Canine cutaneous neosporosis in Brazil.

The clinical signs of infection in dogs with Neospora caninum are usually associated with neurological disorders and are seen in young dogs. In this brief case report we observed multifocal ulcerative and exudative skin nodules on the neck and pelvic limbs of a 10-year-old cocker spaniel dog. Infection with N. caninum was diagnosed on the basis of cytology and examination of skin tissues by PCR. The dog initially responded to treatment with clindamycin and then relapsed; the dog died. Infection with N. caninum may have been due in part to immune suppression due to hyperadrenocorticism; which either allowed for the development of a primary infection or reactivation of a latent infection by N. caninum with the occurrence of skin lesions.

Neospora caninum and Toxoplasma gondii: relationship between hepatic lesions, cytological and biochemical analysis of the cavitary liquid during the acute phase of the diseases in experimental models.

The objective of this study was to evaluate the pathogenesis of ascites in mice infected with Toxoplasma gondii and gerbils infected with Neospora caninum during the acute phase disease. For that, 12 gerbils [Experiment I: not infected/control (n=6) and infected (n=6)] and 12 mice [Experiment II: control (n=6) and infected (n=6)] were used. Infected gerbils and mice showed marked ascites on days 5-7 post-infection (PI), while the not-infected animals had not ascites. Peritoneal liquid was collected from the all mice with uninfected animals receiving 1.5mL of saline solution into their abdominal cavity, allowing the recovery of cavity liquid. As a result, it was possible to observe differences in physics, chemistry and cytological analysis of the fluid cavity of animals infected with N. caninum and T. gondii, when they were compared with uninfected animals, as well as between animals experimentally infected. Additionally both, N. caninum and T gondii, caused an increase in the levels of nitric oxide (NOx-nitrate/nitrite), protein oxidation (AOPP) and lipid peroxidation (TBARS), while serum total protein and albumin were reduced in infected gerbils and mice. Gerbils infected with N. caninum showed multiple large cells with multilobulated nucleus, lytic necrosis and abundant amount of eosinophilic cytoplasm into the hepatic parenchyma. By the other hand, mice infected with T. gondii developed myriad foci of lytic necrosis combined with tachyzoites and cysts containing bradyzoites in liver. Both experimental models for N. caninum and T. gondii showed inflammatory foci and tachyzoites the peritoneum, which could be a major cause of ascites. Toxoplasmosis and neosporosis were able to cause clinical signs in experimental models with similar alterations in peritoneal fluid; however the toxoplasmosis histological changes were much more evident. Therefore, the pathogenesis of ascites appears to be directly related to liver damage, which strongly suggests alteration in the normal production of proteins as observed in this study, along with peritonitis.

Influence of infection by Toxoplasma gondii on purine levels and E-ADA activity in the brain of mice experimentally infected mice.

The aim of this study was to assess the purine levels and E-ADA activity in the brain of mice (BALB/c) experimentally infected with Toxoplasma gondii. In experiment I (n=24) the mice were infected with RH strain of T. gondii, while in experiment II (n=36) they were infected with strain ME-49 of T. gondii. Our results showed that, for RH strain (acute phase), an increase in both periods in the levels of ATP, ADP, AMP, adenosine, hypoxanthine, xanthine (only on day 6 PI) and uric acid (only on day 6 PI). By the other hand, the RH strain led, on days 4 and 6 PI, to a reduction in the concentration of inosine. ME-49, a cystogenic strain, showed some differences in acute and chronic phase, since on day 6 PI the levels of ATP and ADP were increased, while on day 30 these same nucleotides were reduced. On day 60 PI, ME-49 induced a reduction in the levels of ATP, ADP, AMP, adenosine, inosine and xanthine, while uric acid was increased. A decrease of E-ADA activity was observed in brain on days 4 and 6 PI (RH), and 30 PI (ME-49); however on day 60 PI E-ADA activity was increased for infection by ME-49 strain. Therefore, it was possible to conclude that infection with T. gondii changes the purine levels and the activity of E-ADA in brain, which may be associated with neurological signs commonly observed in this disease.

In vitro porphyrin-based photodynamic therapy against mono and polyculture of multidrug-resistant bacteria isolated from integumentary infections in animals.

