granulocytic cell hyperplasia observed in the intestine of L. infantum-infected dogs suggests that these cells may be involved in the cell-mediated immune response for parasite elimination.

T lymphocytes and macrophages in the intestinal tissues of dogs infected with Leishmania infantum.

This study was about a semi-quantitative analysis of T lymphocytes (CD4+ and CD8+, FoxP3+ regulatory T cells), and macrophages in the gut wall of dogs naturally infected with Leishmania infantum. Thirteen dogs were divided into three groups: group 1 (G1, n=5), dogs with canine visceral leishmaniasis (CVL) and infected with L. infantum amastigotes in the intestine; group 2 (G2, n=5), dogs with CVL but without intestinal amastigotes; and group 3 (G3, n=3), uninfected dogs (control group). There was no significant difference (p ≥ 0.05) on CD4+ and Treg cell numbers among the groups, whereas the levels of CD8+ T cells and macrophages were significantly higher in dogs from G1 group than in G2 and G3 (p ≤ 0.05), especially in intestinal segments with high parasite burden. Parasite burden correlated positively with levels of CD8+ T cells and macrophages (p ≤ 0.05), but was inversely correlated to levels of CD4+ T lymphocytes and FoxP3+ Treg cells. In conclusion, in the intestine of dogs with CVL, the increase of CD8+ T cells and macrophages population associated with high parasite burdens, but no changes of CD4+ T cells and FoxP3+ Treg cells suggest a possible immunoregulation by the parasite not dependent on Treg cells.

Correlation study and histopathological description of intestinal alterations in dogs infected with Leishmania infantum.

The aim of this work was a correlation study and histopathological description of alterations associated with the presence of Leishmania infantumamastigote in the intestinal wall of dogs infected with canine visceral leishmaniasis (CVL). Three groups were used: G1 (n = 8), comprising naturally infected dogs with CVL with amastigotes of L. infantum in the small and large intestines; G2 (n = 9), infected dogs with CVL, without intestinal amastigotes; and G3 (n = 3), uninfected dogs. Histochemistry and immunohistochemistry methods were used for histopathology and amastigotes identification. 47.1% (8/17) of dogs from G1 group had amastigotes in the mucosa, submucosa and muscle layers of the small and large intestines and it was observed a prominent inflammatory reaction characterized by chronic infiltration of mononuclear cells: macrophages, lymphocytes and plasma cells. Comparison between the groups showed only a significant difference in relation to mucosal microscopic structural alterations in dogs from G1 in relation to G2 and G3. Parasite burden showed significant correlations with the microscopic alterations and clinical status of dogs in G1. By the conclusion, the inflammatory reactions caused by the parasites in the intestines might have contributed towards alterations in digestive processes, worsening the dogs' clinical status of CVL.

Leishmania infantum in the reproductive organs of dogs / Leishmania infantum em órgãos reprodutivos de cães

ABSTRACT: Leishmania infantum causes canine leishmaniasis. Using parasitological and molecular analyses, we identified L. infantum in the reproductive organs of male and female dogs. Using histochemistry, immunohistochemistry, and PCR, we examined tissue samples from the reproductive organs of 8 male dogs and 16 female dogs diagnosed with leishmaniasis. Despite the absence of macroscopic or microscopic lesions in these organs, we observed L. infantum amastigotes in tissue samples from the testis and the uterus. PCR and sequencing of these tissues revealed sequences that matched 100% with L. infantum DNA available at GenBank. The presence of L. infantum amastigotes and DNA in testicular and uterine tissue samples suggested that these organs can harbor the parasite without associated macroscopic or microscopic lesions, and this can be especially important in the vertical and venereal transmission of leishmaniasis in dogs.

RESUMO: Leishmania infantum é agente etiológico da leishmaniose canina. Por meio de análises parasitológicas e moleculares, a presença do parasita foi investigada em órgãos reprodutivos de cães machos e fêmeas. Amostras de tecidos dos órgãos reprodutivos de 8 cães machos e 16 fêmeas diagnosticados com leishmaniose foram avaliadas por histoquímica, imunohistoquímica e PCR. Apesar de não terem sido observadas lesões macroscópicas ou microscópicas nos órgãos reprodutivos desses cães, formas amastigotas de L. infantum foram observadas em amostras teciduais do testículo e útero. A PCR e o sequenciamento do DNA extraído desses tecidos revelaram sequências 100% idênticas a L. infantum depositadas no GenBank. Nossos resultados sugerem que os testículos e o útero podem abrigar o parasita, sem associação com lesões macroscópicas ou microscópicas, o que pode ter uma grande importância na transmissão venérea e vertical da leishmaniose entre cães.