Detection of Neospora caninum and Toxoplasma gondii in semen of naturally infected rams

Background: Neospora caninum and Toxoplasma gondii are closely related cyst-forming apicomplexan parasites identified as important causes of reproductive failure in cattle. Moreover, abortion cases attributed to N. caninum and T. gondiiinfection have been occasionally reported in sheep. Due to the relatively scarce information on the molecular detection ofN. caninum in the semen of naturally infected rams, this study aimed to detect parasitic DNA in fresh semen samples andin frozen extended semen straws from male sheep from artificial inseminations centers in Southern Brazil.Materials, Methods & Results: Semen samples of 38 rams from artificial insemination centers were evaluated. Eleven ramswere naturally infected (seropositive for anti-N. caninum and/or anti-T. gondii IgG) and were selected for fresh semen collection.We tested all the samples for the closely related protozoan T. gondii to detect a possible cross-reaction and co-infection, due tothe close similarity with N. caninum. The indirect fluorescent antibody test was used to detect IgG antibodies in the 11 serumsamples from rams. Fresh semen samples were collected from 11 rams on days 1, 50, 55, and 58 using an artificial vaginaand ewe in estrus. Other 27 rams had their frozen extended semen straws analyzed. A total of 20 fresh semen samples and27 frozen extended semen straws samples were used to detect the presence of N. caninum and T. gondii DNA by polymerasechain reaction (PCR). Nc-5 and B1 genes were used as target regions to detect N. caninum and T. gondii DNA, respectively.The presence of N. caninum DNA was confirmed in the third collection of a fresh semen sample of one seropositive ram. T.gondii DNA was detected in a fresh semen sample of one seropositive ram. The DNA sequences of 186 bp from N. caninum(GenBank accession: MH806393) and 492 bp from T. gondii (GenBank accession: MH793503)...

Detection of Neospora caninum and Toxoplasma gondii in semen of naturally infected rams

Background: Neospora caninum and Toxoplasma gondii are closely related cyst-forming apicomplexan parasites identified as important causes of reproductive failure in cattle. Moreover, abortion cases attributed to N. caninum and T. gondiiinfection have been occasionally reported in sheep. Due to the relatively scarce information on the molecular detection ofN. caninum in the semen of naturally infected rams, this study aimed to detect parasitic DNA in fresh semen samples andin frozen extended semen straws from male sheep from artificial inseminations centers in Southern Brazil.Materials, Methods & Results: Semen samples of 38 rams from artificial insemination centers were evaluated. Eleven ramswere naturally infected (seropositive for anti-N. caninum and/or anti-T. gondii IgG) and were selected for fresh semen collection.We tested all the samples for the closely related protozoan T. gondii to detect a possible cross-reaction and co-infection, due tothe close similarity with N. caninum. The indirect fluorescent antibody test was used to detect IgG antibodies in the 11 serumsamples from rams. Fresh semen samples were collected from 11 rams on days 1, 50, 55, and 58 using an artificial vaginaand ewe in estrus. Other 27 rams had their frozen extended semen straws analyzed. A total of 20 fresh semen samples and27 frozen extended semen straws samples were used to detect the presence of N. caninum and T. gondii DNA by polymerasechain reaction (PCR). Nc-5 and B1 genes were used as target regions to detect N. caninum and T. gondii DNA, respectively.The presence of N. caninum DNA was confirmed in the third collection of a fresh semen sample of one seropositive ram. T.gondii DNA was detected in a fresh semen sample of one seropositive ram. The DNA sequences of 186 bp from N. caninum(GenBank accession: MH806393) and 492 bp from T. gondii (GenBank accession: MH793503)...(AU)

Concordância na detecção de anticorpos anti-Toxoplasma gondii e anti-Neospora caninum no sangue e no leite bovino pela reação de imunofluorescência indireta / Agreement in detection of antibodies against Toxoplasma gondii and Neospora caninum in blood and milk of cattle by indirect fluorescent antibody test

O objetivo deste estudo foi avaliar a aplicabilidade da pesquisa de anticorpos anti- Toxoplasma gondii e anti- Neospora caninum em amostras de leite bovino pela reação de imunofluorescência indireta (RIFI) e sua concordância com a detecção desses anticorpos em amostras de soro sanguíneo, simultaneamente coletadas das mesmas vacas. No total, foram analisadas amostras correspondentes de soro sanguíneo e de leite de 177 vacas em início de lactação. Para o T. gondii, o diagnóstico no leite demonstrou concordância boa e sensibilidade baixa, quando comparado ao soro sanguíneo com título de anticorpos séricos 64 (ponto de corte). Quanto ao N. caninum, obteve-se concordância excelente entre a detecção de anticorpos no soro sanguíneo com título 50 (considerado ponto de corte para termos de diagnóstico) e no leite, com sensibilidade de 80,7% e especificidade de 100%, no entanto, para as vacas com títulos de anticorpos 100 no soro sanguíneo, a sensibilidade e a especificidade foram de 100%. Nas condições do presente estudo, demonstrou-se que a RIFI não é indicada para diagnóstico da toxoplasmose em amostras de leite bovino, devido ao alto percentual de vacas soropositivas para anticorpos anti-T. gondii no soro sanguíneo diluído 1:64, em cujas amostras de leite não houve detecção desses anticorpos. Por outro lado, a RIFI é um teste viável para o diagnóstico da neosporose bovina, especialmente considerando como ponto de corte um título de anticorpos séricos 100, podendo ser recomendável pela praticidade de coleta da amostra como parte de programas de saúde dos rebanhos e estudos epidemiológicos.(AU)

