High genetic diversity and superinfection by Anaplasma marginale strains in naturally infected Angus beef cattle during a clinical anaplasmosis outbreak in southeastern Brazil.

Anaplasma marginale is an obligate intracellular Gram-negative bacterium that is parasitic to erythrocytes and is the main agent of bovine anaplasmosis. This disease causes severe anemia and reduces weight gain and milk production, thus giving rise to major economic losses relating to livestock worldwide. The genetic diversity of this bacterium has been characterized based on sequences of major surface proteins (MSPs), especially MSP1α. This has enabled identification of several geographical strains, according to different amino acid sequences. The aim of this study was to investigate the genetic diversity of A. marginale in naturally infected Angus beef cattle during a disease outbreak in southeastern Brazil. Four blood samples were collected over a four-month period from each of 20 animals on a cattle farm in Itú, São Paulo, Brazil. Serum samples were subjected to indirect ELISA to detect anti-A. marginale IgG antibodies. The 80 whole-blood samples obtained were subjected to DNA extraction, quantitative real-time PCR (qPCR) for the msp1ß gene, semi-nested PCR (snPCR) for the msp1α gene, cloning of the target fragment and sequencing using the Sanger method. The sequences obtained were analyzed for genetic diversity using the RepeatAnalyzer software. Both iELISA tests, using recombinant MSP5 and the Anaplasma antibody test kit (VMRD), revealed high seroprevalence: 91.25% and 97.5%, respectively. In qPCR, 100% of the samples were positive, with between 103 and 107 DNA copies/µL. In the snPCR based on the msp1α gene, 57.5% (46/80) of the samples were positive. Microsatellite analysis on the 36 sequences obtained showed the presence of genotypes H (58.3%), F (25%), E (19.4%), C (2.7%) and G (2.7%). The RepeatAnalyzer software identified 36 strains in the study region, among which some had not previously been described in the literature (13 27 13 27 13 F; 16 FF; τ 27; 63 29 104 29; LJ1 13 LJ1 13; 16 F 17; 16 F 91). High genetic diversity of A. marginale bacteria was found on this farm in Itú, São Paulo.

Anaplasma marginale in goats from a multispecies grazing system in northeastern Brazil.

Anaplasma marginale, a tick-borne α-proteobacterium that causes significant economic losses for the cattle industry worldwide, has been increasingly detected in other animal species. This agent has been previously detected in buffaloes and goats co-grazed with cattle in Brazil. This study aimed to investigate the occurrence of A. marginale in a multispecies (goats, sheep and cattle) grazing farm in the State of Paraíba, northeastern Brazil. A total of 119 goats, 71 sheep, and five cattle were evaluated. An epidemiological questionnaire was applied to the farm owner addressing age, gender, and presence of ticks. Serum samples from goat, sheep and cattle were tested for anti-Anaplasma marginale antibodies by a commercial MSP5-based on indirect enzyme-linked immunosorbent assay (iELISA). EDTA-blood samples were screened for A. marginale- and A. ovis-infection by PCR using primers targeting Anaplasma spp. msp4 gene. Sequencing of the repeat region of the msp1α gene was used for genotyping A. marginale strains found in the present study. A total of 47/119 (39.5 %, 95 % CI: 31.1-48.4 %) goats and 2/71 (3%, 95 % CI: 0.7-9.7 %) sheep were seroreactive for A. marginale rMSP5 by the commercial iELISA. All cattle were seronegative for A. marginale. Anaplasma spp. msp4 PCR results revealed that two out of 119 (1.7 %; 95 % CI: 0.4-5.9 %) goats tested positive and all sheep and cattle samples were negative. It was not possible to sequence one sample. Therefore, the other sequencing sample found tandem repeats of A. marginale msp1α gene demonstrating that goat was infected with the genotype F/91. Rhipicephalus microplus ticks were found parasitizing goats but not on sheep or cattle. Considering that in Brazil A. marginale genotype F/91 and the MSP1a tandem repeat F has only been detected in goats so far, we hypothesized that this genotype may be related to goats.

New records and genetic diversity of Mycoplasma ovis in free-ranging deer in Brazil.

