RESUMO

To a better insight into the epidemiology and genetic diversity of protozoan hemoparasites infections in wild mammals, this study aimed to the post mortem detection of DNA from species of the order Piroplasmida (Babesia sp., Cytauxzoon sp., and Theileria sp.) and suborder Adelorina (Hepatozoon sp.) using polymerase chain reaction based on the 18S rRNA gene followed by genetic sequencing of blood and spleen samples collected from carcasses of 164 free-ranging and captive wild mammals from Mato Grosso state. Among them, one Leopardus pardalis, three Panthera onca, two Puma concolor were positive for Cytauxzoon sp., and six Tapirus terrestris tested positive for Piroplasmida, while one L. pardalis was positive for Hepatozoon sp. Furthermore, an uncharacterized piroplasmid genetically related to Theileria sp. previously detected in cats from Brazil was described in lowland tapirs. Despite the controversy regarding the epidemiological threat of these protozoa, the detection of these tick-borne agents in wild free-living and captive mammals, even when asymptomatic, demonstrates the importance of monitoring, particularly in hotspots such as the state of Mato Grosso, to verify the circulation and genetic diversity, to anticipate the possible emergence of diseases, and even their consequences to other animals as well as humans.

Assuntos

RESUMO

Background: The enzootic bovine leukosis (EBL) is a viral disease with a chronic aspect and its etiological agent is anoncogenic deltaretrovirus called bovine leukemia virus (VLB). It has a wide distribution in dairy cattle herds, and it maytake several years before the first clinical manifestations occur. Animals seropositive for VLB are more susceptible todeveloping infectious diseases such as mastitis. The objective of this study was to evaluate the seroprevalence of EBL, theprevalence of mastitis, the possible association between leukosis and mastitis, and the risk factors related to leukosis inmunicipalities of the Caparaó region of Espírito Santo, Brazil.Materials, Methods & Results: The prevalence of enzootic bovine leukosis and its association with mastitis were evaluatedin an 854 clinically healthy crossbred (Bos tauros tauros x Bos taurus indicus) dairy cows were used, in different lactationphases, from farms located in the 12 municipalities that compose the Caparaó Capixaba micro-region, in the southernpart of Espírito Santo, Brazil, in the period from February to December 2015. The seroprevalence of EBL was determinedby the agar gel immunodiffusion test, the presence of clinical mastitis was ascertained by the black background mug testand of subclinical mastitis by the CMT. The reuse of needles, type of milking, reproductive management, calf mortality,presence of reproductive problems and milk yield were the risk factors assessed. The association between the variableswas estimated by the odds ratio (95% confidence interval). The significance of the associations was determined by theChi-square test. The prevalence of EBL, clinical and subclinical mastitis was 56.79%, 5.50% and 43.55%, respectively.There was a positive association between the EBL virus and the presence of...(AU)

Assuntos

RESUMO

Background: The enzootic bovine leukosis (EBL) is a viral disease with a chronic aspect and its etiological agent is anoncogenic deltaretrovirus called bovine leukemia virus (VLB). It has a wide distribution in dairy cattle herds, and it maytake several years before the first clinical manifestations occur. Animals seropositive for VLB are more susceptible todeveloping infectious diseases such as mastitis. The objective of this study was to evaluate the seroprevalence of EBL, theprevalence of mastitis, the possible association between leukosis and mastitis, and the risk factors related to leukosis inmunicipalities of the Caparaó region of Espírito Santo, Brazil.Materials, Methods & Results: The prevalence of enzootic bovine leukosis and its association with mastitis were evaluatedin an 854 clinically healthy crossbred (Bos tauros tauros x Bos taurus indicus) dairy cows were used, in different lactationphases, from farms located in the 12 municipalities that compose the Caparaó Capixaba micro-region, in the southernpart of Espírito Santo, Brazil, in the period from February to December 2015. The seroprevalence of EBL was determinedby the agar gel immunodiffusion test, the presence of clinical mastitis was ascertained by the black background mug testand of subclinical mastitis by the CMT. The reuse of needles, type of milking, reproductive management, calf mortality,presence of reproductive problems and milk yield were the risk factors assessed. The association between the variableswas estimated by the odds ratio (95% confidence interval). The significance of the associations was determined by theChi-square test. The prevalence of EBL, clinical and subclinical mastitis was 56.79%, 5.50% and 43.55%, respectively.There was a positive association between the EBL virus and the presence of...