Multidrug-resistant (MDR) organisms have been frequently isolated from integumentary lesions of animals, and these lesions are usually infected by more than one pathogen. This study evaluated an in vitro antimicrobial photodynamic therapy (aPDT) using two water-soluble tetra-cationic porphyrins (3-H2TMeP and 4-H2TMeP) against mono and polyculture of MDR bacteria isolated from dogs, cats, and horses. Ten isolates of MDR bacteria (two of each species: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia marcescens, and Staphylococcus pseudointermedius) were used to evaluate aPDT against the monoculture using a non-cytotoxic concentration of 3-H2TMeP and 4-H2TMeP porphyrins (40 µM), with 30 min of light irradiation in Gram-positive and 90 min for Gram-negative bacteria. The aPDT using the 4-H2TMeP porphyrin was also tested against five different polycultures (Coagulase positive Staphylococcus (CPS) and Pseudomonas sp.; E. coli and Proteus sp.; Pseudomonas sp. and Proteus sp.; CPS and E. coli; and CPS and Proteus sp.) for 90 min. The efficacy of both treatments was evaluated by plating the solution exposed to light or kept in the dark and counting the colonies forming units after 24 h of incubation at 37 °C. Atomic force microscope analysis was used to map bacteria morphological changes and extract adhesion force parameters from the bacteria membranes. Only the 4-H2TMeP porphyrin had antibacterial activity against MDR bacteria in monoculture, especially S. pseudointermedius and P. aeruginosa. In polyculture, the 4-H2TMeP porphyrin reduced bacterial concentrations (p < 0.05) in the associations of E. coli and S. pseudointermedius, P. aeruginosa and S. pseudointermedius, and P. aeruginosa and P. mirabilis. These results showed that aPDT using 4-H2TMeP is a good option for future associations of aPDT and other therapies or in vivo research.

Coinfection with canine distemper virus and canine circovirus in a dog in Brazil.

Canine distemper virus (CDV) and canine circovirus (CanineCV) have been described worldwide in multi-systemic disease in dogs. Both agents may be occasionally associated with other viral pathogens, but reports of coinfection by CDV and CanineCV associated with disease are rare. In this article, we report a coinfection between CDV and CanineCV detected during an investigation of viral agents involved in multisystemic disease in dogs in Southern Brazil. Molecular testing by PCR in lungs and intestines of 77 dogs necropsied in pathology services (2015-2020) revealed several single and mixed viral infections, including a CDV/CanineCV coinfection. In the case reported here, gross and histological findings were compatible with CDV pathology (bronchointerstitial pneumonia and viral intracytoplasmatic inclusions in pneumocytes and transitional epithelial cells of urinary bladder). CanineCV DNA and CDV antigens were detected in lung and intestine fragments by PCR and immunohistochemistry, respectively. CanineCV PCR amplicons subjected to nucleotide sequencing showed > 98.6% nucleotide identity with CanineCV sequences from GenBank. Although the role of CanineCV in the pathogenesis of the reported case could not be determined, our results show that CanineCV may be associated with other viral infections in cases of multisystemic disease in dogs. These results reinforce the circulation of CanineCV in dogs in Brazil and highlight the importance of including this virus in the list of differential diagnoses of respiratory and gastroenteric infectious diseases in dogs.

The Influence of Landscape Structure on the Occurrence of Neospora caninum, Toxoplasma gondii, and Sarcocystis spp. in Free-Living Neotropical Primates.

PURPOSE: Habitat fragmentation is the main threat to primate survival in the world. Additionally, changes in the environments in which they live can also contribute to exposure to pathogens. To investigate some pathogens that free-living primates may be exposed to in Rio Grande do Sul State (RS; southern Brazil) and characterize the forest remnants in which they live, we investigated anti-Neospora caninum, Toxoplasma gondii, and Sarcocystis spp. antibodies in the serum of the animals. METHODS: We analyzed 105 serum samples from 63 black howler monkeys (Alouatta caraya), 39 southern brown howler monkeys (Alouatta guariba clamitans), and 03 capuchin monkeys (Sapajus nigritus cucullatus), which were captured in forest fragments of RS. Indirect fluorescence antibody test (IFAT) and indirect hemagglutination assay (IHA) were used to detect antibodies to the agents. We then characterized the landscapes in a multiscale approach in radii from 200 to 1400 m to investigate the relationship of the presence of the agents with landscape elements. RESULTS: In the IFAT-IgG, 13.3% (14/105) of the samples were seropositive for N. caninum, 4.8% (5/105) for T. gondii, and 5.7% (6/105) for Sarcocystis spp. In the IHA-IgM/IgG, 24.8% (26/105) were seropositive for T. gondii. The metrics that best explained exposure to agents were edge and patch density, forest cover, urban cover, and average Euclidean distance to the nearest patch. CONCLUSIONS: This study indicated that the primates were exposed to the agents studied, demonstrating that some landscape features are associated with exposures to the investigated pathogens.

Occurrence of Sarcocystis gigantea macrocysts and high frequency of S. tenella microcysts in sheep from southern Brazil.

The aim of this study was to investigate the occurrence of sarcocysts in sheep slaughtered in the Rio Grande do Sul state, Brazil. Heart and esophagus samples from 130 sheep were subjected to macroscopic and microscopic examination, followed by molecular analysis. Ten sheep (7.7%) had Sarcocystis gigantea macrocysts in esophagus, as identified by gene sequencing. Microcysts were present in 96.1% of the sheep, with a higher frequency (p < .05) in the heart (91.5%) compared to the esophagus (81.5%) samples. The microcysts were identified as Sarcocystis tenella by gene sequencing. Our results revealed a high frequency of Sarcocystis spp. infection in sheep from southern Brazil. To the authors knowledge, this is the first molecular confirmation of S. gigantea presence in Brazil.