Cervids represent a mammal group which plays an important role in the maintenance of ecological balance. Recent studies have highlighted the role of these species as reservoirs for several arthropods-borne pathogens. Globally, hemotropic mycoplasmas (haemoplasmas) are emerging or remerging bacteria that attach to red blood cells of several mammals species causing hemolytic anaemia. Therefore, the aim of this study was to investigate the occurrence and assess the phylogenetic positioning of Mycoplasma ovis in free-ranging deer from Brazil. Using a polymerase chain reaction targeting the 16S rRNA region, 18 (40%) out of 45 sampled deer were positive to M. ovis. Among the nine sequences analysed, four distinct genotypes were identified. The sequences detected in the present study were closely related to sequences previously identified in deer from Brazil and the USA. On the other hand, the Neighbour-Net network analysis showed that the human-associated M. ovis genotypes were related to genotypes detected in sheep and goats. The present study shows, for the first time, the occurrence of M. ovis in Mazama gouazoubira and Mazama bororo deer species, expanding the knowledge on the hosts harbouring this haemoplasma species. Once several deer species have your population in decline, additional studies are needed to evaluate the pathogenicity of M. ovis among deer populations around the world and assess its potential as reservoir hosts to human infections.

Coxiella burnetii associated with BVDV (Bovine Viral Diarrhea Virus), BoHV (Bovine Herpesvirus), Leptospira spp., Neospora caninum, Toxoplasma gondii and Trypanosoma vivax in reproductive disorders in cattle.

This is a cross-sectional study to assess the presence of antibodies in ruminants against selected pathogens associated with reproductive disorders in cattle in four Brazilian states, including the zoonotic agent Coxiella burnetii. The used tests were Virus Neutralization Assay for IBR and BVD, Microscopic Agglutination Test for Leptospira spp., Indirect Fluorescent Antibody Test (IFAT) for C. burnetii and Toxoplasma gondii, and Enzyme-Linked Immunosorbent Assay for Neospora caninum and Trypanosoma vivax. Seropositivity for C. burnetii was 13.7% with titers from 128 to 131,072; 57.8% for BoHV-1, with titers between 2 and 1,024; 47.1% for BVDV-1a, with titers from 10 to 5,120; 89.2% for N. caninum; 50% for T. vivax; and 52.0% for Leptospira spp., with titers between 100 to 800 (the following serovars were found: Tarassovi, Grippotyphosa, Canicola, Copenhageni, Wolffi, Hardjo, Pomona and Icterohaemorrhagiae); 19.6% for T. gondii with titer of 40. This is the first study that has identified C. burnetii in cattle associated with BoHV and BVDV, N. caninum, Leptospira spp., T. gondii and T. vivax. Thus, future studies should be conducted to investigate how widespread this pathogen is in Brazilian cattle herds.

Coxiella burnetii associated with BVDV (Bovine Viral Diarrhea Virus), BoHV (Bovine Herpesvirus), Leptospira spp, Neospora caninum, Toxoplasma gondii and Trypanosoma vivax in reproductive disorders in cattle / Coxiella burnetii associada ao BVDV (Vírus da Diarreia Viral Bovina), BoHV (Vírus do Herpes Bovino), Leptospira spp, Neospora caninum, Toxoplasma gondii e Trypanosoma vivax em distúrbios reprodutivos em bovinos

Abstract This is a cross-sectional study to assess the presence of antibodies in ruminants against selected pathogens associated with reproductive disorders in cattle in four Brazilian states, including the zoonotic agent Coxiella burnetii. The used tests were Virus Neutralization Assay for IBR and BVD, Microscopic Agglutination Test for Leptospira spp., Indirect Fluorescent Antibody Test (IFAT) for C. burnetii and Toxoplasma gondii, and Enzyme-Linked Immunosorbent Assay for Neospora caninum and Trypanosoma vivax. Seropositivity for C. burnetii was 13.7% with titers from 128 to 131,072; 57.8% for BoHV-1, with titers between 2 and 1,024; 47.1% for BVDV-1a, with titers from 10 to 5,120; 89.2% for N. caninum; 50% for T. vivax; and 52.0% for Leptospira spp., with titers between 100 to 800 (the following serovars were found: Tarassovi, Grippotyphosa, Canicola, Copenhageni, Wolffi, Hardjo, Pomona and Icterohaemorrhagiae); 19.6% for T. gondii with titer of 40. This is the first study that has identified C. burnetii in cattle associated with BoHV and BVDV, N. caninum, Leptospira spp., T. gondii and T. vivax. Thus, future studies should be conducted to investigate how widespread this pathogen is in Brazilian cattle herds.