Assuntos

RESUMO

Abstract To a better insight into the epidemiology and genetic diversity of protozoan hemoparasites infections in wild mammals, this study aimed to the post mortem detection of DNA from species of the order Piroplasmida (Babesia sp., Cytauxzoon sp., and Theileria sp.) and suborder Adelorina (Hepatozoon sp.) using polymerase chain reaction based on the 18S rRNA gene followed by genetic sequencing of blood and spleen samples collected from carcasses of 164 free-ranging and captive wild mammals from Mato Grosso state. Among them, one Leopardus pardalis, three Panthera onca, two Puma concolor were positive for Cytauxzoon sp., and six Tapirus terrestris tested positive for Piroplasmida, while one L. pardalis was positive for Hepatozoon sp. Furthermore, an uncharacterized piroplasmid genetically related to Theileria sp. previously detected in cats from Brazil was described in lowland tapirs. Despite the controversy regarding the epidemiological threat of these protozoa, the detection of these tick-borne agents in wild free-living and captive mammals, even when asymptomatic, demonstrates the importance of monitoring, particularly in hotspots such as the state of Mato Grosso, to verify the circulation and genetic diversity, to anticipate the possible emergence of diseases, and even their consequences to other animals as well as humans.

Resumo Para uma melhor compreensão da epidemiologia e diversidade genética das infecções por hemoprotozoários em mamíferos selvagens, este estudo teve como objetivo a detecção post mortem de DNA de espécies da ordem Piroplasmida (Babesia sp., Cytauxzoon sp. e Theileria sp.) e subordem Adelorina (Hepatozoon sp.), utilizando-se a reação em cadeia pela polimerase, baseada no gene 18S rRNA, seguido de sequenciamento genético de amostras de sangue e baço, coletadas de 164 carcaças de mamíferos selvagens de vida livre e cativos do estado de Mato Grosso. Entre eles, um Leopardus pardalis, três Panthera onca, dois Puma concolor foram positivos para Cytauxzoon sp., e seis Tapirus terrestris testaram positivos para Piroplasmida, enquanto um L. pardalis foi positivo para Hepatozoon sp. Além disso, foi descrito em antas, um piroplasmídeo não caracterizado geneticamente, relacionado à Theileria sp., previamente detectado em gatos do Brasil. Apesar da controvérsia quanto à ameaça epidemiológica desses protozoários, a detecção desses agentes em mamíferos silvestres e cativos, mesmo quando assintomáticos, demonstra a importância do monitoramento, principalmente em hotspots, como no estado de Mato Grosso, para verificar a circulação e a diversidade genética, a fim de antecipar o possível surgimento de doenças e, até mesmo, suas consequências para outros animais, bem como os humanos.

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RESUMO

Cryptosporidium spp. is a protozoan that infects a wide range of vertebrate hosts; it has been reported to be the cause of severe illness or death in livestock worldwide, which leads to decreased performance and production losses, especially in young animals. This study investigated the presence of Cryptosporidiumin calves from beef farms in the state of Mato Grosso, midwestern Brazil. For this purpose, fecal samples from 237 animals aged ≤ 45 days, raised in 20 rural properties were subjected to DNA extraction and nested polymerase chain reaction (nPCR) targeting 18S ribosomal RNA (18S rRNA) gene followed by sequencing. Additionally, positive samples, previously identified as Cryptosporidium parvum by sequencing and phylogenetic analyses based on 18S rRNA gene, were subsequently analyzed focusing the amplification and sequencing using nPCR of a fragment of the 60 kDa glycoprotein (gp60) gene. Of the 237 fecal samples analyzed by PCR (18S rRNA), 50 (21.1%) fecal samples were positive for Cryptosporidium spp., while 14 (70%) of the 20 properties had at least one positive animal. The following Cryptosporidium species were detected: C. bovis, C. parvum, and C. ryanae. Thereafter, two potentially zoonotic subtypes (IIaA15G2R1 and IIaA16G3R1) of C. parvum were identified based on gp60 gene sequences. This study resulted in the detection of subtype IIaA16G3R1 for the first time in South America and showed a wide distribution of the protozoan in beef farms in the studied area of the State.(AU)

