Molecular detection of Leishmania spp. in cattle from Brazil by means of PCR using internal transcribed spacer 1.

Leishmania spp. are important agents of human and animal leishmaniases that have an important impact on public health. In this study, we aimed to detect the circulation of Leishmania spp. in cattle from a visceral leishmaniasis non-endemic area of the state of São Paulo, Brazil. DNA was extracted from blood samples from 100 heifers in the municipality of Pirassununga and was amplified using primers specific for the first internal transcriber spacer (ITS1), to assess the presence of trypanosomatids. The assays revealed that one sample presented bands of between 300 and 350 base pairs. In GenBank, this sample matched 100% with Leishmania infantum (314 base pairs). The results suggest that cattle can be infected by Leishmania infantum in Brazil.

Molecular detection of Leishmania spp. in cattle from Brazil by means of PCR using internal transcribed spacer 1 / Detecção molecular de Leishmania spp. em gado no Brasil por PCR com espaçador interno transcrito 1

Abstract Leishmania spp. are important agents of human and animal leishmaniases that have an important impact on public health. In this study, we aimed to detect the circulation of Leishmania spp. in cattle from a visceral leishmaniasis non-endemic area of the state of São Paulo, Brazil. DNA was extracted from blood samples from 100 heifers in the municipality of Pirassununga and was amplified using primers specific for the first internal transcriber spacer (ITS1), to assess the presence of trypanosomatids. The assays revealed that one sample presented bands of between 300 and 350 base pairs. In GenBank, this sample matched 100% with Leishmania infantum (314 base pairs). The results suggest that cattle can be infected by Leishmania infantum in Brazil.

Resumo Leishmania spp. são agentes causadores das leishmanioses em humanos e em animais, gerando grande impacto à saúde pública. Este estudo objetivou detectar a circulação de Leishmania spp. em área não endêmica para leishmaniose visceral de São Paulo, Brasil. Foram extraídas amostras de DNA de 100 novilhas da cidade de Pirassununga. Estas amostras foram amplificadas com os iniciadores específicos para tripanosomatídeos Internal Transcriber Spacer 1 (ITS1). Os ensaios revelaram uma amostra com bandas entre 300 e 350 pares de base (pb). A amostra demonstrou 100% de identidade com Leishmania infantum (314 pb). Os resultados sugerem que o gado pode ser infectado por L. infantum no Brasil.

Detection of canine visceral leishmaniasis by conjunctival swab PCR.

INTRODUCTION: Conjunctival swab PCR was evaluated as a tool to diagnose visceral leishmaniasis in dogs. METHODS: Conjunctival swab PCR was compared to indirect immunofluorescence antibody test and blood PCR. RESULTS: Indirect immunofluorescence was significantly correlated with conjunctival swab PCR (p < 0.05), but not with blood PCR (p > 0.05). In addition, conjunctival swab PCR was significantly associated with presence of clinical symptoms (p < 0.05), whereas blood PCR was associated with absence of clinical symptoms (p < 0.05). CONCLUSIONS: Results indicate that conjunctival swab PCR is useful in epidemiological surveys of canine visceral leishmaniasis.

Toxoplasma gondii, Neospora caninum and Leishmania spp. serology and Leishmania spp. PCR in dogs from Pirassununga, SP.

We examined the presence of antibodies against the parasites Toxoplasma gondii, Neospora caninum, and Leishmania spp., as well the presence of DNA from Leishmania spp., in dogs from Pirassununga - SP. The seropositivity rate was compared with the animals' originating location. Three hundred seventy-three blood samples from the county's kennel and local veterinary clinics were collected and analyzed. A total of 300 samples were tested for T. gondii and N. caninum using an indirect immunofluorescence antibody test (IFAT); 45% (135/300) were positive for T. gondii and 24.3% (73/300) were positive for N. caninum. Three hundred seventy-three samples were tested for Leishmania spp. using the IFAT. Of these, 4.6% (17/373) were positive. Additionally, 145 samples were tested using a polymerase chain reaction (PCR); of these samples, 0.7% (1/145) was positive. Considering the results, we conclude that these parasites are present in the city of Pirassununga - SP and that the animals have contact with the protozoan. It is therefore necessary to create methods for disease prevention to maintain both animal and human health in regard to leishmaniasis and toxoplasmosis.