Resumo Este é um estudo transversal para avaliar a presença de anticorpos em ruminantes contra patógenos selecionados e associados a distúrbios reprodutivos em bovinos de quatro estados brasileiros, incluindo o agente zoonótico Coxiella burnetii. Os testes utilizados foram Teste de Vírus-Neutralização para BoHV e BVDV, teste de Aglutinação Microscópica para Leptospira spp., Reação de Imunofluorescência Indireta for C. burnetii e Toxoplasma gondii, e Ensaio de Imunoabsorção Enzimática para Neospora caninum e Trypanosoma vivax. A soropositividade para C. burnetii foi de 13,7% com títulos de 128 a 131.072; 57,8% para BoHV-1, com títulos entre 2 a 1.024; 47,1% para BVDV-1a, com títulos de 10 a 5.120; 89,2% para N. caninum; 50% para T. vivax; e 52,0% para Leptospira spp., com títulos entre 100 a 800 (sorovares encontrados: Tarassovi, Grippotyphosa, Canicola, Copenhageni, Wolffi, Hardjo, Pomona e Icterohaemorrhagiae) 19,6% para T. gondii com título de 40. Este é o primeiro estudo que evidencia a participação de C. burnetii em bovinos associada ao Vírus da Rinotraqueíte bovina infecciosa e da diarreia viral bovina, N. caninum, Leptospira spp., T. gondii e T. vivax em bovinos. Desta forma, futuros estudos devem ser conduzidos a fim de investigar o quão disseminado se encontra este patógeno em rebanhos bovinos brasileiros.

Evaluation of dot-blot test for serological diagnosis of bovine brucellosis

Abstract The objective of this study was to standardize and validate the dot-blot test for the serological diagnosis of bovine brucellosis, compare the results with those found in the 2-mercaptoethanol (2-ME) and complement fixation test (CF), and estimate the relative sensitivity and specificity of the dot-blot compared to these tests. Fifty bovine blood serum samples were used for the test standardization, and 1315 samples were used for evaluation and comparison between the tests; the results were compared using the Kappa indicator. At the end of standardization, it was established as optimal for the antigen obtained from Brucella abortus B19 after passing through a microorganism rupture process, the blood serum samples diluted at 1:100, and the conjugate at 1:30,000. The comparison of the dot-blot results with 2-ME showed Kappa index of 0.9939, sensitivity of 99.48%, and specificity 99.91%, with CF, Kappa index of 0.8226, sensitivity 100% and specificity 95.32%. Using the combination of the test results 2-ME and CF to establish the true condition of the animal, the dot-blot showed relative sensitivity of 100%, and relative specificity of 99.91%. The evaluated test proved to be effective and reliable, besides being easy to handle and interpret the results.

Evaluation of dot-blot test for serological diagnosis of bovine brucellosis.

The objective of this study was to standardize and validate the dot-blot test for the serological diagnosis of bovine brucellosis, compare the results with those found in the 2-mercaptoethanol (2-ME) and complement fixation test (CF), and estimate the relative sensitivity and specificity of the dot-blot compared to these tests. Fifty bovine blood serum samples were used for the test standardization, and 1315 samples were used for evaluation and comparison between the tests; the results were compared using the Kappa indicator. At the end of standardization, it was established as optimal for the antigen obtained from Brucella abortus B19 after passing through a microorganism rupture process, the blood serum samples diluted at 1:100, and the conjugate at 1:30,000. The comparison of the dot-blot results with 2-ME showed Kappa index of 0.9939, sensitivity of 99.48%, and specificity 99.91%, with CF, Kappa index of 0.8226, sensitivity 100% and specificity 95.32%. Using the combination of the test results 2-ME and CF to establish the true condition of the animal, the dot-blot showed relative sensitivity of 100%, and relative specificity of 99.91%. The evaluated test proved to be effective and reliable, besides being easy to handle and interpret the results.

Expression of a recombinant protein, A2 family, from Leishmania infantum (Jaboticabal strain) and its evaluation in Canine Visceral Leishmaniasis serological test.

This study aimed to express a recombinant A2 family protein of Leishmania chagasi, Jaboticabal strain; test this protein as an antigen in serological assays; and investigate its antigenicity and immunogenicity. A protein coded by an allele of the A2 gene isolated from L. chagasi was expressed in three different strains of Escherichia coli. We used 29 sera samples from Leishmune-vaccinated dogs, 482 sera samples from dogs from endemic areas (positive controls), and 170 sera samples from dogs from non-endemic areas (negative controls) in ELISA tests using soluble Leishmaniaantigen (SLA) and His-A2 as antigen. Expressed proteins showed, by western blotting, the expression of an 11 KDa protein. Sixty-three percent (303/482) of the samples from endemic areas were positive by ELISA His-A2, whereas 93.1% (27/29) of Leishmune®-vaccinated animals were negative by His-A2-ELISA. Anti-A2 antibodies from mice inoculated with the A2 protein were detected in slides containing amastigote forms, but not in slides containing promastigote forms. The A2 recombinant protein from L. chagasi may be a useful tool in the diagnosis of CVL, and further tests regarding the infection stage and the specie of parasite at which the dogs are sampled should provide a better understanding of our results.

