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. gondii infection have been occasionally reported in sheep. Due to the relatively scarce information on the molecular detection of N. caninum in the semen of naturally infected rams, this study aimed to detect parasitic DNA in fresh semen samples and in 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 rams were 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 to the close similarity with N. caninum. The indirect fluorescent antibody test was used to detect IgG antibodies in the 11 serum samples from rams. Fresh semen samples were collected from 11 rams on days 1, 50, 55, and 58 using an artificial vagina and ewe in estrus. Other 27 rams had their frozen extended semen straws analyzed. A total of 20 fresh semen samples and 27 frozen extended semen straws samples were used to detect the presence of N. caninum and T. gondii DNA by polymerase chain 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) were obtained by sequencing, and analysis revealed 99% and 100% identity, respectively, compared with other sequences deposited at GenBank. N. caninum and T. gondii DNAs were not detected in any of the 27 frozen extended semen straws used for artificial insemination.Discussion: This study demonstrated the presence of N. caninum and T. gondii DNA in fresh semen samples of naturally infected rams. The non-detection of N. caninum and T. gondii DNA in frozen semen samples of rams could be due to the dilution that was used to prepare the semen straws (GGL diluent and 5% glycerol), since fresh semen samples were not diluted prior to the test. Moreover, in our study, the volume of frozen semen samples (0.25 mL) used for PCR was lower than the volume of sediment obtained from fresh semen (0.5 mL), and the fresh semen centrifugation to obtain the sediment may have grouped the tachyzoites, increasing the sensitivity of the technique employed. No high IgG serological titers were detected in the rams at the time they were eliminating the parasite through fresh semen. The final titer of anti-N. caninum and anti-T. gondii IgGs in serum was 1:100, suggesting chronic infection. It is suggested that a new parasite elimination pathway is occurring among rams used for reproduction, due to the presence of N. caninum and T. gondii DNA in fresh semen samples from seropositive animals. Although the detection of genomic DNA of N. caninum and T. gondii in semen does not necessarily imply the presence of infectious stages of the parasites and does not determine their viability, these results demonstrate the need for further studies. Our study also indicates the need to reinforce preventive measures for sheep in artificial insemination centers until the risks are evaluated, by performing serological examinations with anti-N. caninum and anti-T. gondii antibodies, for instance, to select the rams that will be used for breeding

Resistência mecânica do solo à penetração associado à umidade, densidade, Granulometria e Macronutrientes em Ji-Paraná-RO / Soil mechanical resistance to penetration associated with humidity, density, Granulometry and Macronutrients in Ji-Paraná-RO

A resistência à penetração do solo associado à umidade são parâmetros estudados  para indicar o grau de compactação em diferentes tipos e condições de solo. O objetivo da pesquisa foi verificar o nível de compactação do solo na área da bacia hidrográfica do Igarapé Dois de abril em Ji-Paraná-RO, a partir dos parâmetros, resistência do solo à penetração, umidade, densidade, granulométria e macronutrientes para analisar se os resultados atendem ao desenvolvimento radicular de plantas. O construto da pesquisa foi realizado com base em material cartográfico, geoprocessamento e as técnicas que o integram como imagem de satélite, Sistema de Informação Geográfica-SIG e para o trabalho de campo foram utilizados o trado holandês, anel de copec e Sistema de Posicionamento Global-GPS. A resistência à penetração foi aferida em condições de campo com uso de penetrômetro de impacto, modelo IAA/Planalsucar-Solf. As análises de solo foram realizadas no Instituto Federal de Educação, Ciência e Tecnologia de Rondônia-IFRO, campus Ji-Paraná, Empresa Brasileira de Pesquisa Agropecuária- Embrapa em Porto Velho e no Laboratório de solos CEUJI/ULBRA, atual Centro Universitário São Lucas Ji-Paraná. Os resultados demonstraram que valores de RP apresentam restrições para o desenvolvimento radicular  em seis pontos onde ultrapassaram 2 MPa e estão associados a valores baixos de umidade e valores de até 1,98 g/cm3 de densidade. Já os seis pontos analisados de macronutrientes precisam de calagem e adubação para corrigir a acidez do solo e propiciar o desenvolvimento de plantas

Occurrence of anti-Neospora caninum and anti-Toxoplasma gondii antibodies in horses in the Pantanal of Mato Grosso, Brazil