Molecular detection of Apicomplexa protozoa in tissues from Alouatta guariba clamitans

ABSTRACT: The brown howler monkey (Alouatta guariba clamitans) is a primate species widely distributed in South America. Infections by protozoa are common in primates. However, studies on protozoa in primates in Brazil are scarce, so the goal of this study was to investigate DNA from the apicomplexan protozoa Neospora caninum, Sarcocystis spp. and Toxoplasma gondii in tissues of A. guariba clamitans. DNA extraction was performed on tissue samples from the heart, brain, liver, spleen, lung and intestine of six A. guariba clamitans from Santa Maria, Central Region of Rio Grande do Sul, Brazil. Conventional PCR was performed using 18S rRNA gene general primers for Apicomplexa and also specific primers to amplify Neosporaspp. and Toxoplasma gondii DNA. All animals were positive in the 18S PCR and the genetic sequencing confirmed the presence of Sarcocystis spp. DNA in the tissues of four animals belonging to at least two species (S. neurona and S. gigantea) and T. gondii DNA in the other two animals. One positive sample for T. gondii was genotypically characterized as atypical by the restriction fragment length polymorphism technique. N. caninum DNA was not detected in the tested samples. The presence of Apicomplexa protozoan DNA in the tissues of the six animals tested in this study highlights the importance of howler monkeys as maintainers of these pathogens in nature.

RESUMO: O bugio ruivo (Alouatta guariba clamitans) é uma espécie de primata amplamente distribuída na América do Sul. As infecções por protozoários são comuns em primatas. Entretanto, estudos sobre protozoários em primatas no Brasil são escassos, portanto o objetivo deste estudo foi pesquisar DNA dos protozoários Apicomplexa Neospora caninum, Sarcocystisspp. e Toxoplasma gondii em tecidos de A. guariba clamitans. A extração de DNA foi realizada em amostras de tecido do coração, cérebro, fígado, baço, pulmão e intestino de seis A. guariba clamitans oriundos de Santa Maria, Região Central do Rio Grande do Sul, Brasil. Foi realizada PCR convencional utilizando primers geral do gene 18S rRNA para Apicomplexa e também primers específicos para amplificação de DNA de Neospora spp.e Toxoplasma gondii. Todos os animais foram positivos no PCR geral para Apicomplexa e no sequenciamento genético confirmou-se a presença de DNA de Sarcocystis nos tecidos de quatro animais pertencentes a pelo menos duas espécies (S. neurona e S. gigantea), e DNA de T. gondii foi detectado nos outros dois animais. Uma amostra positiva para T. gondii foi caracterizada genotipicamente como atípico pela técnica de polimorfismo do comprimento do fragmento de restrição. Não foi detectado DNA de N. caninum nas amostras testadas. A presença de DNA de protozoários apicomplexa nos tecidos dos seis animais testados neste estudo destaca a importância dos bugios ruivos como mantenedores desses patógenos na natureza.

Molecular detection of Apicomplexa protozoa in tissues from Alouatta guariba clamitans / Detecção molecular de protozoários Apicomplexa em tecidos de Alouatta guariba clamitans

The brown howler monkey (Alouatta guariba clamitans) is a primate species widely distributed in South America. Infections by protozoa are common in primates. However, studies on protozoa in primates in Brazil are scarce, so the goal of this study was to investigate DNA from the apicomplexan protozoa Neospora caninum, Sarcocystis spp. and Toxoplasma gondii in tissues of A. guariba clamitans. DNA extraction was performed on tissue samples from the heart, brain, liver, spleen, lung and intestine of six A. guariba clamitans from Santa Maria, Central Region of Rio Grande do Sul, Brazil. Conventional PCR was performed using 18S rRNA gene general primers for Apicomplexa and also specific primers to amplify Neosporaspp. and Toxoplasma gondii DNA. All animals were positive in the 18S PCR and the genetic sequencing confirmed the presence of Sarcocystis spp. DNA in the tissues of four animals belonging to at least two species (S. neurona and S. gigantea) and T. gondii DNA in the other two animals. One positive sample for T. gondii was genotypically characterized as atypical by the restriction fragment length polymorphism technique. N. caninum DNA was not detected in the tested samples. The presence of Apicomplexa protozoan DNA in the tissues of the six animals tested in this study highlights the importance of howler monkeys as maintainers of these pathogens in nature.(AU)