Expression of a recombinant protein, A2 family, from Leishmania infantum (Jaboticabal strain) and its evaluation in Canine Visceral Leishmaniasis serological test / Expressão de uma proteína recombinante, da família A2, de Leishmania infantum (amostra Jaboticabal) e sua avaliação no teste sorológico da Leishmaniose Visceral Canina

This study aimed to express a recombinant A2 family protein of Leishmania chagasi, Jaboticabal strain; test this protein as an antigen in serological assays; and investigate its antigenicity and immunogenicity. A protein coded by an allele of the A2 gene isolated from L. chagasi was expressed in three different strains of Escherichia coli. We used 29 sera samples from Leishmune-vaccinated dogs, 482 sera samples from dogs from endemic areas (positive controls), and 170 sera samples from dogs from non-endemic areas (negative controls) in ELISA tests using soluble Leishmaniaantigen (SLA) and His-A2 as antigen. Expressed proteins showed, by western blotting, the expression of an 11 KDa protein. Sixty-three percent (303/482) of the samples from endemic areas were positive by ELISA His-A2, whereas 93.1% (27/29) of Leishmune®-vaccinated animals were negative by His-A2-ELISA. Anti-A2 antibodies from mice inoculated with the A2 protein were detected in slides containing amastigote forms, but not in slides containing promastigote forms. The A2 recombinant protein from L. chagasi may be a useful tool in the diagnosis of CVL, and further tests regarding the infection stage and the specie of parasite at which the dogs are sampled should provide a better understanding of our results.

Este estudo teve como objetivos expressar uma proteína recombinante da família A2 de Leishmania chagasi, amostra de Jaboticabal-SP; testar essa proteína como antígeno em testes sorológicos; e investigar a antigenicidade e imunogenicidade dessa proteína. Uma proteína codificada por um alelo do gene A2 isolado de L. chagasi foi expressa em três diferentes amostras de Escherichia coli. Foram utilizadas 29 amostras de soro de cães vacinados com Leishmune, 482 amostras de soro de cães de áreas endêmicas (controles positivos), e 170 amostras de soro de cães de áreas não-endêmicas (controles negativos) no ELISA-teste utilizando-se antígeno solúvel total de Leishmania (AST) e His-A2 como antígenos. As proteínas expressas, detectadas pelo western blotting, mostraram a expressão de uma proteína de 11 KDa. Sessenta e três por cento (303/482) das amostras de áreas endêmicas foram positivas pelo ELISA-teste, utilizando-se antígeno His-A2; e 93,1% (27/29) dos animais vacinados com a Leishmune foram negativos. Anticorpos anti-A2 de camundongos inoculados com a proteína A2 foram detectados em lâminas contendo formas amastigotas, enquanto em lâminas contendo formas promastigotas não houve detecção de anticorpos anti-A2. A proteína recombinante A2 pode ser uma ferramenta útil no diagnóstico da LVC, e maiores estudos sobre o estágio de infecção e a espécie de parasita dos cães amostrados devem prover melhor entendimento dos resultados encontrados.

Lack of acquired resistance in dogs to successive infestations of Rhipicephalus sanguineus ticks from Brazil and Argentina.

Comparative studies between brown dog tick Rhipicephalus sanguineus populations from Brazil (Jaboticabal, São Paulo) and Argentina (Rafaela, Santa Fé) showed significant biological, morphological and genetic differences between them. This work aimed to study, in a comparative way, the acquisition of resistance in domestic dogs to R. sanguineus from Jaboticabal and Rafaela, after successive and controlled infestations. Ticks were kept in a BOD incubator under controlled conditions (27 °C, 80 % relative humidity, 12-h photoperiod). Ten dogs, Dachshund breed, males and females, 6 months old, short- or long-haired, without prior contact with ticks, were used as hosts. They were distributed into two experimental groups composed of five animals each: G1 infested with ten adult couples of R. sanguineus (Jaboticabal) per animal, and G2 infested with ten adult couples of R. sanguineus (Rafaela) per animal. Ticks' biological parameters and titration of antibodies from the dogs' sera by ELISA test were used for comparison between the strains. Results of the biological parameters showed that the dogs did not acquire immunity to either of the R. sanguineus strains after repeated infestations. The ELISA test showed low antibody titers in sera of dogs from G2, in successive infestations, and higher antibody responses post second and third infestations in G1. It also demonstrated cross-reactivity between sera of dogs infested with R. sanguineus (Jaboticabal) and antigens from R. sanguineus (Rafaela) and vice versa. We conclude that Dachshund dogs did not develop resistance against neither Jaboticabal nor Rafaela strains of R. sanguineus.

