Density of sand flies (diptera: psychodidae) in domestic and wild animal shelters in an area of visceral leishmaniasis in the state of Rio Grande do Norte, Brazil

XIMENES, Maria de Fátima Freire de Melo; SOUZA, Maria de Fátima de; CASTELLON, Eloy Guilhermo. Density of sand flies (Diptera: Psychodidae) in domestic and wild animal shelters in an area of visceral Leishmaniasis in the State of Rio Grande do Norte, Brazil. Memorias do Instituto Oswaldo Cruz, Rio de Janeiro, v.94, n.4, p.427-432, jul./ago. 1999. Disponivel em: Acesso em: 4 out. 2010.

Sensitivity and specificity of parallel or serial serological testing for detection of canine Leishmania infection

In Brazil, human and canine visceral leishmaniasis (CVL) caused by Leishmania infantum has undergone urbanisation since 1980, constituting a public health problem, and serological tests are tools of choice for identifying infected dogs. Until recently, the Brazilian zoonoses control program recommended enzyme-linked immunosorbent assays (ELISA) and indirect immunofluorescence assays (IFA) as the screening and confirmatory methods, respectively, for the detection of canine infection. The purpose of this study was to estimate the accuracy of ELISA and IFA in parallel or serial combinations. The reference standard comprised the results of direct visualisation of parasites in histological sections, immunohistochemical test, or isolation of the parasite in culture. Samples from 98 cases and 1,327 noncases were included. Individually, both tests presented sensitivity of 91.8% and 90.8%, and specificity of 83.4 and 53.4%, for the ELISA and IFA, respectively. When tests were used in parallel combination, sensitivity attained 99.2%, while specificity dropped to 44.8%. When used in serial combination (ELISA followed by IFA), decreased sensitivity (83.3%) and increased specificity (92.5%) were observed. Serial testing approach improved specificity with moderate loss in sensitivity. This strategy could partially fulfill the needs of public health and dog owners for a more accurate diagnosis of CVL.

Leishmaniose visceral canina em cães atendidos em hospital veterinário particular do Distrito Federal, Brasil

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Medicina, Programa de Pós-Graduação em Medicina Tropical, 2016.

Adaptive Immunity against Leishmania Nucleoside Hydrolase Maps Its C-Terminal Domain as the Target of the CD4+ T Cell-Driven Protective Response

Nucleoside hydrolases (NHs) show homology among parasite protozoa, fungi and bacteria. They are vital protagonists in the establishment of early infection and, therefore, are excellent candidates for the pathogen recognition by adaptive immune responses. Immune protection against NHs would prevent disease at the early infection of several pathogens. We have identified the domain of the NH of L. donovani (NH36) responsible for its immunogenicity and protective efficacy against murine visceral leishmaniasis (VL). Using recombinant generated peptides covering the whole NH36 sequence and saponin we demonstrate that protection against L. chagasi is related to its C-terminal domain (amino-acids 199-314) and is mediated mainly by a CD4+ T cell driven response with a lower contribution of CD8+ T cells. Immunization with this peptide exceeds in 36.73 +/- 12.33% the protective response induced by the cognate NH36 protein. Increases in IgM, IgG2a, IgG1 and IgG2b antibodies, CD4+ T cell proportions, IFN-gamma secretion, ratios of IFN-gamma/IL-10 producing CD4+ and CD8+ T cells and percents of antibody binding inhibition by synthetic predicted epitopes were detected in F3 vaccinated mice. The increases in DTH and in ratios of TNF alpha/IL-10 CD4+ producing cells were however the strong correlates of protection which was confirmed by in vivo depletion with monoclonal antibodies, algorithm predicted CD4 and CD8 epitopes and a pronounced decrease in parasite load (90.5-88.23%; p = 0.011) that was long-lasting. No decrease in parasite load was detected after vaccination with the N-domain of NH36, in spite of the induction of IFN-gamma/IL-10 expression by CD4+ T cells after challenge. Both peptides reduced the size of footpad lesions, but only the C-domain reduced the parasite load of mice challenged with L. amazonensis. The identification of the target of the immune response to NH36 represents a basis for the rationale development of a bivalent vaccine against leishmaniasis and for multivalent vaccines against NHs-dependent pathogens.