O bugio ruivo (Alouatta guariba clamitans) é uma espécie de primata amplamente distribuída na América do Sul. As infecções por protozoários são comuns em primatas. Entretanto, estudos sobre protozoários em primatas no Brasil são escassos, portanto o objetivo deste estudo foi pesquisar DNA dos protozoários Apicomplexa Neospora caninum, Sarcocystisspp. e Toxoplasma gondii em tecidos de A. guariba clamitans. A extração de DNA foi realizada em amostras de tecido do coração, cérebro, fígado, baço, pulmão e intestino de seis A. guariba clamitans oriundos de Santa Maria, Região Central do Rio Grande do Sul, Brasil. Foi realizada PCR convencional utilizando primers geral do gene 18S rRNA para Apicomplexa e também primers específicos para amplificação de DNA de Neospora spp.e Toxoplasma gondii. Todos os animais foram positivos no PCR geral para Apicomplexa e no sequenciamento genético confirmou-se a presença de DNA de Sarcocystis nos tecidos de quatro animais pertencentes a pelo menos duas espécies (S. neurona e S. gigantea), e DNA de T. gondii foi detectado nos outros dois animais. Uma amostra positiva para T. gondii foi caracterizada genotipicamente como atípico pela técnica de polimorfismo do comprimento do fragmento de restrição. Não foi detectado DNA de N. caninum nas amostras testadas. A presença de DNA de protozoários apicomplexa nos tecidos dos seis animais testados neste estudo destaca a importância dos bugios ruivos como mantenedores desses patógenos na natureza.(AU)

Occurrence and molecular characterization of Giardia duodenalis from naturally infected dogs in the municipality of Santa Maria, Rio Grande do Sul, Brazil / Ocorrência e caracterização molecular de Giardia duodenalis em cães naturalmente infectados no município de Santa Maria, Rio Grande do Sul, Brasil

Giardiasis is an important and prevalent zoonosis in dogs and humans caused by Giardia spp. The close relationship between pets and humans has physical, emotional and social benefits. The dogs have an important role in Giardia duodenalis cycle and transmission. This study aimed to verify the occurrence of the parasite in dogs from Central Region, in Santa Maria, Rio Grande do Sul State, Brazil, from April to October 2018. Dog feces (230) were submitted to Faust coproparasitological and molecular analyses. The positive samples in the nested-PCR (β-giardin gene) were sent for DNA sequencing and phylogenetic analyses (Neighbor-Joining). The occurrence of G. duodenalis, was 5.6% (13/230) and 4.3% (10/230) detected by coproparasitological technique and nested-PCR, respectively. There was no difference in the sensitivity of the tests used. From the faecal samples analyzed, there were no differences among the variables: diagnostic techniques, local, sex, and age of the animals (p>0.05). Only in the stool examination methodology a difference was observed between the ages (p<0.05). G. duodenalis assemblages were C and D, frequently reported in dogs. The close relationship between dogs and people may allow co-infections of circulating parasites in the population, including Giardia spp. and increasing the risk of transmission of zoonotic agents.(AU)

A giardíase é uma zoonose importante e prevalente em cães e humanos, sendo causada por Giardia spp. A estreita relação entre animais de estimação e seres humanos traz benefícios físicos, emocionais e sociais. Os cães têm um papel importante no ciclo e transmissão de Giardia duodenalis. Este estudo teve como objetivo verificar a ocorrência do parasita em cães da Região Central, em Santa Maria, RS, Brasil, de abril a outubro de 2018. As fezes de cães (230) foram submetidas a técnica coproparasitológica de Faust e análises moleculares. As amostras positivas no nested-PCR (gene β-giardin) foram enviadas para sequenciamento de DNA e posterior análise filogenética (Neighbor-Joining). A ocorrência de G. duodenalis foi de 5,6% (13/230) e 4,3% (10/230) detectados pela técnica coproparasitológica e nested-PCR, respectivamente. Não houve diferença na sensibilidade dos testes utilizados. Das amostras fecais analisadas, não houve diferenças entre as variáveis: técnicas de diagnóstico, local, sexo e idade dos animais (p>0,05). Somente na metodologia de exame de fezes observou-se diferença entre as idades (p<0,05). As assemblages de G. duodenalis encontradas foram C e D, frequentemente relatadas em cães. A estreita relação entre cães e pessoas pode permitir co-infecções de parasitas circulantes na população, incluindo Giardia spp. e aumentando o risco de transmissão de agentes zoonóticos.(AU)

Occurrence and molecular characterization of Giardia duodenalis from naturally infected dogs in the municipality of Santa Maria, Rio Grande do Sul, Brazil

ABSTRACT: Giardiasis is an important and prevalent zoonosis in dogs and humans caused by Giardia spp. The close relationship between pets and humans has physical, emotional and social benefits. The dogs have an important role in Giardia duodenalis cycle and transmission. This study aimed to verify the occurrence of the parasite in dogs from Central Region, in Santa Maria, Rio Grande do Sul State, Brazil, from April to October 2018. Dog feces (230) were submitted to Faust coproparasitological and molecular analyses. The positive samples in the nested-PCR (-giardin gene) were sent for DNA sequencing and phylogenetic analyses (Neighbor-Joining). The occurrence of G. duodenalis, was 5.6% (13/230) and 4.3% (10/230) detected by coproparasitological technique and nested-PCR, respectively. There was no difference in the sensitivity of the tests used. From the faecal samples analyzed, there were no differences among the variables: diagnostic techniques, local, sex, and age of the animals (p>0.05). Only in the stool examination methodology a difference was observed between the ages (p 0.05). G. duodenalis assemblages were C and D, frequently reported in dogs. The close relationship between dogs and people may allow co-infections of circulating parasites in the population, including Giardia spp. and increasing the risk of transmission of zoonotic agents.