Molecular and serological detection of tick-borne pathogens in dogs from an area endemic for Leishmania infantum in Mato Grosso do Sul, Brazil / Detecção sorológica e molecular de patógenos transmitidos por carrapatos em cães de uma área endêmica para Leishmania infantum do estado do Mato Grosso do Sul

Tick-borne pathogens affect a wide range of vertebrate hosts. To identify tick-borne pathogens among dogs from Campo Grande, MS, Brazil testing seropositive for Leishmania infantum (syn. L. chagasi), a serological and molecular study was conducted to detect Ehrlichia canis, Anaplasma platys and Babesia vogeli in 60 serum and spleen samples. A confirmatory diagnosis of L. infantum based on serological and molecular assays was also performed, as was sequence alignment and phylogenetic analysis to assess the identity of the parasite species infecting these animals. IgG antibodies to Ehrlichia spp., B. vogeli and L. infantum were found, respectively, in 39 (65%), 49 (81.6%) and 60 (100%) of the sampled dogs. Twenty-seven (45%), fifty-four (90%), fifty-three (88.3%), two (3.3%) and one (1.6%) dog were positive, respectively, for E. canis, Leishmania spp., Leishmania donovani complex, Babesia sp. and Anaplasma sp. in PCR assays. After sequencing, the amplicons showed 99% of identity with E. canis, B. vogeli, A. platys and Leishmania chagasi isolates. The findings of this study indicate that L. infantum-seropositive dogs from Campo Grande are exposed to multiple tick-borne pathogens, which should therefore be included in the differential diagnosis of dogs with clinical suspicion of leishmaniasis.

Patógenos transmitidos por carrapatos atingem uma variedade de hospedeiros vertebrados. Para identificar os agentes patogênicos transmitidos por carrapatos entre cães soropositivos para Leishmania infantum no município Campo Grande-MS, foi realizado um estudo sorológico e molecular para a detecção de Ehrlichia canis, Anaplasma platys e Babesia vogeli em 60 amostras de soro e baço, respectivamente. Adicionalmente, foi realizado o diagnóstico confirmatório de L. infantum por meio de técnicas sorológicas e moleculares. Também foi realizado o alinhamento e análise filogenética das sequências para indicar a identidade das espécies de parasitas que infectam esses animais. Anticorpos IgG anti-Ehrlichia spp., anti-B. vogeli e anti-L. infantum foram detectados em 39 (65%), 49 (81,6%) e 60 (100%) dos cães amostrados, respectivamente. Vinte e sete (45%), cinquenta e quatro (90%), cinquenta e três (88,3%), dois (3,3%) e um (1,6%) cães mostraram-se positivos na PCR para E. canis, Leishmania spp., Leishmania donovani complex, Babesia sp. e Anaplasma sp., respectivamente. Após o seqüenciamento, os amplicons mostraram 99% de similaridade com isolados de E. canis, B. vogeli e A. platys e Leishmania chagasi. Os resultados deste estudo indicaram que os cães soropositivos para L. infantum de Campo Grande, MS, são expostos a vários agentes transmitidos por carrapatos, e, portanto, devem ser incluídos no diagnóstico diferencial em cães com suspeita clínica de leishmaniose.

Molecular and serological detection of tick-borne pathogens in dogs from an area endemic for Leishmania infantum in Mato Grosso do Sul, Brazil.

Tick-borne pathogens affect a wide range of vertebrate hosts. To identify tick-borne pathogens among dogs from Campo Grande, MS, Brazil testing seropositive for Leishmania infantum (syn. L. chagasi), a serological and molecular study was conducted to detect Ehrlichia canis, Anaplasma platys and Babesia vogeli in 60 serum and spleen samples. A confirmatory diagnosis of L. infantum based on serological and molecular assays was also performed, as was sequence alignment and phylogenetic analysis to assess the identity of the parasite species infecting these animals. IgG antibodies to Ehrlichia spp., B. vogeli and L. infantum were found, respectively, in 39 (65%), 49 (81.6%) and 60 (100%) of the sampled dogs. Twenty-seven (45%), fifty-four (90%), fifty-three (88.3%), two (3.3%) and one (1.6%) dog were positive, respectively, for E. canis, Leishmania spp., Leishmania donovani complex, Babesia sp. and Anaplasma sp. in PCR assays. After sequencing, the amplicons showed 99% of identity with E. canis, B. vogeli, A. platys and Leishmania chagasi isolates. The findings of this study indicate that L. infantum-seropositive dogs from Campo Grande are exposed to multiple tick-borne pathogens, which should therefore be included in the differential diagnosis of dogs with clinical suspicion of leishmaniasis.