TESA-blot for the diagnosis of Chagas disease in dogs from co-endemic regions for Trypanosoma cruzi, Trypanosoma evansi and Leishmania chagasi

We standardized serodiagnosis of dogs infected with Trypanosoma cruzi using TESA (trypomastigote excreted-secreted antigen)-blot developed for human Chagas disease. TESA-blot showed 100% sensitivity and specificity. In contrast, ELISA using TESA (TESA-ELISA) or epimastigotes (epi-ELISA) as antigen yielded 100% sensitivity but specificity of 94.1% and 49.4%, respectively. When used in field studies in an endemic region for Chagas disease, visceral leishmaniasis and Trypanosoma evansi (Mato Grosso do Sul state, Central Brazil), positivities were 9.3% for TESA-blot, 10.7% for TESA-ELISA and 32% for epi-ELISA. Dogs from a non-endemic region for these infections (Rondonia state, western Amazonia) where T cruzi is enzootic showed positivity of 4.5% for TESA-blot and epi-ELISA and 6.8% for TESA-ELISA. Sera from urban dogs from Santos, Sao Paulo, where these diseases are absent, yielded negative results. TESA-blot was the only method that distinguished dogs infected with T cruzi from those infected with Leishmania chagasi and/or Trypanosoma evansi. (C) 2009 Published by Elsevier B.V.

Histopathology and immunocytochemical study of type 3 and type 4 complement receptors in the liver and spleen of dogs naturally and experimentally infected with Leishmania (Leishmania) chagasi

The objective of this study was to compare the histopathological changes and expression of CR3 and CR4 in the liver and spleen of dogs naturally and experimentally infected with L. chagasi. The basic histopathological lesions observed mainly in naturally infected dogs were: epithelioid hepatic granulomas, hyperplasia and hypertrophy of Kupffer cells, Malpigui follicles and mononucleated cells of the red pulp of the spleen. Sections from the liver and spleen by immunocytochemistry technique showed the presence of CD11b,c\CD 18 antigens in the control and infected animals and no qualitative or quantitative differences in the liver. Nevertheless, CD18 was always increased in the spleen of naturally and experimentally infected dogs. These results indicate that there is a difference in the activaton of CD 18 in both experimental and natural cases of canine visceral leishmaniasis that should play an important role in the immunological response to Leishmania chagasi infection.

THE SERO-CONVERSION AND EVALUATION OF RENAL ALTERATIONS IN DOGS INFECTED BY Leishmania (Infantum) chagasi

This study investigated the sero-conversion period in which dogs from endemic areas test positive for visceral leishmaniasis (VL) as well as the early post-infection period in which renal alterations are observed. Dogs that were initially negative for Canine Visceral Leishmaniasis (CVL) were clinically evaluated every three months by serological, parasitological and biochemical tests until sero-conversion was confirmed, and six months later a subsequent evaluation was performed. Samples of kidney tissues were processed and stained with Hematoxylin and Eosin (H&E), Periodic Acid Schiff (PAS) and Masson’s trichrome stain and lesions were classified based on the WHO criteria. Of the 40 dogs that initially tested negative for VL, 25 (62.5%) exhibited positive serological tests during the study period. Of these 25 dogs, 15 (60%) tested positive within three months, five (20%) tested positive within six months and five (20%) tested positive within nine months. The dogs exhibited antibody titers between 1:40 and 1:80 and 72% of the dogs exhibited clinical symptoms. The Leishmania antigen was present in the kidneys of recently infected dogs. We found higher levels of total protein and globulin as well as lower levels of albumin in the infected dogs when compared to the control dogs. Additionally, infected dogs presented levels of urea and creatinine that were higher than those of the uninfected dogs. Glomerulonephritis was detected in some of the dogs examined in this study. These data suggest that in Teresina, the sero-conversion for VL occurs quickly and showed that the infected dogs presented abnormal serum proteins, as well as structural and functional alterations in the kidneys during the early post-infection period.

EVIDENCE OF Leishmania (Leishmania) infantum INFECTION IN DOGS FROM JUIZ DE FORA, MINAS GERAIS STATE, BRAZIL, BASED ON IMMUNOCHROMATOGRAPHIC DUAL-PATH PLATFORM (DPP®) AND PCR ASSAYS

In Brazil, domestic dogs are branded as the primary reservoir for zoonotic visceral leishmaniasis, due to the clear positive correlation observed between human and canine infection rates. This study aimed to carry out a serological survey of canine visceral leishmaniasis (CVL) in dogs housed at a public kennel in the municipality of Juiz de Fora, Minas Gerais State, Brazil, using the immunochromatographic TR DPP® CVL rapid test. Additionally, conventional and/or real time PCR assay was used to detect and confirm L. infantum infection in the DPP positive dogs only. Of the 400 dogs studied, most did not present clinical signs for CVL (p < 0.05), and fifteen (3.8%) were seropositive in the DPP test. There was no statistically significant difference between the DPP seropositive dogs and the clinical signs of the disease (p > 0.05). Both conventional and real time PCR tests confirmed L. infantum infection in nine (75.0%) of the twelve DPP seropositive dogs that remained alive during the follow-up period. This study is the first seroepidemiologic survey of CVL held in the city of Juiz de Fora, and the results reinforce the idea that this disease is currently in a process of expansion and urbanization in Brazil. Furthermore, this study highlights the use of the DPP test as an alternative for diagnosing CVL in large and mid-sized cities, due to its ease of implementation.