RESUMO: A giardíase é uma zoonose importante e prevalente em cães e humanos, sendo causada por Giardia spp. A estreita relação entre animais de estimação e seres humanos traz benefícios físicos, emocionais e sociais. Os cães têm um papel importante no ciclo e transmissão de Giardia duodenalis. Este estudo teve como objetivo verificar a ocorrência do parasita em cães da Região Central, em Santa Maria, RS, Brasil, de abril a outubro de 2018. As fezes de cães (230) foram submetidas a técnica coproparasitológica de Faust e análises moleculares. As amostras positivas no nested-PCR (gene -giardin) foram enviadas para sequenciamento de DNA e posterior análise filogenética (Neighbor-Joining). A ocorrência de G. duodenalis foi de 5,6% (13/230) e 4,3% (10/230) detectados pela técnica coproparasitológica e nested-PCR, respectivamente. Não houve diferença na sensibilidade dos testes utilizados. Das amostras fecais analisadas, não houve diferenças entre as variáveis: técnicas de diagnóstico, local, sexo e idade dos animais (p>0,05). Somente na metodologia de exame de fezes observou-se diferença entre as idades (p 0,05). As assemblages de G. duodenalis encontradas foram C e D, frequentemente relatadas em cães. A estreita relação entre cães e pessoas pode permitir co-infecções de parasitas circulantes na população, incluindo Giardia spp. e aumentando o risco de transmissão de agentes zoonóticos.

Information technology by mobile communication for the notification of canine visceral leishmaniasis

ABSTRACT: Visceral leishmaniasis is the most severe form of a human and animal disease complex entitled leishmaniasis, which is endemic to 70 countries. It is imperative to develop and offer technologies capable of increasing the resolution ability of control programs of this zoonosis. In the search for technological innovations in health, especially in environmental surveillance, the objective is to develop a mobile application (App) for smartphones in order to facilitate and systematize the notification of positive cases of canine visceral leishmaniasis (CVL) by veterinarians working in clinics for assisting the municipal health surveillance in the management of this zoonosis. Thus, we developed an App, C7 LVC - Canine Visceral Leishmaniasis Notification System, with formatting based on the CR Campeiro 7® software. The technology created enables the filling of important gaps in information systems, facilitating the transmission of data and the use of this data by public management bodies to take CVL prevention and control actions.

RESUMO: A leishmaniose visceral é a forma mais grave de um complexo de doenças humanas e animais, denominado leishmaniose, endêmica em 70 países. É imprescindível desenvolver e oferecer tecnologias capazes de aumentar a capacidade de resolução dos programas de controle desta zoonose. Na busca por inovações tecnológicas em saúde, principalmente na vigilância ambiental, o objetivo é desenvolver um aplicativo móvel (App) para smartphones a fim de facilitar e sistematizar a notificação de casos positivos de leishmaniose visceral canina (LVC) por médicos veterinários que atuam em clínicas auxiliando a vigilância sanitária municipal, na gestão desta zoonose. Para tanto, foi desenvolvido um App, C7 LVC - Sistema de Notificação da Leishmaniose Visceral Canina, com formatação baseada no software CR Campeiro 7®. A tecnologia criada possibilita o preenchimento de lacunas importantes nos sistemas de informação, facilitando a transmissão de dados e a utilização desses dados pelos órgãos da gestão pública para a tomada de ações de prevenção e controle da LVC.

Information technology by mobile communication for the notification of canine visceral leishmaniasis / Tecnologia da informação por comunicação móvel para notificação de leishmaniose visceral canina

Visceral leishmaniasis is the most severe form of a human and animal disease complex entitled leishmaniasis, which is endemic to 70 countries. It is imperative to develop and offer technologies capable of increasing the resolution ability of control programs of this zoonosis. In the search for technological innovations in health, especially in environmental surveillance, the objective is to develop a mobile application (App) for smartphones in order to facilitate and systematize the notification of positive cases of canine visceral leishmaniasis (CVL) by veterinarians working in clinics for assisting the municipal health surveillance in the management of this zoonosis. Thus, we developed an App, C7 LVC - Canine Visceral Leishmaniasis Notification System, with formatting based on the CR Campeiro 7® software. The technology created enables the filling of important gaps in information systems, facilitating the transmission of data and the use of this data by public management bodies to take CVL prevention and control actions.(AU)

A leishmaniose visceral é a forma mais grave de um complexo de doenças humanas e animais, denominado leishmaniose, endêmica em 70 países. É imprescindível desenvolver e oferecer tecnologias capazes de aumentar a capacidade de resolução dos programas de controle desta zoonose. Na busca por inovações tecnológicas em saúde, principalmente na vigilância ambiental, o objetivo é desenvolver um aplicativo móvel (App) para smartphones a fim de facilitar e sistematizar a notificação de casos positivos de leishmaniose visceral canina (LVC) por médicos veterinários que atuam em clínicas auxiliando a vigilância sanitária municipal, na gestão desta zoonose. Para tanto, foi desenvolvido um App, C7 LVC - Sistema de Notificação da Leishmaniose Visceral Canina, com formatação baseada no software CR Campeiro 7®. A tecnologia criada possibilita o preenchimento de lacunas importantes nos sistemas de informação, facilitando a transmissão de dados e a utilização desses dados pelos órgãos da gestão pública para a tomada de ações de prevenção e controle da LVC.(AU)