Polymerase chain reaction and real-time PCR for diagnosing of Leishmania infantum chagasi in dogs.

The importance of dogs as a reservoir for Leishmania infantumchagasi in urban environments has stimulated numerous studies assessing diagnostic techniques. When performed properly, such procedures are an important step in preventing leishmaniasis in humans. Molecular methods have become prominent for this purpose. The aim of the present study was to determine the performance of the polymerase chain reaction (PCR) and real-time PCR (qPCR) for diagnosing of canine visceral leishmaniasis (CVL) using different biological samples. For this, 35 dogs from an area endemic for CVL were used. Bone marrow aspirate and lymph node and spleen fragments from these dogs were used for the molecular diagnosis. In the present study, qPCR was able to detect a greater number of positive animals than seen with PCR. Among the different biological samples used, there was no significant difference in L. infantumchagasi DNA detection between PCR and qPCR. However, considering that lymph nodes are easy to acquire, these can be considered to be the best samples for making molecular diagnoses of L. infantum chagasi infection.

Polymerase chain reaction and real-time PCR for diagnosing of Leishmania infantum chagasi in dogs / Reação em Cadeia da Polimerase e PCR em tempo real para diagnóstico de Leishmania infantum chagasi em cães

The importance of dogs as a reservoir for Leishmania infantumchagasi in urban environments has stimulated numerous studies assessing diagnostic techniques. When performed properly, such procedures are an important step in preventing leishmaniasis in humans. Molecular methods have become prominent for this purpose. The aim of the present study was to determine the performance of the polymerase chain reaction (PCR) and real-time PCR (qPCR) for diagnosing of canine visceral leishmaniasis (CVL) using different biological samples. For this, 35 dogs from an area endemic for CVL were used. Bone marrow aspirate and lymph node and spleen fragments from these dogs were used for the molecular diagnosis. In the present study, qPCR was able to detect a greater number of positive animals than seen with PCR. Among the different biological samples used, there was no significant difference in L. infantumchagasi DNA detection between PCR and qPCR. However, considering that lymph nodes are easy to acquire, these can be considered to be the best samples for making molecular diagnoses of L. infantum chagasi infection.

A importância do cão como reservatório de L. infantum chagasi no meio urbano tem estimulado a realização de inúmeros trabalhos de avaliação de técnicas de diagnóstico, uma vez que este procedimento, quando realizado corretamente, torna-se um importante passo na prevenção da doença em humanos. Dentre os métodos de diagnóstico, as técnicas moleculares têm adquirido destaque. Objetivou-se neste trabalho verificar o desempenho da Reação em Cadeia da Polimerase (PCR) e da PCR em tempo real (qPCR) para diagnóstico da Leishmaniose Visceral Canina (LVC) utilizando diferentes amostras biológicas. Para tanto foram utilizados 35 cães provenientes de uma área endêmica para LVC, onde foram utilizados para o diagnóstico molecular, aspirado de medula óssea, fragmentos de linfonodo e baço. Neste estudo a qPCR foi capaz de detectar um maior número de animais positivos quando comparada com a PCR. Já entre as diferentes amostras biológicas utilizadas não foi observada diferença significativa na detecção de DNA de L. infantumchagasi por meio da PCR e qPCR. Mesmo assim, considerando a facilidade de obtenção, o linfonodo pode ser considerada como a melhor amostra para diagnóstico molecular da infecção por L. infantum chagasi.

Occurrence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in cats with outdoor access in São Luís, Maranhão, Brazil.

The present study aimed to investigate the frequency of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in cats with outdoor access in São Luís, Maranhão, Brazil. The presence of IgG anti-T. gondii and anti-N. caninum antibodies was tested using the Indirect Immunofluorescent Antibody Test (IFAT). IgG anti-T. gondii and anti-N. caninum antibodies were detected in 101 (50.5%) and 54 (27%) sampled cats, respectively. The titers of anti-T. gondii antibodies ranged from 40 (cut-off) to 2560. On the other hand, the titers of anti-N. caninum antibodies ranged from 25 (cut-off) to 400. Twenty-seven cats (13.5%) were shown to be seropositive for both parasites. Seventy-four cats (34%) were seropositive only for T. gondii. Twenty-two cats (11%) were seropositive only for N. caninum. The present study showed that cats with outdoor access in São Luís, Maranhão, are exposed to T. gondii and N. caninum.