Characterization of Leishmania infantum species in dogs from the urban area of Cuiabá, State of Mato Grosso, Brazil

INTRODUCTION: Visceral leishmaniasis presents urban behavior in some Brazilian cities, with domestic dogs as the main infection source. In Cuiabá, MT, canine visceral leishmaniasis was diagnosed and characterized as recommended by the Ministry of Health. METHODS: Biological samples from suspected canine carriers were analyzed by the isoenzyme electrophoresis technique. The 6PGDH enzyme and reference strain IOC/L0566 (MHOM/BR/1975/M2903) of Leishmania (Leishmania) infantum was used as one of the controls. RESULTS: Electrophoresis analysis revealed that the canine isolates belonged to the species L. (L.) infantum. CONCLUSIONS: The authors emphasize the importance of species characterization, particularly in areas of mixed infection like Cuiabá.

Diagnosis of Leishmania (Leishmania) chagasi infection in dogs and the relationship with environmental and sanitary aspects in the municipality of Palmas, state of Tocantins, Brazil

INTRODUCTION: The aim of the present study was to identify the presence of Leishmania (Leishmania) chagasi infection in dogs in the City of Palmas, Tocantins, Brazil, using the PCR technique to list the hot spots of infected dogs in the city and associate their occurrence to significant environmental changes at capture sites. METHODS: DNA was extracted from blood of dogs, and the PCR were performed with primers RV1/RV2. After screening the population studied, the regions of the city that had the highest occurrence of canine infection were detected. These sites were visited, and ecological parameters denoting anthropogenic disturbance were evaluated. RESULTS: Some important features were listed in the regions visited, such as low urbanization, lack of public collection of sewage, limited garbage collection, vacant lots with tall vegetation, decaying organic matter, and, most importantly, the occurrence of stray dogs and poultry in homes. CONCLUSIONS: The methodology for screening the population was very efficient, especially in evaluating a large number of individuals in a short time, with a high degree of automation. The results indicate an association between the observed parameters and the occurrence of infection in dogs. The model presented in the city is ideal for studies of disease progression and expansion and for the evaluation of control measures adopted for canine VL.

Clinical and laboratory alterations in dogs naturally infected by Leishmania chagasi

INTRODUCTION: Canine visceral leishmaniasis (CVL) is a zoonotic disease with different clinical manifestations. Parasitism often occurs in bone marrow, but changes have been observed in peripheral blood and serum biochemical parameters. The aim of this study was to evaluate the hematological and biochemical parameters in dogs naturally infected by Leishmania chagasi. METHODS: Eighty-five adult dogs of both sexes and various weights and ages from the Zoonosis Control Center of Fortaleza (CCZ) were used, selected by immunofluorescence assay (IFA) and considered positive with IFA titers greater than 1:40 and by visualizing amastigotes of Leishmania chagasi in smears obtained by bone marrow aspiration. The dogs (n = 85) were grouped according to clinical signs: negative (CN = 7), subclinical (CS = 10), and clinical (CC = 68). Blood samples were collected for determination of hematological and biochemical serum values. The experimental protocol was approved by the CEUA/UECE. RESULTS: The most frequent clinical signs were cachexia (77.9%), keratitis (61.8%), and lymphadenopathy (55.9%), and 86.8% of the animals showed more than one clinical sign characteristic of CVL. In CC were observed reductions in red blood cells (63%), hematocrit (72%), and hemoglobin (62%), as well as leukocytosis (33%), neutropenia (28%), thrombocytopenia (50%), uremia (45%), hyperproteinemia (53%, p<0.05), hypergammaglobulinemia (62%, p<0.01), and hypoalbuminemia (58%). CONCLUSIONS: Animals with the clinical form of the disease demonstrate hematological and biochemical changes consistent with anemia, uremia, hyperproteinemia, and hyperglobulinemia, which present themselves as strong clinical markers of visceral leishmaniasis associated with the signs previously reported.

