Isolamento in vitro de Neospora caninum e toxoplasma gondii do sêmen de câes naturalmente infectados

Orientador : Prof. Dr. Romildo Romualdo WeissCo-orientadora : Profª. Drª. Rosangela Locatelli DittrichDissertação (mestrado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduação em Engenharia de Bioprocessos e Biotecnologia. Defesa: Curitiba, 26/03/2014Inclui referênciasÁrea de concentração: Saúde humana e animalResumo: Neospora caninum e Toxoplasma gondii são protozoários que infectam vários animais. O DNA dos parasitos foi detectado no sêmen de bovinos e homens, respectivamente, mas não há comprovação da presença desses protozoários no sêmen de cão. O cão pode ser um transmissor assintomático dos protozoários. O presente estudo aborda no capítulo I uma revisão de literatura sobre a Neosporose e Toxoplasmose, com ênfase nas vias de transmissão e o impacto na reprodução canina, e no capítulo II a investigação dos protozoários Neospora caninum e Toxoplasma gondii em amostras de sêmen de cães naturalmente infectados por análise citológica, isolamento em cultivo celular e reação da cadeia em polimerase (PCR). Neste trabalho foram realizadas colheitas de sêmen de 11 cães clinicamente saudáveis (quatro cães soropositivos para N. caninum, cinco soropositivos para T. gondii, dois soropositivos para ambos e dois soronegativos) para realizar o isolamento in vitro. Os isolados obtidos em cultivo celular e amostras do sêmen in natura foram submetidos à amplificação por PCR utilizando iniciadores que delimitam o segmento do DNA da região Nc5 de N. caninum (Np6/Np21) e da região de 529 pb repetida 200 a 300 vezes no genoma do T. gondii (TOX4/TOX5). Foram obtidos dois isolados de N. caninum e cinco de T. gondii confirmados pela PCR. Obteve-se amplificação do DNA de N. caninum em quatro amostras de sêmen, e amplificação do DNA de T. gondii somente nos isolados. Os parasitas estão presentes no sêmen e o isolamento e manutenção em cultivo celular comprovou sua viabilidade. Palavras-chave: isolamento in vitro, sêmen, cão.Abstract: Neospora caninum and Toxoplasma gondii are protozoa that infect many animals. The DNA of parasites was detected in the semen of cattle and men, respectively, but there is no evidence of the presence of these protozoa in dog semen. The dog may be an asymptomatic protozoan transmitter. The chapter I of the present study provides a literature review about neosporosis and toxoplasmosis, with emphasis on routes of transmission and the impact on canine reproduction, and the chapter II brings the investigation of the protozoa Neospora caninum and Toxoplasma gondii in semen samples of dogs naturally infected by cytological analysis, isolation in cell culture and polymerase chain reaction (PCR). In this study semen were collected in 11 clinically healthy dog (four dogs seropositive for N. caninum, five seropositive for T. gondii, two seropositive for both and two seronegative) to perform the isolation in vitro. The strains obtained in cell culture and the samples of nature semen was submitted to PCR amplification using primers that define the Nc5 DNA sequence of N. caninum (Np6/Np21) and the 529 bp repeated 200 to 300 times the genome of T. gondii (TOX4/TOX5). There was obtained two strains of N. caninum and five strains of T. gondii confirmed by PCR. Was obtained four amplification of N. caninum DNA in semen samples and five amplification of T. gondii DNA only in isolated strains. The parasites are present in the semen and the isolation and maintenance in cell culture have proven their viability. Keywords: isolation in vitro, semen, dog

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

Isolamento in vitro de Neospora caninum e toxoplasma gondii do sêmen de câes naturalmente infectados

