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table.MsoNormalTable {mso-style-name:"Tabela normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman","serif";} --> <!--[endif] --> <p class="MsoNormal" style="text-align: justify; line-height: 150%; background: none repeat scroll 0% 0% white;"><span lang="EN-US">Many established protocols are available to produce antigens for ELISA tests but expensive equipments, the purification methods or recombinant technologies are not always available to veterinary diagnostic laboratories. The aim of this work was to develop an alternative ELISA test to detect antibodies against Lentivirus Small Ruminant Virus (Caprine arthritis-encephalitis virus-CAEV) in goats, simple to elaborate with a low cost of production. The antigen was obtain from a whole cellular lysates of goat synovial membrane cells (GSMC) infected with CAEV and treated with SDS 0,1%. The whole-viral antigen was capable of detecting antibodies against several viral proteins including the p28 kDa (viral capsid), the p33 kDa, the p44 kDa (capsid precursors) and p97 kDa (transmembrane glycoprotein). The comparison with others assays as indirect immunofluorescence showed a high correlation with the ELISA results and the polimerasse chain reaction (PCR) demonstrated that most of the goat seropositives by ELISA, had proviral DNA in the milk cells. In conclusion, the viral antigen obtained from GSMC was simple to elaborate, low cost of production. The ELISA test was capable of detecting antibodies against CAEV, to detect early infection.<span style="letter-spacing: -0.3pt;"></span></span></p>
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table.MsoNormalTable {mso-style-name:"Tabela normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman","serif";} --> <!--[endif] --> <p class="MsoNormal" style="text-align: justify; line-height: 150%; background: none repeat scroll 0% 0% white;"><span lang="EN-US">Many established protocols are available to produce antigens for ELISA tests but expensive equipments, the purification methods or recombinant technologies are not always available to veterinary diagnostic laboratories. The aim of this work was to develop an alternative ELISA test to detect antibodies against Lentivirus Small Ruminant Virus (Caprine arthritis-encephalitis virus-CAEV) in goats, simple to elaborate with a low cost of production. The antigen was obtain from a whole cellular lysates of goat synovial membrane cells (GSMC) infected with CAEV and treated with SDS 0,1%. The whole-viral antigen was capable of detecting antibodies against several viral proteins including the p28 kDa (viral capsid), the p33 kDa, the p44 kDa (capsid precursors) and p97 kDa (transmembrane glycoprotein). The comparison with others assays as indirect immunofluorescence showed a high correlation with the ELISA results and the polimerasse chain reaction (PCR) demonstrated that most of the goat seropositives by ELISA, had proviral DNA in the milk cells. In conclusion, the viral antigen obtained from GSMC was simple to elaborate, low cost of production. The ELISA test was capable of detecting antibodies against CAEV, to detect early infection.<span style="letter-spacing: -0.3pt;"></span></span></p>
ResumoO vírus da Artrite encefalite caprina é o agente causal de uma doença progressiva e debilitante em caprinos, em que as células da linhagem monócito/macrófago são as principais hospedeiras do vírus in vivo. Essas células estão presentes no colostro, no leite ou no sangue. Estudos demonstram que a replicação viral é dependente do nível de maturação ou diferenciação da célula monocítica, influenciando na sensibilidade de detecção do vírus. Neste estudo, utilizamos o cocultivo de monócitos/macrófagos com células de membrana sinovial de cabras, para o isolamento do vírus circulante no campo; a técnica de double nested PCR (dn PCR) dos co-cultivos e sangue, viabilizou a confirmação do isolamento viral, e a detecção direta do vírus no sangue. Através dessa técnica, detectou-se a presença do DNA proviral em animais soronegativos por Imunodifusão em Gel de Agarose (IDGA); esses achados confirmam que o tempo entre o inicio da infecção e o aparecimento de anticorpos no sangue é variável, facilitando a permanência de animais falsos-negativos no rebanho. Nas amostras processadas, tivemos uma divergência de resultados entre amostras sorologicamente positivas, entretanto, negativas por dn PCR, quando se utilizou a amplificação direta do sangue. Quanto ao cultivo de monócitos/ macrófagos in vitro e posterior co-cultivo com células de MSC obteve-se êxito pelo isolamento do vírus em quatro animais, havendo sido um deles soronegativo. O presente estudo demonstra o primeiro isolamento do vírus nos rebanhos do estado da Bahia, além da implementação da técnica de dn PCR, em co-cultivo de células.Palavras-chave: CAEV-Isolamento; monócito; PCR.
RESUMO: A Artrite Encefalite Caprina se caracteriza por ser multissistêmica e infecciosa, causada por um lentivírus. O estudo teve como objetivo avaliar a transmissibilidade do Lentivírus Caprino, para fêmeas e sua prole, por meio de sêmen infectado experimentalmente. Para tanto, onze fêmeas livres de CAEV foram inseminadas artificialmente com sêmen de bode livre de CAEV ao qual foi adicionado CAEV-Cork para obter título infectante com carga viral em 105 TCID50/ml. (grupo experimental 1). Destas, seis obtiverem prenhez confirmada, e a sua prole (n=6) constituiu o grupo experimental 2. Duas cabras livres de CAEV foram inseminadas artificialmente com sêmen do mesmo bode, sem o inócuo viral, constituindo-se o grupo controle. O diagnóstico da infecção pelo Lentivírus Caprino, foi realizado por IDGA, cELISA e nested-PCR. As fêmeas foram monitoradas durante 210 dias pós inseminação artificial. Já as proles foram imediatamente separadas das mães após o nascimento, e monitoradas nos momentos hora zero, aos quinze dias de idade e mensalmente, até doze meses de idade. Em relação às cabras, 56,96%(9/158) apresentaram positividade para cELISA, 24,05% (38/158) foram positivas a IDGA e nenhuma para nested-PCR. Em relação aos cabritos, 11,28% (15/133) amostras positivas para nested-PCR, 5,26% (7/133) amostras positivas para IDGA e nenhum para cELISA. As proles do grupo controle apresentaram resultados negativos para as três técnicas. A positividade encontrada em nested-PCR pode indicar grande importância para identificação de animais infectados, porém soronegativos, em situações de soroconversão tardia. De acordo com os resultados, concluiu-se que há a transmissão do Lentivírus caprino para a prole e para as mães pelo sêmen infectado.
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