Quality Control of Biotechnological Inputs Detecting Mycoplasma

The aim of this work was to study the Polymerase Chain Reaction (PCR) as a tool of quality control of bovine sera and cellular cultures used in the biotechnological industry. A total of 46 samples of bovine sera derived from two slaughterhouses and 33 samples of BHK21 cells derived from two biotechnological industries were evaluated using the primers GPO-3 (sense) and MGSO (antisense). The PCR technique sensibility analysis showed that 280 bp were amplified for the quantities of 50 ng to 0.006 ng of Micoplasma DNA. The primers specificity was confirmed in the test using Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Candida albicans; except by the positive control, none of the samples showed amplification. The presence of Mycoplasma in bovine sera and in the cultures of BHK21 cells showed that 56.5 and 15.2%, respectively, were contaminated. Thus, it was possible to conclude that PCR was a fast and confident technique to detect mycoplasma and that it could be used to control the quality of immunobiological products and inputs, such as sera and cultures of BHK21 cells.

Experimental infection of BHK21 and Vero cell lines with different Mycoplasma spp.

Mycoplasma spp, belongs to the class Mollicutes and is capable to produce alterations in cellular cultures causing damages to the biotechnological industry. Bioproducts generally require two essential inputs, bovine serum and cells. The study herein aims to evaluate the mycoplasma concentrations that affect the growing of BHK21 and Vero cells. The species used were: Mycoplasma orale, M. salivarium, M. arginini and M. hyorhinis, cultivated in a SP4 media. Two contamination tests were performed with BHK21 and Vero cells and one of them applied different concentrations of mycoplasma. In the first one, mycoplasma was applied at the day zero and, in the second one, the contamination was performed after the monolayer establishment. The both cellular cultures presented cytopathic effects with mycoplasma contamination, but the Vero cells suffered more damages than the BHK21 ones. It was also observed that the severity of the cytopathic effect depended on the mycoplasma specie, on the concentration and on the time of contact with the cellular culture, which evidences the importance of controlling the presence of mycoplasma in biotechnological industries.

Experimental infection of BHK21 and Vero cell lines with different Mycoplasma spp

Mycoplasma spp, belongs to the class Mollicutes and is capable to produce alterations in cellular cultures causing damages to the biotechnological industry. Bioproducts generally require two essential inputs, bovine serum and cells. The study herein aims to evaluate the mycoplasma concentrations that affect the growing of BHK21 and Vero cells. The species used were: Mycoplasma orale, M. salivarium, M. arginini and M. hyorhinis, cultivated in a SP4 media. Two contamination tests were performed with BHK21 and Vero cells and one of them applied different concentrations of mycoplasma. In the first one, mycoplasma was applied at the day zero and, in the second one, the contamination was performed after the monolayer establishment. The both cellular cultures presented cytopathic effects with mycoplasma contamination, but the Vero cells suffered more damages than the BHK21 ones. It was also observed that the severity of the cytopathic effect depended on the mycoplasma specie, on the concentration and on the time of contact with the cellular culture, which evidences the importance of controlling the presence of mycoplasma in biotechnological industries.

Experimental infection of BHK21 and Vero cell lines with different Mycoplasma spp.

Mycoplasma spp, belongs to the class Mollicutes and is capable to produce alterations in cellular cultures causing damages to the biotechnological industry. Bioproducts generally require two essential inputs, bovine serum and cells. The study herein aims to evaluate the mycoplasma concentrations that affect the growing of BHK21 and Vero cells. The species used were: Mycoplasma orale, M. salivarium, M. arginini and M. hyorhinis, cultivated in a SP4 media. Two contamination tests were performed with BHK21 and Vero cells and one of them applied different concentrations of mycoplasma. In the first one, mycoplasma was applied at the day zero and, in the second one, the contamination was performed after the monolayer establishment. The both cellular cultures presented cytopathic effects with mycoplasma contamination, but the Vero cells suffered more damages than the BHK21 ones. It was also observed that the severity of the cytopathic effect depended on the mycoplasma specie, on the concentration and on the time of contact with the cellular culture, which evidences the importance of controlling the presence of mycoplasma in biotechnological industries.