Cryptosporidium spp. é um protozoário que infecta uma grande variedade de hospedeiros vertebrados, sendo descrito como causa de doenças graves ou morte na pecuária em todo o mundo, o que leva à diminuição do desempenho e à perda de produção, especialmente em animais jovens. Este estudo investigou a presença de Cryptosporidium em bezerros de fazendas de bovinocultura de corte no estado de Mato Grosso, Centro-Oeste do Brasil. Para esse fim, amostras fecais de 237 animais com idade ≤ 45dias, criados em 20 propriedades rurais foram submetidas à extração de DNA e uma "nested" da reação em cadeia pela polimerase (nPCR) direcionada ao gene 18S RNA ribossomal (18S rRNA), seguido do sequenciamento. Adicionalmente, amostras positivas, previamente identificadas como Cryptosporidium parvum pelo sequenciamento e análises filogenéticas baseadas no gene 18S rRNA, foram posteriormente analisadas usando a nPCR buscando a amplificação e sequenciamento de um fragmento do gene de uma glicoproteína de 60 kDa (gp60). Das 237 amostras fecais analisadas pela PCR (18S rRNA), 50 (21,1%)amostras fecais foram positivas para Cryptosporidium spp., enquanto 14 (70%) das 20 propriedades tinham ao menos um animal positivo. As seguintes espécies de Cryptosporidium foram detectadas: C. bovis, C. parvum e C. ryanae. Posteriormente, dois subtipos potencialmente zoonóticos (IIaA15G2R1e IIaA16G3R1) foram identificados. Este estudo resultou na detecção do subtipo IIaA16G3R1 pela primeira vez na América do Sul e mostrou uma ampla distribuição do protozoário em rebanhos de corte na área estudada do Estado.(AU)

Assuntos

RESUMO

Cryptosporidium spp. is a protozoan that infects a wide range of vertebrate hosts; it has been reported to be the cause of severe illness or death in livestock worldwide, which leads to decreased performance and production losses, especially in young animals. This study investigated the presence of Cryptosporidiumin calves from beef farms in the state of Mato Grosso, midwestern Brazil. For this purpose, fecal samples from 237 animals aged ≤ 45 days, raised in 20 rural properties were subjected to DNA extraction and nested polymerase chain reaction (nPCR) targeting 18S ribosomal RNA (18S rRNA) gene followed by sequencing. Additionally, positive samples, previously identified as Cryptosporidium parvum by sequencing and phylogenetic analyses based on 18S rRNA gene, were subsequently analyzed focusing the amplification and sequencing using nPCR of a fragment of the 60 kDa glycoprotein (gp60) gene. Of the 237 fecal samples analyzed by PCR (18S rRNA), 50 (21.1%) fecal samples were positive for Cryptosporidium spp., while 14 (70%) of the 20 properties had at least one positive animal. The following Cryptosporidium species were detected: C. bovis, C. parvum, and C. ryanae. Thereafter, two potentially zoonotic subtypes (IIaA15G2R1 and IIaA16G3R1) of C. parvum were identified based on gp60 gene sequences. This study resulted in the detection of subtype IIaA16G3R1 for the first time in South America and showed a wide distribution of the protozoan in beef farms in the studied area of the State.

Cryptosporidium spp. é um protozoário que infecta uma grande variedade de hospedeiros vertebrados, sendo descrito como causa de doenças graves ou morte na pecuária em todo o mundo, o que leva à diminuição do desempenho e à perda de produção, especialmente em animais jovens. Este estudo investigou a presença de Cryptosporidium em bezerros de fazendas de bovinocultura de corte no estado de Mato Grosso, Centro-Oeste do Brasil. Para esse fim, amostras fecais de 237 animais com idade ≤ 45dias, criados em 20 propriedades rurais foram submetidas à extração de DNA e uma "nested" da reação em cadeia pela polimerase (nPCR) direcionada ao gene 18S RNA ribossomal (18S rRNA), seguido do sequenciamento. Adicionalmente, amostras positivas, previamente identificadas como Cryptosporidium parvum pelo sequenciamento e análises filogenéticas baseadas no gene 18S rRNA, foram posteriormente analisadas usando a nPCR buscando a amplificação e sequenciamento de um fragmento do gene de uma glicoproteína de 60 kDa (gp60). Das 237 amostras fecais analisadas pela PCR (18S rRNA), 50 (21,1%)amostras fecais foram positivas para Cryptosporidium spp., enquanto 14 (70%) das 20 propriedades tinham ao menos um animal positivo. As seguintes espécies de Cryptosporidium foram detectadas: C. bovis, C. parvum e C. ryanae. Posteriormente, dois subtipos potencialmente zoonóticos (IIaA15G2R1e IIaA16G3R1) foram identificados. Este estudo resultou na detecção do subtipo IIaA16G3R1 pela primeira vez na América do Sul e mostrou uma ampla distribuição do protozoário em rebanhos de corte na área estudada do Estado.