Occurrence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in cats with outdoor access in São Luís, Maranhão, Brazil / Ocorrência de anticorpos anti-Toxoplasma gondii e anti-Neospora caninum em gatos com acesso à rua em São Luís, Maranhão, Brasil

The present study aimed to investigate the frequency of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in cats with outdoor access in São Luís, Maranhão, Brazil. The presence of IgG anti-T. gondii and anti-N. caninum antibodies was tested using the Indirect Immunofluorescent Antibody Test (IFAT). IgG anti-T. gondii and anti-N. caninum antibodies were detected in 101 (50.5%) and 54 (27%) sampled cats, respectively. The titers of anti-T. gondii antibodies ranged from 40 (cut-off) to 2560. On the other hand, the titers of anti-N. caninum antibodies ranged from 25 (cut-off) to 400. Twenty-seven cats (13.5%) were shown to be seropositive for both parasites. Seventy-four cats (34%) were seropositive only for T. gondii. Twenty-two cats (11%) were seropositive only for N. caninum. The present study showed that cats with outdoor access in São Luís, Maranhão, are exposed to T. gondii and N. caninum.

O presente estudo objetivou verificar a frequência de anticorpos anti-Toxoplasma gondii e anti-Neospora caninum em gatos com acesso à rua em São Luís, Maranhão, Brasil. A presença de anticorpos IgG anti-T. gondii e anti-N. caninum foi verificado pela Reação de Imunofluorescência Indireta (RIFI). Anticorpos IgG anti-T. gondii e anti-N. caninum foram detectados em 101 (50,5%) e 54 (27%) gatos amostrados, respectivamente. Os títulos de anticorpos anti-T. gondii variaram de 40 (ponto de corte) a 2560. Por outro lado, anticorpos anti-N. caninum variaram de 25 (ponto de corte) a 400. Vinte e sete gatos (13,5%) mostraram-se soropositivos para ambos os parasitas. Setenta e quatro gatos (34%) foram soropositivos somente para T. gondii. Vinte e dois gatos (11%) foram soropositivos somente para N. caninum. O presente estudo demonstrou que gatos com acesso à rua em São Luís, Maranhão, são expostos ao T. gondii e N. caninum.

Visceral leishmaniasis in a captive crab-eating fox Cerdocyon thous.

Visceral leishmaniasis (VL) is a zoonotic disease with worldwide distribution. The crab-eating fox (Cerdocyon thous) is considered a wild reservoir of many zoonotical diseases, particularly VL. This study reported the presence of Leishmania infantum amastigotes in different organs of one captive C. thous found dead in a zoo. This animal was positive by the indirect fluorescence antibody test and had many clinical signs of VL. Intracellular amastigote forms of L. infantum were seen in neutrophils and macrophages in sample tissues from skin, lymph nodes (popliteal, submandibular, prescapular, and mesenteric), spleen, and liver. The numbers of positive cells and intracellular parasites were higher in macrophages than in neutrophils. In addition, polymerase chain reaction demonstrated extensive distribution of Leishmania DNA in C. thous tissues from multiple organs. The presence of intracellular amastigotes in neutrophils and macrophages as well as DNA of the parasite in tissues, specifically skin demonstrate that this crab-eating fox is an adequate host for L. infantum and reinforce the importance of VL for symptomatic wild canids kept in captivity in endemic areas.

Molecular and serological detection of Leishmania spp. in captive wild animals from Ilha Solteira, SP, Brazil.

Leishmaniasis is a zoonotic disease that affects 12 million people worldwide. Several mammalian species can serve as a reservoir for this disease. Dogs are the main reservoir for visceral leishmaniasis in urban areas, which has become a serious public health concern in Brazil. The aim of this study was to evaluate the presence of Leishmania spp. in captive wild animals from Ilha Solteira, São Paulo, Brazil. Blood and various tissues samples were collected from animals of five different species: Speothos venaticus, Chrysocyon brachyurus, Cerdocyon thous, Pseudalopex vetulus, and Procyon cancrivorus. Antibodies against Leishmania spp. were detected in three wild canids by indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). PCR analyses of blood and bone marrow from all animals were negative, but Leishmania DNA was found in the tissues and skin of seropositive animals. Positive PCR samples were also positive for Leishmania donovani complex. Analysis of sequenced PCR products showed similarities with different regions of Leishmania (Leishmania) infantum and Leishmania (Leishmania) chagasi kinetoplastids. Measures to control visceral leishmaniasis in wild animals kept in Brazilian zoos should be established, as no disease control programs are currently available.