Performance of the Dot-blot test method for detecting antibodies to Sarcocystis spp. in cattle / Desempenho do teste Dot-blot para detecção de anticorpos para Sarcocystis spp. em bovinos

Serological techniques can detect antibodies against Sarcocystis spp., Neospora caninum and Toxoplasma gondii antigens in single or mixed infections. Immunofluorescent antibody tests (IFAT) is considered the gold standard technique for Sarcocystosis diagnostic in cattle serum and a positive IFAT result reflects Sarcocystis spp. infection. Therefore, the aims of the present study were to compare IFAT and Dot-blot for sarcocystosis diagnostic in experimentally infected mice and to investigate serological cross-reactions with N. caninum and T. gondii in these methods. Mice (Mus musculus) were inoculated intraperitoneally with bradizoites of Sarcocystis spp. or tachyzoites of N. caninum or T. gondii. Serum samples were obtained and analyzed by IFAT and Dot-blot for the three protozoa. Serum from N. caninum and T. gondii experimentally infected mice were tested by IFAT and reacted only to N. caninum or T. gondii antigens, respectively. Specific antibodies against Sarcocystis spp. were present in all animals experimentally infected with this protozoan, with IFAT titers from 10 to 800. Serum samples from mice experimentally infected with Sarcocystis spp., N. caninum and T. gondii and tested by Dot-blot demonstrated no cross reaction between protozoa. A Dot-blot using Sarcocystis spp. antigen appears to be a good alternative to IFAT in the serological diagnosis of Sarcocystosis.(AU)

As técnicas sorológicas podem detectar anticorpos contra os antígenos de Sarcocystis spp., Neospora caninum e Toxoplasma gondii em infecções únicas ou mistas. O teste de anticorpos imunofluorescentes (IFAT) é considerado a técnica padrão-ouro para o diagnóstico de sarcocistose no soro de bovinos e um resultado positivo de IFAT reflete Sarcocystis spp. infecção. Portanto, os objetivos do presente estudo foram comparar IFAT e Dot-blot para diagnóstico de sarcocistose em camundongos infectados experimentalmente e investigar reações cruzadas sorológicas com N. caninum e T. gondii nesses métodos. Os camundongos (Mus musculus) foram inoculados intraperitonealmente com bradizoítos de Sarcocystis spp. ou taquizoítos de N. caninum ou T. gondii. As amostras de soro foram obtidas e analisadas por IFAT e Dot-blot para os três protozoários. O soro de N. caninum e T. gondii infectados experimentalmente foram testados por IFAT e reagiram apenas aos antígenos de N. caninum ou T. gondii, respectivamente. Anticorpos específicos contra Sarcocystis spp. estavam presentes em todos os animais experimentalmente infectados com este protozoário, com títulos de IFAT de 10 a 800. Amostras de soro de camundongos infectados experimentalmente com Sarcocystis spp., N. caninum e T. gondii e testadas por Dot-blot não demonstraram reação cruzada entre protozoários. Um Dot-blot usando Sarcocystis spp. O antígeno parece ser uma boa alternativa ao IFAT no diagnóstico sorológico da sarcocistose.(AU)

DNA extraction methods for molecular detection of Eimeria spp. in cattle and sheep / Métodos de extração de DNA para detecção molecular de Eimeria spp. em bovinos e ovinos

Molecular detection of Eimeria species in fecal samples can be useful for experimental and diagnostic purposes. However, the parasite quantity presence in feces and the oocyst wall are an obstacle in DNA extraction protocols. Therefore, adequate sampling and effective disruption of the oocysts are essential to improve the accuracy of DNA detection by PCR. The aims of this study were to evaluate the suitability of six protocols for DNA extraction from Eimeria spp. present in bovine and sheep. Twenty pools of fecal samples from cattle (10 pools) and sheep (10 pools) were distributed to six DNA extraction protocols: commercial kit, commercial kit with modification, DNAzol, cetyl-trimethyl ammonium bromide (CTAB), glass beads and commercial kit for fecal samples. Fecal samples were submitted to DNA extraction and PCR. Among the protocols tested, CTAB was determined to be most suitable for DNA extraction from oocysts (90% of DNA detection by PCR); DNAzol and CTAB resulted in higher DNA detection from bovine samples (80%). CTAB and commercial kit with modification improved PCR detection of Eimeria spp. in sheep samples, with positive amplification of DNA in all tested samples.(AU)