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RESUMO

American cutaneous leishmaniasis (ACL) is a zoonosis caused by Leishmania, a protozoan. Commonantigens occur in the strains found in America, which allow antigenic cross-reactivity. Therefore,multivalent vaccines can be used for this pathogen. In this study, we investigated the efficacy of twodifferent commercial vaccines for visceral leishmaniasis to induce an immune response to the soluble L.(Viannia) braziliensis antigens. In 2014, 70 seronegative dogs from the municipality of Iúna (EspíritoSanto State, Brazil) were vaccinated and serologically evaluated by ELISA and immunoblotting by usingthe soluble antigen of L. braziliensis. Of the 121 dogs initially selected, only 70 received vaccinationbecause 51 dogs tested positive by ELISA, yielding a positive frequency of 42.14% in the asymptomaticgroup. These 70 dogs were divided into two equal groups and administered three doses of each vaccine,according to the manufacturers instructions. We found that the sera of dogs immunized with threedoses of both vaccines A and B had antibodies against the soluble antigens of L. (V.) braziliensis, asdetermined by ELISA and immunoblotting 120 days post vaccination. Antibodies produced in responseto vaccines A and B were found in 22/35 and 18/35 serum samples, respectively, at T1 (120 days), while7/35 and 4/35 serum samples tested positive at T2 (240 days). Furthermore, immunoblotting allowed usto differentiate between vaccinated and asymptomatic dogs.(AU)

A Leishmaniose Tegumentar Americana (LTA) é uma zoonose causada por protozoários do gêneroLeishmania. Existem antígenos comuns entre as várias espécies de Leishmania da América determinandoreações antigênicas cruzadas o que possibilita vacinas multivalentes. Este estudo avaliou soros de cãesvacinados com duas vacinas comerciais diferentes para leishmaniose visceral na indução de respostaimunitária cruzada para antígenos solúveis de Leishmania (Viannia) braziliensis. Durante o ano de2014, 70 cães soronegativos para Leishmania spp. do município de Iúna (Espírito Santo, Brasil) foramvacinados e examinadossorologicamente por ELISA e imunoblotting utilizando o antígeno solúvel de L.braziliensis. Para 121 cães inicialmente selecionados, apenas 70 foram submetidos a vacinação porque 51 dos animais foram positivos no teste sorológico ELISA para LTA, indicando uma frequência positivade 42,14% para os animais LTA assintomáticos. Estes setenta animais foram divididos em dois gruposde tamanhos iguais e foram aplicadas três doses de cada vacina segundo recomendações dos fabricantes.Como resultado inédito verificamos que os soros de cães imunizados com as três doses de vacinas A e Bapresentaram anticorpos contra antígenos solúveis de L. (V.) braziliensis quando avaliados por ELISAe immunoblotting com 120 dias pós-vacinação. As vacinas A e B apresentaram resultados positivoscom presença de anticorpos nos testes serológicos, respectivamente, 22/35 e 18/35 positivos em T1(120 dias), enquanto que em T2 (240 dias), 7/35 e 4/35 soros positivos. Além disso, o immunoblottingpermitiu diferenciar o soro de cães de diferentes vacinas e cães assintomáticos.(AU)

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RESUMO

American cutaneous leishmaniasis (ACL) is a zoonosis caused by Leishmania, a protozoan. Commonantigens occur in the strains found in America, which allow antigenic cross-reactivity. Therefore,multivalent vaccines can be used for this pathogen. In this study, we investigated the efficacy of twodifferent commercial vaccines for visceral leishmaniasis to induce an immune response to the soluble L.(Viannia) braziliensis antigens. In 2014, 70 seronegative dogs from the municipality of Iúna (EspíritoSanto State, Brazil) were vaccinated and serologically evaluated by ELISA and immunoblotting by usingthe soluble antigen of L. braziliensis. Of the 121 dogs initially selected, only 70 received vaccinationbecause 51 dogs tested positive by ELISA, yielding a positive frequency of 42.14% in the asymptomaticgroup. These 70 dogs were divided into two equal groups and administered three doses of each vaccine,according to the manufacturers’ instructions. We found that the sera of dogs immunized with threedoses of both vaccines A and B had antibodies against the soluble antigens of L. (V.) braziliensis, asdetermined by ELISA and immunoblotting 120 days post vaccination. Antibodies produced in responseto vaccines A and B were found in 22/35 and 18/35 serum samples, respectively, at T1 (120 days), while7/35 and 4/35 serum samples tested positive at T2 (240 days). Furthermore, immunoblotting allowed usto differentiate between vaccinated and asymptomatic dogs.