Molecular and serological detection of Leishmania spp. in captive wild animals from Ilha Solteira, SP, Brazil / Detecção sorológica e molecular de Leishmania spp. em animais selvagens do zoológico de Ilha Solteira, SP, Brasil

Leishmaniasis is a zoonotic disease that affects 12 million people worldwide. Several mammalian species can serve as a reservoir for this disease. Dogs are the main reservoir for visceral leishmaniasis in urban areas, which has become a serious public health concern in Brazil. The aim of this study was to evaluate the presence of Leishmania spp. in captive wild animals from Ilha Solteira, São Paulo, Brazil. Blood and various tissues samples were collected from animals of five different species: Speothos venaticus, Chrysocyon brachyurus, Cerdocyon thous, Pseudalopex vetulus, and Procyon cancrivorus. Antibodies against Leishmania spp. were detected in three wild canids by indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). PCR analyses of blood and bone marrow from all animals were negative, but Leishmania DNA was found in the tissues and skin of seropositive animals. Positive PCR samples were also positive for Leishmania donovani complex. Analysis of sequenced PCR products showed similarities with different regions of Leishmania (Leishmania) infantum and Leishmania (Leishmania) chagasi kinetoplastids. Measures to control visceral leishmaniasis in wild animals kept in Brazilian zoos should be established, as no disease control programs are currently available.

Leishmaniose é uma doença zoonótica que afeta cerca de 12 milhões de pessoas no mundo todo. Várias espécies mamíferas podem servir de reservatório para a doença. Os cães são considerados os principais reservatórios para a leishmaniose visceral em áreas urbanas, o que tem se tornado um sério problema de saúde pública no Brasil. O objetivo deste trabalho foi avaliar a presença de Leishmania spp. em animais selvagens mantidos no zoológico de Ilha Solteira, São Paulo, Brasil. Foram coletados amostras de sangue e tecidos de cinco espécies diferentes: Speothos venaticus, Chrysocyon brachyurus, Cerdocyon thous, Pseudalopex vetulus, e Procyon cancrivorus. Anticorpos contra Leishmania spp. foram detectados em três canídeos pelo teste de imunofluorescência indireta (RIFI) e pelo ensaio imunoenzimático indireto (ELISA-teste). A análise de PCR das amostras de sangue e medula óssea foi negativa para todas as amostras, mas DNA de Leishmania foi encontrado em tecidos e pele de animais soropositivos. As amostras de PCR positivas também foram positivas para o complexo Leishmania donovani. Análise de sequenciamento dos produtos de PCR mostrou similaridade com diferentes regiões do cinetoplasto de Leishmania (Leishmania) infantum e Leishmania (Leishmania) chagasi. Medidas de controle de leishmaniose visceral em animais selvagens mantidos em zoológicos brasileiros devem ser estabelecidas, uma vez que não há nenhum programa de controle disponível.

A novel A2 allele found in Leishmania (Leishmania) infantum chagasi.

Visceral leishmaniasis (VL) is a widely spread zoonotic disease. In Brazil the disease is caused by Leishmania (Leishmania)infantum chagasi. Peridomestic sandflies acquire the etiological agent by feeding on blood of infected reservoir animals, such as dogs or wildlife. The disease is endemic in Brazil and epidemic foci have been reported in densely populated cities all over the country. Many clinical features of Leishmania infection are related to the host-parasite relationship, and many candidate virulence factors in parasites that cause VL have been studied such as A2 genes. The A2 gene was first isolated in 1994 and then in 2005 three new alleles were described in Leishmania (Leishmania) infantum. In the present study we amplified by polymerase chain reaction (PCR) and sequenced the A2 gene from the genome of a clonal population of L. (L.) infantum chagasi VL parasites. The L. (L.) infantum chagasi A2 gene was amplified, cloned, and sequenced in. The amplified fragment showed approximately 90% similarity with another A2 allele amplified in Leishmania (Leishmania) donovani and in L. (L.) infantum described in literature. However, nucleotide translation shows differences in protein amino acid sequence, which may be essential to determine the variability of A2 genes in the species of the L. (L.) donovani complex and represents an additional tool to help understanding the role this gene family may have in establishing virulence and immunity in visceral leishmaniasis. This knowledge is important for the development of more accurate diagnostic tests and effective tools for disease control.