Factors associated with Leishmania chagasi infection in domestic dogs from Teresina, State of Piauí, Brazil

INTRODUCTION: Many studies have evaluated risk factors for human visceral leishmaniasis, but few have focused on the infection among dogs. The objective of this study was to assess the association between peridomestic socioeconomic and environmental factors and the presence of dogs seropositive for Leishmania chagasi in the City of Teresina, Brazil. METHODS: This case-control study was based on the results of a routine seroepidemiological survey among domestic dogs carried out in 2007. Serological tests were performed by means of indirect immunofluorescence antibody test. All dwellings in which at least one seropositive dog was detected were considered cases, and controls were a random sample of dwellings in which only seronegative dogs were identified. Associations between variables were expressed as odds ratios (OR) and their respective 95% confidence intervals (95%CI) estimated using multivariate logistic regression. RESULTS: Dwellings with a history of dogs removed by the visceral leishmaniasis control program in the last 12 months had five-fold higher odds of having at least one seropositive dog as compared with dwellings having no history of dog removal (OR = 5.19; 95%CI = 3.20-8.42). Dwellings with cats had 58% increased odds of dog infection as compared with those having no cats (OR = 1.58; 95%CI = 1.01-2.47). CONCLUSIONS: Identification of factors associated with canine visceral leishmaniasis might be used for the delimitation of areas of higher risk for human visceral leishmaniasis, since infection in dogs generally precedes the appearance of human cases.

Serological cross-reactivity of Trypanosoma cruzi, Ehrlichia canis, Toxoplasma gondii, Neospora caninum and Babesia canis to Leishmania infantum chagasi tests in dogs

Introduction: The aim of this study was to evaluate the serological cross-reactivity between Leishmania sp. and other canine pathogens. Methods: Positive serum samples for Ehrlichia canis, Babesia canis, Toxoplasma gondii, Neospora caninum and Trypanosoma cruzi were tested using three serological methods enzyme linked immunosorbent assay (ELISA), indirect immunofluorescent antibody test (IFAT) and Kalazar Detect™, for canine visceral leishmaniasis. Results: Of the 57 dog samples tested, 24 (42.1%) tested positive using one of the three serological methods: 10/57 (17.5%) for ELISA, 11/57 (19.3%) for IFAT and 3/57 (5.3%) for Kalazar Detect™. Conclusions: Our results demonstrated that the presence of other infectious agents may lead to cross-reactivity on leishmaniasis serological tests.

Coinfection by Toxoplasma gondii and Leishmania spp. in domestic cats (Felis catus) in State of Mato Grosso do Sul

Introduction Leishmaniasis and toxoplasmosis are important to public health. Methods Antibodies for Toxoplasma gondii and Leishmania spp. were evaluated in cats from Campo Grande, State of Mato Grosso do Sul, Brazil, a region endemic for canine visceral leishmaniasis. Serum samples from 50 asymptomatic cats were titrated for T. gondii by the immunofluorescence antibody test and modified agglutination test and for Leishmania spp. by the immunofluorescence antibody test. Results These two agents coinfected two (4%) of the 50 tested animals. Conclusions These findings demonstrate the concomitant presence of two important zoonoses in cats from Brazilian endemic regions for canine visceral leishmaniasis.

Leishmania, Babesia and Ehrlichia in urban pet dogs: co-infection or cross-reaction in serological methods?

INTRODUCTION: The present study was designed to assess the occurrence of co-infection or cross-reaction in the serological techniques used for detecting the anti-Leishmania spp., -Babesia canis vogeli and -Ehrlichia canis antibodies in urban dogs from an area endemic to these parasites. METHODS: The serum samples from dogs were tested for the Babesia canis vogeli strain Belo Horizonte antigen and Ehrlichia canis strain São Paulo by immunofluorescence antibody test (IFAT) and by anti-Leishmania immunoglobulin G (IgG) antibody detection to assess Leishmania infection. We used the following four commercial kits for canine visceral leishmaniasis: ELISA, IFAT, Dual Path Platform (DPP) (Bio Manguinhos(r)/FIOCRUZ/MS) and a rK39 RDT (Kalazar Detect Canine Rapid Test; Inbios). RESULTS : Of 96 serum samples submitted to serological assays, 4 (4.2%) were positive for Leishmania as determined by ELISA; 12 (12.5%), by IFAT; 14 (14.6%) by rK39 RDT; and 20 (20.8%), by DPP. Antibodies against Ehrlichia and Babesia were detected in 23/96 (23.9%) and 30/96 (31.2%) samples, respectively. No significant association was identified between the results of tests for detecting Babesia or Ehrlichia and those for detecting Leishmania (p-value>0.05). CONCLUSIONS: In the present study, we demonstrated co-infection with Ehrlichia or Babesia and Leishmania in dogs from Minas Gerais (Brazil); we also found that the serological tests that were used did not cross-react.