A Leishmaniose Tegumentar Americana (LTA) é uma zoonose causada por protozoários do gêneroLeishmania. Existem antígenos comuns entre as várias espécies de Leishmania da América determinandoreações antigênicas cruzadas o que possibilita vacinas multivalentes. Este estudo avaliou soros de cãesvacinados com duas vacinas comerciais diferentes para leishmaniose visceral na indução de respostaimunitária cruzada para antígenos solúveis de Leishmania (Viannia) braziliensis. Durante o ano de2014, 70 cães soronegativos para Leishmania spp. do município de Iúna (Espírito Santo, Brasil) foramvacinados e examinadossorologicamente por ELISA e imunoblotting utilizando o antígeno solúvel de L.braziliensis. Para 121 cães inicialmente selecionados, apenas 70 foram submetidos a vacinação porque 51 dos animais foram positivos no teste sorológico ELISA para LTA, indicando uma frequência positivade 42,14% para os animais LTA assintomáticos. Estes setenta animais foram divididos em dois gruposde tamanhos iguais e foram aplicadas três doses de cada vacina segundo recomendações dos fabricantes.Como resultado inédito verificamos que os soros de cães imunizados com as três doses de vacinas A e Bapresentaram anticorpos contra antígenos solúveis de L. (V.) braziliensis quando avaliados por ELISAe immunoblotting com 120 dias pós-vacinação. As vacinas A e B apresentaram resultados positivoscom presença de anticorpos nos testes serológicos, respectivamente, 22/35 e 18/35 positivos em T1(120 dias), enquanto que em T2 (240 dias), 7/35 e 4/35 soros positivos. Além disso, o immunoblottingpermitiu diferenciar o soro de cães de diferentes vacinas e cães assintomáticos.

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Abstract Fasciolosis is caused by Fasciola hepatica that affects the bile ducts and liver parenchyma of ruminants, which can result in economic loss. This study aimed to carry out the validity of the commercial kit ELISA® indirect front of the simple fecal sedimentation test used as the standard. 143 samples were collected blood and feces of cattle from Jerome, south of the Espírito Santo. Serum samples were left at -80 °C and used to perform the ELISA kit IDEXX®. All animals to stool examinations were also positive to the ELISA (22) and negative samples to test stool (121), 52 animals reacted positively against the antibody research. The frequency of fasciolosis was 15.4% in the stool examinations and 51.8% by ELISA. The validity was calculated by sensitivity (100%), specificity (57%), positive predictive value (29%) and negative predictive value (100%), and the correlation between the tests calculated using the kappa index of 0.35. The better sensitivity of the ELISA commercial kit should not be a separately evaluated, since the cost benefit and the technical facility must be considered.

Resumo Fasciolose é causada pela Fasciola hepatica, parasito que acomete os ductos biliares e o parênquima hepático dos ruminantes e pode resultar em perdas econômicas. Objetivou-se realizar a validade do kit comercial ELISA® indireto frente ao teste de sedimentação fecal simples utilizado como padrão. Foram coletadas 143 amostras de sangue e fezes de vacas provenientes do Sul do Espírito Santo. As amostras de sangue foram centrifugadas para separação do soro. As amostras de soro foram congeladas a -80ºC e utilizadas para análise com o kit de ELISA IDEXX®. Todos os 22 animais positivos ao exame coproparasitológico também foram positivos ao ELISA e, das 121 amostras negativas ao exame de fezes, 52 reagiram positivamente frente à pesquisa de anticorpos. A frequência de fasciolose foi de 15,4% no exame coproparasitológico e 51,8% pelo ELISA. A validade foi calculada pela sensibilidade (100%), especificidade (57%), valor preditivo positivo (29%) e valor preditivo negativo (100%), sendo considerada medíocre a concordância entre os testes, calculada pelo índice kappa (0,35). A maior sensibilidade obtida para o kit comercial ELISA não deve ser avaliada isoladamente, uma vez que o custo benefício e a facilidade da técnica devem ser considerados.

Assuntos

RESUMO

Fasciolosis is caused by Fasciola hepatica that affects the bile ducts and liver parenchyma of ruminants, which can result in economic loss. This study aimed to carry out the validity of the commercial kit ELISA® indirect front of the simple fecal sedimentation test used as the standard. 143 samples were collected blood and feces of cattle from Jerome, south of the Espírito Santo. Serum samples were left at -80 °C and used to perform the ELISA kit IDEXX®. All animals to stool examinations were also positive to the ELISA (22) and negative samples to test stool (121), 52 animals reacted positively against the antibody research. The frequency of fasciolosis was 15.4% in the stool examinations and 51.8% by ELISA. The validity was calculated by sensitivity (100%), specificity (57%), positive predictive value (29%) and negative predictive value (100%), and the correlation between the tests calculated using the kappa index of 0.35. The better sensitivity of the ELISA commercial kit should not be a separately evaluated, since the cost benefit and the technical facility must be considered.

Assuntos