A detecção molecular de espécies de Eimeria em amostras fecais pode ser útil para fins experimentais e de diagnóstico. No entanto, a quantidade de parasitas nas fezes e a parede do oocisto são um obstáculo nos protocolos de extração de DNA. Portanto, uma amostragem adequada e a ruptura efetiva dos oocistos são essenciais para melhorar a precisão da detecção de DNA por PCR. Os objetivos deste estudo foram avaliar seis protocolos para extração de DNA de Eimeria spp. em amostras de bovinos e ovinos. Foram distribuídos 20 grupos de amostras fecais de bovinos (10 grupos) e ovinos (10 grupos) em seis protocolos de extração de DNA: kit comercial, kit comercial com modificação, DNAzol, brometo de cetil-trimetil amônio (CTAB), pérolas de vidro e kit comercial para amostras fecais. As amostras fecais foram submetidas à extração de DNA e PCR. Entre os protocolos testados, CTAB foi considerado o mais adequado para extração de DNA de oocistos (90% de detecção de DNA por PCR); DNAzol e CTAB resultaram em maior detecção de DNA em amostras de bovinos (80%). CTAB e kit comercial com modificação melhoraram a detecção por PCR de Eimeria spp. em amostras de ovinos, amplificação positiva de DNA em todas as amostras testadas.(AU)

Co-infection by Neopora caninum and bovine viral diarrhea virus in cattle from Rio Grande do Sul, Brazil, destined to exportation / Coinfecção por Neospora caninum e vírus da diarreia viral bovina em bovinos do Rio Grande do Sul, Brasil, destinados à exportação

Reproductive tests in cattle are of great economic importance, given the impact it can have on the production system and may be caused by agents. Neospora caninum and Bovine Viral Diarrhea virus (BVDV) are considered of great importance as reproductive and should be considered responsible for keeping animals persistently infected. The present study included 479 calf serum samples for export in the state of Rio Grande do Sul (RS). All samples were screened for BVDV by an ELISA antigen. BVDV antigen-positive ELISA samples were isolated from BVDV in cell culture. An indirect immunofluorescence (IFT) technique was used to detect anti-N. caninum antibodies. Of the 479 export-treated serum samples, 361 were positive for BVDV antigens by ELISA and/or viral isolation test (361/479-75.36%), and 109 IFT-positive samples for N. caninum (109/479-22.75%). Despite detection of antibodies anti-N. caninum did not differ statistically between naturally infected BVDV and non-BVDV infected animals suggesting that there is no interference of BVDV infection on infection or detection rate of animals with N. caninum, positive animals in viral isolation and high DO in BVDV-Ag ELISA. may present active disease and consequent immunosuppression, contributing to a potential reactivation of N. caninum.(AU)

Testes reprodutivos em bovinos são de grande importância econômica, dado o impacto que podem ter no sistema de produção e podem ser causados por agentes. O Neospora caninum e o vírus da Diarreia Viral Bovina (BVDV) são considerados de grande importância como reprodutivos e devem ser considerados responsáveis por manter os animais persistentemente infectados. O presente estudo incluiu 479 amostras de soro de bezerro para exportação no estado do Rio Grande do Sul (RS). Todas as amostras foram rastreadas para BVDV por um antígeno ELISA. As amostras de ELISA positivas para o antigénio BVDV foram isoladas a partir de BVDV em cultura de células. Uma técnica de imunofluorescência indireta (IFT) foi utilizada para detectar anticorpos anti-N caninum. Das 479 amostras de soro tratadas para exportação, 361 foram positivas para antígenos de BVDV por ELISA e/ou teste de isolamento viral (361/479-75,36%) e 109 amostras positivas para IFT para N. caninum (109/479-22,75%). Apesar da detecção de anticorpos anti-N. caninum não diferiu estatisticamente entre animais infectados naturalmente BVDV e não BVDV sugerindo que não há interferência da infecção pelo BVDV na infecção ou taxa de detecção de animais com N. caninum, animais positivos em isolamento viral e alta DO em BVDV-Ag ELISA, pode apresentar doença ativa e consequente imunossupressão, contribuindo para uma potencial reativação de N. caninum.(AU)

Phylogenetic analysis of Brazilian bovine viral diarrhea virus type 2 (BVDV-2) isolates: evidence for a subgenotype within BVDV-2.