This study aimed to evaluate: the applicability search for anti-Toxoplasma gondii and anti-Neospora caninum antibodies in cattle milk samples by indirect fluorescent antibody test (IFAT), and their concordance with detection of the same antibodies in blood serum samples, simultaneously collected from the same cows. A total of 177 cows in the beginning of lactation provided corresponding samples of blood serum and milk. To T. gondii, milk diagnosis demonstrated good conformity and low sensitivity in comparison with blood samples whose serum antibody titer ranged 64 (cut-off point). As for N. caninum it was obtained optimum conformity between the detection of blood serum antibodies, with titer ranging 50 (diagnose-aimed cut-off point) and milk, with sensitivity reaching 80.7% and 100% specificity. However, sensitivity and specificity reached 100% for cows whose antibodies titers in blood serum ranged 100. The study was able to demonstrate that IFAT is not indicate to diagnose toxoplasmosis in cattle milk samples due to the high percentage of cows that showed seropositivity to anti-T. gondii antibodies at the 1:64 blood serum dilution, in whose blood samples there was no such antibody detection. On the other hand, IFAT is a feasible test for diagnosing cattle neosporosis, especially when cut-off point is a serum antibody titer 100. It can be recommended as part of herd health programs and epidemiologic studies due to sampling practicality.(AU)

Detection of Neospora sp. antibodies in cart horses from urban areas of Curitiba, Southern Brazil / Detecção de anticorpos anti-Neospora sp. em cavalos de carroceiros de áreas urbanas de Curitiba, Sul do Brasil

Neospora caninum is a protozoan parasite which affects dogs as definitive hosts and several mammalian species as intermediate hosts mainly causing abortions and central nervous system disorders. The reemerging population of cart horses for carrying recycling material in urban areas of major cities in Brazil may have an impact on disease spreading, and these animals may be used as sentinels for environmental surveillance. Thus, the present study investigated the frequency of Neospora sp. antibodies in cart horses from Curitiba and surrounding areas, Paraná State, Southern Brazil. IgG antibodies against Neospora sp. were detected using indirect fluorescence antibody test (IFAT), and titers equal to or higher than 1:50 were considered reactive. Of all samples, 14/97 (14.4%) were positive: 2/29 (6.9%) were younger than 5; 5/26 (19.2%) between 6 and 9; and 6/31 (19.4%) older than 10 years of age. One of the 11 animals with unknown age was positive (9.1%). Cart horses are likely to be more exposed to dog feces and to Neospora sp. oocyst contamination in urban settings and a lower frequency of disease in dogs may have a negative impact on horse infection risk in these areas.(AU)

Neospora caninum é um protozoário parasita que afeta cães como hospedeiros definitivos e diversos mamíferos como hospedeiros intermediários, envolvido em abortos e distúrbios do sistema nervoso central. A população reemergente de cavalos de carroceiros utilizados para transportar material reciclável em áreas urbanas de grandes cidades brasileiras na disseminação de doenças, e estes animais podem ser utilizados como sentinelas para vigilância ambiental. Deste modo, no presente estudo foi investigada a frequência de anticorpos anti-Neospora sp. em cavalos de carroceiros da Região Metropolitana de Curitiba, Estado do Paraná, Sul do Brasil. Anticorpos da classe IgG anti-Neospora sp. foram detectados utilizando a reação de imunofluorescência indireta (RIFI), e títulos maiores ou iguais a 1:50 foram considerados reagentes. Do total de amostras testadas, 14/97 (14,4%) foram positivas: 2/29 (6,9%) tinham menos do que 5,5/26 (19,2%) entre 6 and 9, e 6/31 (19,4%) mais de 10 anos de idade. Um dos 11 animais positivos não tinha a idade conhecida (9,1%). Como cavalos de carroceiros possivelmente estejam mais expostos a oocistos de Neospora sp. em ambientes urbanos, uma baixa freqüência da doença em cães pode ter impacto negativo no risco de infecção de cavalos nestas áreas.(AU)