Toxoplasma gondii, Neospora caninum and Leishmania spp. serology and Leishmania spp. PCR in dogs from Pirassununga, SP / Sorologia para Toxoplasma gondii, Neospora caninum e Leishmania spp. e PCR para Leishmania spp. em cães de Pirassununga, SP

Abstract We examined the presence of antibodies against the parasites Toxoplasma gondii, Neospora caninum, and Leishmania spp., as well the presence of DNA from Leishmania spp., in dogs from Pirassununga - SP. The seropositivity rate was compared with the animals’ originating location. Three hundred seventy-three blood samples from the county’s kennel and local veterinary clinics were collected and analyzed. A total of 300 samples were tested for T. gondii and N. caninum using an indirect immunofluorescence antibody test (IFAT); 45% (135/300) were positive for T. gondii and 24.3% (73/300) were positive for N. caninum. Three hundred seventy-three samples were tested for Leishmania spp. using the IFAT. Of these, 4.6% (17/373) were positive. Additionally, 145 samples were tested using a polymerase chain reaction (PCR); of these samples, 0.7% (1/145) was positive. Considering the results, we conclude that these parasites are present in the city of Pirassununga - SP and that the animals have contact with the protozoan. It is therefore necessary to create methods for disease prevention to maintain both animal and human health in regard to leishmaniasis and toxoplasmosis.

Resumo Avaliou-se a presença de anticorpos contra Toxoplasma gondii, Neospora caninum e Leishmania spp.; assim como a presença de DNA de Leishmania spp. em cães de Pirassununga-SP, e associou-se sua soropositividade ao local de origem dos animais. Foram coletadas 373 amostras de sangue do canil municipal e de clínicas veterinárias locais, que foram analisadas pelo teste de Imunofluorescência Indireta (RIFI). Do total, 300 amostras foram testadas para T. gondii e N. caninum, das quais 45% (135/300) foram positivas para T. gondii e 24,3% (73/300) para N. caninum. Para Leishmania spp. foram avaliadas 373 amostras pela RIFI, sendo 4,6% (17/373) positivas. Adicionalmente, 145 amostras foram testadas utilizando-se a PCR e, dessas amostras, 0,7% (1/145) foi positiva. Considerando-se os resultados, pode-se concluir que esses parasitos estão presentes na cidade de Pirassununga - SP e que os animais tiveram contato com os protozoários. Faz-se, dessa forma, necessária a divulgação de meios de prevenção às doenças, com o intuito de manter o controle sobre as mesmas, tanto na saúde animal quanto na saúde humana, em relação à leishmaniose e à toxoplasmose.

Conjunctival swab PCR to detect Leishmania spp. in cats.

The relevance of the dog as a source of visceral leishmaniasis infection is known, but the role of cats as reservoir hosts for leishmaniasis is not yet fully clear. This study assessed the efficacy of conjunctival swab PCR (CS-PCR) in the detection of cats infected by Leishmania spp. The results were seven (13.5%) cats positive for Leishmania spp. in the PCR, in 52 cats tested from Pirassunuga-SP and Ilha Solteira-SP. From the city of Pirassununga - SP 28.6% (2/7) were positive and from the city of Ilha Solteira - SP 11.1% (5/45) were positive. The results showed that CS-PCR was capable of detecting cats infected by this protozoan. Conjunctival swab samples proved easier to perform in cats, which might facilitate studies on the frequency and distribution of feline leishmaniasis.

Conjunctival swab PCR to detect Leishmania spp. in cats / Uso da PCR de suabe conjuntival para detecção de Leishmania spp. em gatos

The relevance of the dog as a source of visceral leishmaniasis infection is known, but the role of cats as reservoir hosts for leishmaniasis is not yet fully clear. This study assessed the efficacy of conjunctival swab PCR (CS-PCR) in the detection of cats infected by Leishmania spp. The results were seven (13.5%) cats positive for Leishmania spp. in the PCR, in 52 cats tested from Pirassunuga-SP and Ilha Solteira-SP. From the city of Pirassununga SP 28.6% (2/7) were positive and from the city of Ilha Solteira SP 11.1% (5/45) were positive. The results showed that CS-PCR was capable of detecting cats infected by this protozoan. Conjunctival swab samples proved easier to perform in cats, which might facilitate studies on the frequency and distribution of feline leishmaniasis.

A importância do cão como fonte de infecção da leishmaniose visceral já é conhecida, mas o papel dos gatos como reservatórios das leishmanioses ainda não está totalmente esclarecido. O presente estudo avaliou a eficácia da PCR de suabe conjuntival (PCR-SC) na detecção de gatos infectados por Leishmania spp. Foram encontrados sete (13,5%) gatos positivos para Leishmania spp. na PCR de suabe conjuntival, dentre 52 animais de Pirassununga - SP e Ilha Solteira - SP testados. Sendo positivos 28,6% (02/07) dos gatos do município de Pirassununga e 11,1% (5/45) dos gatos do município de Ilha Solteira. Os resultados demonstraram que o suabe de conjuntiva ocular foi capaz de detectar gatos infectados por esse protozoário. A coleta de amostras da conjuntiva mostrou ser um método simples, menos invasivo e pouco estressante para os gatos e seus proprietários, o que pode facilitar estudos sobre a frequência e distribuição da leishmaniose felina.

Toxoplasma gondii: humoral and cellular immune response of BALB/c mice immunized via intranasal route with rTgROP2.