Phylogenetic analysis divides bovine viral diarrhea viruses (BVDV) into two different genotypes (BVDV1 and BVDV2). BVDV1 strains have been further subdivided into two to 11 subgenotypes. Phylogenetic analysis of BVDV2 isolates, however, has not been able to identify discrete subgenotypes. In this study, we identified six South American BVDV2 strains and one North American BVDV2 strain that cluster to a separate genetic group within BVDV2, thus representing a distinct subgenotype. The 5' untranslated region (UTR) sequence homology between these six strains and other BVDV2 from North America, Europe and Asia (81.7%) is lower than the homology used to segregate BVDV1 into BVDV1a and BVDV1b (83.6%). Most nucleotide differences observed between the two subgroups of BVDV2 were concentrated in two regions, which also harbor most of the differences seen between BVDV1a and BVDV1b. To determine if this segregation was real, an additional analysis was performed comparing NS2/3 sequences. Analysis of a conserved sequence located between nucleotides 6670 and 7186 of the NS2/3 coding region also segregated these isolates to a separate group. The sequence homology between the two subgroups (86.3%) was higher than the homology in the 5'UTR (81.7%), with mean sequence homologies of 91 and 87.2% within the proposed subgroups. In contrast to the 5'UTR, alignment of the NS2/3 sequences revealed nucleotide differences distributed across the region. These results demonstrate that BVDV2 isolates cluster to two genetically distinct subgroups within BVDV2. The differences in both the 5'UTR and NS2/3 are consistent and justify this segregation. We suggest that BVDV2 may thereafter be subgenotyped into BVDV2a and BVDV2b. The existence of subgroups within the BVDV2 genotype with genetic heterogeneity similar to that seen among BVDV1 subgroups argues against BVDV2 isolates arising from BVDV1 in a recent evolutionary event. Unless the evolutionary clocks for BVDV1 and BVDV2 isolates tick along at different rates, these results indicate that BVDV2 have existed as long as BVDV1.

Influence of toxoplasmosis on acetylcholinesterase activity, nitric oxide levels and cellular lesion on the brain of mice.

The objective of this study was to investigate the activity of acetylcholinesterase (AChE), nitrite/nitrate (NOx) levels, as well as the biomarkers of cellular damage in the brain of mice experimentally infected with Toxoplasma gondii. Sixty mice were divided into two experiments: in experiment I the mice were infected with T. gondii/RH strain, while in experiment II they were infected with T. gondii, strains VEG and ME-49. Our evaluations were carried out on brain homogenized samples, assessing the AChE and glutathione reductase (GR) activities, and NOx, TBARS and AOPP levels in all the infected animals, compared with the control group. In both experiments, I and II, it was observed an increase in the activity of AChE and GR, as well as in the levels of NOx in the brain of infected mice with T. gondii. TBARS levels were increased in mice infected with the three different strains, RH, ME-49, and VEG. AOPP concentration was increased only in mice infected with the RH strain. Animals infected with the strains VEG and ME-49 showed histological lesions, associated with the presence of the parasite in the brain. Therefore, the infection by T. gondii is able to interfere in cholinesterase activity and NO levels, in association with oxidative stress and histological lesion.

Virulência e multiplicação de isolados de Toxoplasma gondii da região central do Rio Grande do Sul / Virulence and multiplication of Toxoplasma gondii isolates from the central region of Rio Grande do Sul, Brazil

O protozoário Toxoplasma gondii possui a capacidade de infectar diversas espécies animais, geralmente causando distúrbios reprodutivos. Quatro diferentes isolados de T. gondii - Pains #1 (P1), Pains #2 (P2), Santa Flora #1 (SF1) e Santa Flora #306 (SF306) - foram avaliados, após prévia genotipagem. A capacidade de multiplicação e virulência destes isolados foi analisada in vitro (células Vero) e in vivo (camundongos), sendo realizadas 3 passagens para cada isolado em cada modo avaliado, sendo sempre inoculada a dose de 1x104 taquizoítos em todas as passagens. Os camundongos eram observados diariamente, quanto à presença de sinais clínicos e ocorrência de mortalidade após inoculação dos taquizoítos. Os isolados SF1 e SF306, foram os que apresentaram maior multiplicação média do número total de taquizoítos em cada uma das 3 diferentes passagens realizadas para cada um dos isolados tanto in vitro quanto in vivo. Os primeiros sinais clínicos observados nos camundongos ocorreram entre os dias 5 a 11, após inoculação, com mortalidade acontecendo entre os dias 6 a 15, após inoculação. Assim, a multiplicação parasitária in vitro é semelhante à multiplicação in vivo destes isolados de T. gondii; diferentes isolados com o mesmo genótipo apresentam comportamento de virulência semelhante, caracterizando o isolado SF1 como mais virulento para camundongos.(AU)

The protozoan Toxoplasma gondii has the ability to infect several animal species, usually causing reproductive disorders. Four different isolates of T. gondii - Pains #1 (P1), Pains #2 (P2), Santa Flora #1 (SF1) and Santa Flora #306 (SF306) were evaluated with prior genotyping. Their virulence and multiplication capacity were analyzed in vitro (Vero cells) and in vivo (mice). For each isolate, three passages were performed with dose inoculation 1x104 tachyzoites at each passage. The mice were daily observed to verify of clinical signs and occurrence of mortality after tachyzoites inoculation. The SF1 isolate and SF306 isolate, presented the highest multiplication of the total tachyzoites number in each of the 3 different passages performed in vitro and in vivo. The initial clinical signs in mice were observed between 5 and 11 days, and occurring mortality between 6 and 15 days, after inoculation. Thus, the parasitic multiplication of theses isolates is similar in vitro and in vivo; different isolates within the same genotype have a similar virulence and the SF1 isolate is the most virulent for mice.(AU)