PESQUISA VETERINÁRIA BRASILEIRA

Abstract in English:

Simionatto S., Lima-Rosa C.A.V., Rubin L.L. & Canal C.W. 2005. [A nested-PCR protocol for detection of the chicken anemia virus.] Um protocolo de “nested-PCR” para detecção do virus da anemia das galinhas. Pesquisa Veterinária Brasileira 25(2):106-110. Laboratório de Virologia, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9090, Porto Alegre, RS 91540-000, Brazil. E-mail: claudio.canal@ufrgs.br This paper reports a nested polymerase chain reaction (nested-PCR) protocol for detection of chicken anemia virus (CAV), the causal agent of infectious chicken anemia. For DNA extraction from clinical samples, a method based on guanidine thiocyanate was found more sensitive and practical than other extraction protocols tested. The pair of primers used in the initial PCR targeted a 664 bp fragment on the VP1 gene. The primers for the internal PCR targeted a fragment of 520 bp. The specificity of the primers was evaluated on samples of CAV controlled flocks. Thirty different viruses and bacteria isolated from chickens did not give rise to any amplification product in the assay. The sensitivity of the nested-PCR was determined on serial dilutions of a CAV vaccine. The nested-PCR was more sensitive than a one step PCR and was able to detect at least 0.16 TCID50 of the vaccine strain. In addition, the protocol employed here detected viral DNA from tissues, sera and litter from flocks with or without clinical signs of disease. It is concluded that the nested-PCR protocol described here is more sensitive, faster and less cumbersome than virus isolation in cell culture as a diagnostic technique for detection of CAV.

• Chicken anemia virus CAV molecular diagnosis nested-PCR.

Abstract in Portuguese:

Simionatto S., Lima-Rosa C.A.V., Rubin L.L. & Canal C.W. 2005. [A nested-PCR protocol for detection of the chicken anemia virus.] Um protocolo de “nested-PCR” para detecção do virus da anemia das galinhas. Pesquisa Veterinária Brasileira 25(2):106-110. Laboratório de Virologia, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9090, Porto Alegre, RS 91540-000, Brazil. E-mail: claudio.canal@ufrgs.br This paper reports a nested polymerase chain reaction (nested-PCR) protocol for detection of chicken anemia virus (CAV), the causal agent of infectious chicken anemia. For DNA extraction from clinical samples, a method based on guanidine thiocyanate was found more sensitive and practical than other extraction protocols tested. The pair of primers used in the initial PCR targeted a 664 bp fragment on the VP1 gene. The primers for the internal PCR targeted a fragment of 520 bp. The specificity of the primers was evaluated on samples of CAV controlled flocks. Thirty different viruses and bacteria isolated from chickens did not give rise to any amplification product in the assay. The sensitivity of the nested-PCR was determined on serial dilutions of a CAV vaccine. The nested-PCR was more sensitive than a one step PCR and was able to detect at least 0.16 TCID50 of the vaccine strain. In addition, the protocol employed here detected viral DNA from tissues, sera and litter from flocks with or without clinical signs of disease. It is concluded that the nested-PCR protocol described here is more sensitive, faster and less cumbersome than virus isolation in cell culture as a diagnostic technique for detection of CAV.

Abstract in English:

Simionatto S., Vaz E.K., Michelon A., Seixas F.K., Dellagostin O.A. 2005. [Development and evaluation of new strategies for immunization against swine colibacillosis.] Desenvolvimento e avaliação de novas estratégias de imunização contra colibacilose suína. Pes-quisa Veterinária Brasileira 25(2):84-90. Laboratório de Biologia Molecular, Centro de Bio-tecnologia, UFPel, Campus Capão do Leão, Cx. Postal 354, Pelotas, RS 96010-900, Brazil. E-mail: ssimionatto@bol.com.br Swine colibacillosis caused by enterotoxigenic Escherichia coli remains one of the main sanitary problems in pig farms. The recombinant DNA technology offers the possibility of developing new immunization strategies. This paper describes the development of a subunit vaccine through the expression and purification of the E. coli K88 FaeC fimbrial protein. The gene that codes for this antigen was amplified by PCR and cloned into an E. coli expression vector fused to a 6X histidine tag. The recombinant protein was purified by affinity chromatography and used for mice immunization. In parallel, the same gene was cloned into an eucariotic expression vector with the addition of the Kozak sequence for improving translation of this gene in muscle cells. The resulting plasmid named pUP310 was purified in large scale and used to immunize mice. The immune response afforded by both forms of immunization was monitored by ELISA. There was an immune response in mice inoculated with pUP310 and purified FaeC. It was possible to detect anti-FaeC antibodies 42 days after the first inoculation. The antibody titer increased with time, being still detectable 7 months after the first inoculation. It is concluded that recombinant FaeC and pUP310 are potential tools for immunization of swine against E. coli K88.

• Swine colibacillosis FaeC Escherichia coli DNA vaccine recombinant vaccine.

Abstract in Portuguese:

Simionatto S., Vaz E.K., Michelon A., Seixas F.K., Dellagostin O.A. 2005. [Development and evaluation of new strategies for immunization against swine colibacillosis.] Desenvolvimento e avaliação de novas estratégias de imunização contra colibacilose suína. Pes-quisa Veterinária Brasileira 25(2):84-90. Laboratório de Biologia Molecular, Centro de Bio-tecnologia, UFPel, Campus Capão do Leão, Cx. Postal 354, Pelotas, RS 96010-900, Brazil. E-mail: ssimionatto@bol.com.br Swine colibacillosis caused by enterotoxigenic Escherichia coli remains one of the main sanitary problems in pig farms. The recombinant DNA technology offers the possibility of developing new immunization strategies. This paper describes the development of a subunit vaccine through the expression and purification of the E. coli K88 FaeC fimbrial protein. The gene that codes for this antigen was amplified by PCR and cloned into an E. coli expression vector fused to a 6X histidine tag. The recombinant protein was purified by affinity chromatography and used for mice immunization. In parallel, the same gene was cloned into an eucariotic expression vector with the addition of the Kozak sequence for improving translation of this gene in muscle cells. The resulting plasmid named pUP310 was purified in large scale and used to immunize mice. The immune response afforded by both forms of immunization was monitored by ELISA. There was an immune response in mice inoculated with pUP310 and purified FaeC. It was possible to detect anti-FaeC antibodies 42 days after the first inoculation. The antibody titer increased with time, being still detectable 7 months after the first inoculation. It is concluded that recombinant FaeC and pUP310 are potential tools for immunization of swine against E. coli K88.