This study aimed to evaluate the occurrence of anti-Neospora caninum and anti-Toxoplasma gondii antibodies in horses from Pantanal, in Mato Grosso state. Two hundred blood samples were collected from horses in Pantanal of Mato Grosso, Brazil. The samples were analyzed by IFAT for the detection of anti-Neospora caninum and anti-Toxoplasma gondii antibodies. Antibodies to N. caninum were found in 30 (15%) of 200 horses in titers of 50 (25 horses), 100 (two horses), 200 (two horses), and 400 (one horse). Antibodies to T. gondii were found in five (2.5%) of 200 horses in titers of 50 (three horses), 200 (one horse), and 400 (one horse). One animal showed antibody titers for both coccidian (titers of 200 for N. caninum e 400 for T. gondii). The pantaneiros horses were exposed to Neospora spp. and T. gondii

Detecção de anticorpos contra Sarcocystis neurona, Neospora caninum e Toxoplasma gondii em cavalos, cães e gatos do estado do Paraná, Brasil

O presente trabalho investigou a ocorrência e distribuição de anticorpos contra Sarcocystis neurona, Neospora caninum e Toxoplasma gondii em cavalos, cães e gatos de Curitiba, estado do Paraná, Brasil. Amostras de soro de 100 cavalos, 100 cães e 100 gatos da rotina do Laboratório de Patologia Clínica Veterinária do Hospital Veterinário da Universidade Federal do Paraná (UFPR) foram selecionadas. As 100 amostras de cães foram divididas em dois grupos: 35 amostras de animais com sinal neurológico (convulsão) e 65 sem sinais neurológicos. Os animais eram adultos de diferentes raças, machos e fêmeas. As amostras foram analisadas pelo teste de reação de imunofluorescência indireta (RIFI) para os protozoários S. neurona, N. caninum e T. gondii nas seguintes diluições de corte: cavalos: 1:50, 1:50 e 1:16; cães: 1:50, 1:50 e 1:16; gatos: 1:50, 1:50 e 1:50, respectivamente. Os resultados obtidos foram 42% dos cavalos, 7% dos cães e 5% dosgatos soropositivos para S. neurona; 58% dos cavalos, 68% dos cães e 42% dos gatos soropositivos para N. caninum; e 36% dos cavalos, 20% dos cães e 21% dos gatos soropositivos para T. gondii. Entre os cães com sinal neurológico, 8.6%, 68.6% e 25.7% deles foram soropositivos para S. neurona, N. caninum e T. gondii, respectivamente. Entre os cães sem sinais neurológicos, 6.2% 67.7% e 16.9% foram soropositivos para S. neurona, N. caninum e T. gondii, respectivamente. Não foi encontrada diferença estatística entre os grupos de cães soropositivos para os três protozoários com sinal neurológico e sem sinais neurológicos. Coinfecção e altos títulos de anticorpos foram detectados. Os anticorpos contra Sarcocystis neurona, Neospora caninum e Toxoplasma gondii foram encontrados amplamente distribuídos entre cavalos, cães e gatos na região de Curitiba, estado do Paraná, Brasil. The occurrence and distribution of antibodies against Sarcocystis neurona, Neospora caninum and Toxoplasma gondii was investigated in horses, dogs and cats from Curitiba, Paraná state, Brazil. Serum samples were selected from 100 horses, 100 dogs and 100 cats from the routine of the Veterinary Clinical Pathology Laboratory in the Veterinary Hospital of the University of Paraná (UFPR). The 100 dog samples were divided into two groups: 35 samples from dogs with neurological sign (convulsion) and 65 samples from dogs without neurological signs. The animals were adults of different breeds, males and females. Samples were analyzed by indirect fluorescent antibody test (IFAT) for protozoa S. neurona, N. caninum andT. gondii at the following cut-off dilutions: horses: 1:50, 1:50 and 1:16; dogs: 1:50, 1:50 and 1:16; cats: 1:50, 1:50 and 1:50, respectively. The obtained results were 42% of horses, 7% of dogs and 5% of cats seropositive for S. neurona; 58% of horses, 68% of dogs and 42% of cats seropositive to N. caninum, and 36% of horses, 20% of dogs and 21% of cats seropositive for T. gondii. Among the dogs with neurological signs, 8.6%, 68.6% and 25.7% were seropositive for S. neurona, N. caninum and T. gondii, respectively. Among the dogs without neurological signs, 6.2% 67.7% and 16.9% were seropositive for S. neurona, N. caninum and T. gondii, respectively. No statistical difference was found between groups of seropositive dogs for the three protozoa with neurological signs and without neurological signs. Co-infection and high antibody titers were detected. The antibodies against Sarcocystis neurona, Neospora caninum and Toxoplasma gondii were found widely distributed among horses, dogs and cats in the region of Curitiba, state of Paraná, Brazil.  &nbsp