Orientador : Prof. Dr. Romildo Romualdo WeissCo-orientadora : Profª. Drª. Rosangela Locatelli DittrichDissertação (mestrado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduação em Engenharia de Bioprocessos e Biotecnologia. Defesa: Curitiba, 26/03/2014Inclui referênciasÁrea de concentração: Saúde humana e animalResumo: Neospora caninum e Toxoplasma gondii são protozoários que infectam vários animais. O DNA dos parasitos foi detectado no sêmen de bovinos e homens, respectivamente, mas não há comprovação da presença desses protozoários no sêmen de cão. O cão pode ser um transmissor assintomático dos protozoários. O presente estudo aborda no capítulo I uma revisão de literatura sobre a Neosporose e Toxoplasmose, com ênfase nas vias de transmissão e o impacto na reprodução canina, e no capítulo II a investigação dos protozoários Neospora caninum e Toxoplasma gondii em amostras de sêmen de cães naturalmente infectados por análise citológica, isolamento em cultivo celular e reação da cadeia em polimerase (PCR). Neste trabalho foram realizadas colheitas de sêmen de 11 cães clinicamente saudáveis (quatro cães soropositivos para N. caninum, cinco soropositivos para T. gondii, dois soropositivos para ambos e dois soronegativos) para realizar o isolamento in vitro. Os isolados obtidos em cultivo celular e amostras do sêmen in natura foram submetidos à amplificação por PCR utilizando iniciadores que delimitam o segmento do DNA da região Nc5 de N. caninum (Np6/Np21) e da região de 529 pb repetida 200 a 300 vezes no genoma do T. gondii (TOX4/TOX5). Foram obtidos dois isolados de N. caninum e cinco de T. gondii confirmados pela PCR. Obteve-se amplificação do DNA de N. caninum em quatro amostras de sêmen, e amplificação do DNA de T. gondii somente nos isolados. Os parasitas estão presentes no sêmen e o isolamento e manutenção em cultivo celular comprovou sua viabilidade. Palavras-chave: isolamento in vitro, sêmen, cão.Abstract: Neospora caninum and Toxoplasma gondii are protozoa that infect many animals. The DNA of parasites was detected in the semen of cattle and men, respectively, but there is no evidence of the presence of these protozoa in dog semen. The dog may be an asymptomatic protozoan transmitter. The chapter I of the present study provides a literature review about neosporosis and toxoplasmosis, with emphasis on routes of transmission and the impact on canine reproduction, and the chapter II brings the investigation of the protozoa Neospora caninum and Toxoplasma gondii in semen samples of dogs naturally infected by cytological analysis, isolation in cell culture and polymerase chain reaction (PCR). In this study semen were collected in 11 clinically healthy dog (four dogs seropositive for N. caninum, five seropositive for T. gondii, two seropositive for both and two seronegative) to perform the isolation in vitro. The strains obtained in cell culture and the samples of nature semen was submitted to PCR amplification using primers that define the Nc5 DNA sequence of N. caninum (Np6/Np21) and the 529 bp repeated 200 to 300 times the genome of T. gondii (TOX4/TOX5). There was obtained two strains of N. caninum and five strains of T. gondii confirmed by PCR. Was obtained four amplification of N. caninum DNA in semen samples and five amplification of T. gondii DNA only in isolated strains. The parasites are present in the semen and the isolation and maintenance in cell culture have proven their viability. Keywords: isolation in vitro, semen, dog

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

Brazilian Flora 2020: Leveraging the power of a collaborative scientific network

International audienceThe shortage of reliable primary taxonomic data limits the description of biological taxa and the understanding of biodiversity patterns and processes, complicating biogeographical, ecological, and evolutionary studies. This deficit creates a significant taxonomic impediment to biodiversity research and conservation planning. The taxonomic impediment and the biodiversity crisis are widely recognized, highlighting the urgent need for reliable taxonomic data. Over the past decade, numerous countries worldwide have devoted considerable effort to Target 1 of the Global Strategy for Plant Conservation (GSPC), which called for the preparation of a working list of all known plant species by 2010 and an online world Flora by 2020. Brazil is a megadiverse country, home to more of the world's known plant species than any other country. Despite that, Flora Brasiliensis, concluded in 1906, was the last comprehensive treatment of the Brazilian flora. The lack of accurate estimates of the number of species of algae, fungi, and plants occurring in Brazil contributes to the prevailing taxonomic impediment and delays progress towards the GSPC targets. Over the past 12 years, a legion of taxonomists motivated to meet Target 1 of the GSPC, worked together to gather and integrate knowledge on the algal, plant, and fungal diversity of Brazil. Overall, a team of about 980 taxonomists joined efforts in a highly collaborative project that used cybertaxonomy to prepare an updated Flora of Brazil, showing the power of scientific collaboration to reach ambitious goals. This paper presents an overview of the Brazilian Flora 2020 and provides taxonomic and spatial updates on the algae, fungi, and plants found in one of the world's most biodiverse countries. We further identify collection gaps and summarize future goals that extend beyond 2020. Our results show that Brazil is home to 46,975 native species of algae, fungi, and plants, of which 19,669 are endemic to the country. The data compiled to date suggests that the Atlantic Rainforest might be the most diverse Brazilian domain for all plant groups except gymnosperms, which are most diverse in the Amazon. However, scientific knowledge of Brazilian diversity is still unequally distributed, with the Atlantic Rainforest and the Cerrado being the most intensively sampled and studied biomes in the country. In times of “scientific reductionism”, with botanical and mycological sciences suffering pervasive depreciation in recent decades, the first online Flora of Brazil 2020 significantly enhanced the quality and quantity of taxonomic data available for algae, fungi, and plants from Brazil. This project also made all the information freely available online, providing a firm foundation for future research and for the management, conservation, and sustainable use of the Brazilian funga and flora