Rapid diagnosis of vesicular stomatitis virus in Ecuador by the use of polymerase chain reaction

Vesicular Stomatitis (VS) is a viral disease that has a great impact in animal health, as infected animals present marked decrease in meat and milk production. Its presence is a limiting factor for international animal trade. Besides the damage in the livestock productivity, such disease assumes an important role in animal health programs since it is clinically indistinguishable from Foot-and-Mouth Disease. The diagnosis of the VS has been made, mainly, through Complement Fixation, ELISA and Virus Neutralization tests, assays that allow not only for viral detection but also for differentiation of the two serotypes described for Vesicular Stomatitis Virus (VSV): New Jersey (NJ) and Indiana (Ind). In this work, a molecular diagnostic approach, the polymerase chain reaction performed after reverse transcription (RT - PCR), based on the specific partial amplification of NS gene of VSV was used, as an alternative method for the detection of the virus. A total of 10 VSV reference samples and 12 specimens collected from animals with clinical signs of vesicular disease obtained from field episodes in Ecuador were tested. The method allowed for the specific partial amplification of the region coding for protein P, both for VSV serotypes New Jersey (642 bp) and Indiana 1 (614 bp). The results were compatible with data obtained by Complement Fixation test and the identity of the amplified products was confirmed by nucleotide sequencing.

A Estomatite Vesicular (EV) é uma enfermidade viral de grande impacto na saúde animal. O animal enfermo apresenta queda na produtividade em rebanho de carne e na produção leiteira, sendo um fator limitante para o comércio internacional de animais. Além dos danos à produtividade essa enfermidade assume importante papel nos programas de saúde animal por ser indistinguível clinicamente da Febre Aftosa. As técnicas para o diagnóstico da EV são, principalmente, a Fixação de Complemento, a ELISA e a Virusneutralização, testes que permitem a detecção viral e a diferenciação dos dois sorotipos descritos para o vírus da Estomatite Vesicular (VEV): New Jersey (NJ) e Indiana (Ind). Neste trabalho a metodologia molecular da reação em cadeia da polimerase após transcrição reversa (RT - PCR) baseada na amplificação parcial específica do gene NS do VEV foi utilizada como um método alternativo para a detecção do vírus. Um total de 10 amostras de referência do VEV e 12 espécimes coletados de animais com sinais clínicos de enfermidade vesicular obtidas de episódios de campo em Equador foi testado. O método permitiu a amplificação parcial da região que codifica para proteína P, tanto para NJ (642 pb) quanto para Ind (614 pb). Os resultados foram concordantes com os dados obtidos por Fixação de Complemento e a identidade dos produtos amplificados foi confirmada por meio de seqüenciamento nucleotídico.

Rapid diagnosis of vesicular stomatitis virus in Ecuador by the use of polymerase chain reaction

Vesicular Stomatitis (VS) is a viral disease that has a great impact in animal health, as infected animals present marked decrease in meat and milk production. Its presence is a limiting factor for international animal trade. Besides the damage in the livestock productivity, such disease assumes an important role in animal health programs since it is clinically indistinguishable from Foot-and-Mouth Disease. The diagnosis of the VS has been made, mainly, through Complement Fixation, ELISA and Virus Neutralization tests, assays that allow not only for viral detection but also for differentiation of the two serotypes described for Vesicular Stomatitis Virus (VSV): New Jersey (NJ) and Indiana (Ind). In this work, a molecular diagnostic approach, the polymerase chain reaction performed after reverse transcription (RT - PCR), based on the specific partial amplification of NS gene of VSV was used, as an alternative method for the detection of the virus. A total of 10 VSV reference samples and 12 specimens collected from animals with clinical signs of vesicular disease obtained from field episodes in Ecuador were tested. The method allowed for the specific partial amplification of the region coding for protein P, both for VSV serotypes New Jersey (642 bp) and Indiana 1 (614 bp). The results were compatible with data obtained by Complement Fixation test and the identity of the amplified products was confirmed by nucleotide sequencing.

A Estomatite Vesicular (EV) é uma enfermidade viral de grande impacto na saúde animal. O animal enfermo apresenta queda na produtividade em rebanho de carne e na produção leiteira, sendo um fator limitante para o comércio internacional de animais. Além dos danos à produtividade essa enfermidade assume importante papel nos programas de saúde animal por ser indistinguível clinicamente da Febre Aftosa. As técnicas para o diagnóstico da EV são, principalmente, a Fixação de Complemento, a ELISA e a Virusneutralização, testes que permitem a detecção viral e a diferenciação dos dois sorotipos descritos para o vírus da Estomatite Vesicular (VEV): New Jersey (NJ) e Indiana (Ind). Neste trabalho a metodologia molecular da reação em cadeia da polimerase após transcrição reversa (RT - PCR) baseada na amplificação parcial específica do gene NS do VEV foi utilizada como um método alternativo para a detecção do vírus. Um total de 10 amostras de referência do VEV e 12 espécimes coletados de animais com sinais clínicos de enfermidade vesicular obtidas de episódios de campo em Equador foi testado. O método permitiu a amplificação parcial da região que codifica para proteína P, tanto para NJ (642 pb) quanto para Ind (614 pb). Os resultados foram concordantes com os dados obtidos por Fixação de Complemento e a identidade dos produtos amplificados foi confirmada por meio de seqüenciamento nucleotídico.