TgROP2 is an intracellular protein associated with rhoptries of Toxoplama gondii and an antigen component of a candidate vaccine for toxoplasmosis. The purpose of the present study was to evaluate the efficacy of rTgROP2 to stimulate humoral and cellular immune responses in BALB/c mice via intranasal injection. TgROP2 partial coding sequence was (196-561) amplified by PCR from genomic T. gondii RH strain DNA and cloned into the pTrcHis expression vector. Escherichia coli Rosetta 2 cells transformed with pTrcHis-TgROP2 showed high levels (~1 mg.mL(-1)) of recombinant protein after 4 hours of IPTG induction. Recombinant TgROP2 exhibited an apparent Mr equal to 54 kDa. In order to test immunogenicity of the recombinant protein, 10 BALB/c mice received 10 µg of rROP2 protein + 10 µg of Quil-A via intranasal injection. Doses were administered at days 0, 21, and 42. Three animals were euthanized and used to evaluate cellular immune response on day 62. Five (50%) and two (20%) out of ten animals produced IgG (DO mean = 0.307; cut-off = 0.240) and IgA (DO mean = 0.133, cut-off = 0.101), respectively, by ELISA on day 62. The proliferation of splenocytes revealed high stimulation index (SI) when co-cultured with 5, 10 and 15 µg.mL(-1) of rTgROP2. These results indicate that intranasal immunization with recombinant protein ROP2 plus Quil-A can elicit both cellular and humoral immune responses in BALB/c mice.

Toxoplasma gondii: humoral and cellular immune response of BALB/c mice immunized via intranasal route with rTgROP2 / Toxoplasma gondii: avaliação da resposta imune humoral e celular de camundongos BALB/c imunizados pela via nasal com rTgROP2

TgROP2 is an intracellular protein associated with rhoptries of Toxoplama gondii and an antigen component of a candidate vaccine for toxoplasmosis. The purpose of the present study was to evaluate the efficacy of rTgROP2 to stimulate humoral and cellular immune responses in BALB/c mice via intranasal injection. TgROP2 partial coding sequence was (196-561) amplified by PCR from genomic T. gondii RH strain DNA and cloned into the pTrcHis expression vector. Escherichia coli Rosetta 2 cells transformed with pTrcHis-TgROP2 showed high levels (~1 mg.mL-1) of recombinant protein after 4 hours of IPTG induction. Recombinant TgROP2 exhibited an apparent Mr equal to 54 kDa. In order to test immunogenicity of the recombinant protein, 10 BALB/c mice received 10 µg of rROP2 protein + 10 µg of Quil-A via intranasal injection. Doses were administered at days 0, 21, and 42. Three animals were euthanized and used to evaluate cell-ular immune response on day 62. Five (50 percent) and two (20 percent) out of ten animals produced IgG (DO mean = 0.307; cut-off = 0.240) and IgA (DO mean = 0.133, cut-off = 0.101), respectively, by ELISA on day 62. The proliferation of splenocytes revealed high stimulation index (SI) when co-cultured with 5, 10 and 15 µg.mL-1 of rTgROP2. These results indicate that intranasal immunization with recombinant protein ROP2 plus Quil-A can elicit both cellular and humoral immune responses in BALB/c mice.

TgROP2 é uma proteína localizada nas roptrias do Toxoplasma gondii, sendo um antígeno candidato a componente de uma vacina contra a toxoplasmose. O objetivo do presente estudo foi avaliar a eficácia da TgROP2 recombinante em estimular a resposta imune celular e humoral de camundongos BALB/c após estímulo intranasal. A sequência da TgROP2 foi amplificada pela PCR a partir da cepa RH e clonada em vetor de expressão pTrc-His. Após a transformação em Escherichia coli- Rosetta 2, a pTrcHis-TgROP2 exibiu alto nível de expressão após 4 horas de indução com IPTG. A proteína recombinante apresentou uma massa molecular aparente de aproximadamente 54 kDa. Para avaliar a imunogenicidade dessa proteína recombinante, 10 camundongos receberam, pela via intranasal, 10 µg da rROP2 associado a 10 µg de Quil-A. Três doses foram realizadas nos dias 0, 21 e 42. No dia 62 do experimento, três animais foram eutanasiados para avaliar as respostas imune celular e humoral. Cinco (50 por cento) e dois (20 por cento) dos 10 animais apresentaram níveis de IgG (DO média = 0,307; ponto de corte = 0,240) e IgA (DO média = 0,133; ponto de corte = 0,101) acima do ponto de corte no ELISA no dia 62. A proliferação de esplenócitos revelou altos Índices de Estimulação (SI), quando as células foram cultivadas com 5, 10 e 15 µg.mL-1 de rTgROP2. Os resultados obtidos indicam que a via nasal pode estimular tanto a resposta imune celular como a humoral.