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Detection and decontamination of Mycoplasma in cultured cells and viral strains.

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Title: Detection and decontamination of Mycoplasma in cultured cells and viral strains.
Alternate Title: Detecção e descontaminação de Mycoplasma em cultivos celulares e cepas virais.
Authors: Prado de Andrade, Glaucia1,2, Hettwer Pedroso, Natália1,2, Becker, Alice1,2, Soares dos Santos, Vinicius2, Bonella, Juciane3, Joaquim Silva Júnior, José Valter2,4,5,6,7, Weiblen, Rudi2, Furtado Flores, Eduardo2 eduardofurtadoflores@gmail.com
Source: Ciência Rural. 2025, Vol. 55 Issue 4, p1-9. 9p.
Subject Terms: *BOVINE viral diarrhea virus, *PRIMARY cell culture, *BIOLOGICAL products, *PARAINFLUENZA viruses, *RESPIRATORY syncytial virus
Abstract (English): Contamination of biological products with Mycoplasma spp. represents a major problem for laboratories and vaccine industry. Herein, we investigated Mycoplasma spp. contamination in cell cultures and viral strains maintained at the Virology Section of the Universidade Federal de Santa Maria and evaluated drugs and protocols for decontamination. Among 25 cell lines and primary cultures tested by PCR, 16 (64%) were contaminated. Fourteen bovine viral strains were also found contaminated, including three bovine viral diarrhea virus 1 (BVDV-1), two BVDV-2, four bovine alphaherpesvirus 1 (BoHV-1), one each: HoBiPeV, BoHV-2, BoHV-5, bovine parainfluenza virus 3 (bPI-3V) and bovine respiratory syncytial virus (BRSV). Two drugs (Plasmocin® [P] and gentamicin [G]) were used individually or mixed for decontamination. Bovine (MDBK and CRIB cells), porcine (PK-15), rabbit (RK-13) and hamster (BHK-21) cell lineages were maintained in media containing the drugs and tested by PCR at weekly intervals. The combined P + G treatment was effective in eliminating Mycoplasma spp. from four cultures between days 35 and 42, followed by treatment with Plasmocin® (3/4) and gentamicin alone (1/4). The CRIB cell line could not be decontaminated. After, a decontamination protocol of viral seeds was carried out, consisting of amplification in cell culture, centrifugation of the viral suspension, filtration, limiting dilution and culture in mycoplasma-free cells. The fourteen viral strains were decontaminated after 1 to 4 cycles of the protocol. Overall, we detected a high frequency of Mycoplasma spp. contamination in cell cultures and viral strains and successfully applied drugs and protocols for decontamination. [ABSTRACT FROM AUTHOR]
Abstract (Portuguese): A contaminação de produtos biológicos com Mycoplasma spp. representa um grande problema para laboratórios e indústria de vacinas. Este trabalho investigou contaminação por Mycoplasma spp. em cultivos celulares e cepas virais do Setor de Virologia da Universidade Federal de Santa Maria e avaliou drogas e protocolos de descontaminação. Dentre 25 cultivos celulares testados por PCR, 16 (64%) estavam contaminados. Quatorze cepas virais também estavam contaminadas, incluindo três do vírus da diarreia viral bovina 1 (BVDV-1), dois BVDV-2, quatro do alfaherpesvírus bovino tipo 1 (BoHV-1) e um de HoBiPeV, BoHV-2, BoHV-5, vírus parainfluenza bovino 3 (bPI-3V) e vírus sincicial respiratório bovino (BRSV). Duas drogas (Plasmocin® [P] e gentamicina [G]) foram utilizadas individualmente ou combinadas para a descontaminação celular. Linhagens celulares bovinas (MDBK e CRIB), suínas (PK-15), de coelho (RK-13) e de hamster (BHK-21) foram mantidas em meio contendo as drogas e testadas por PCR a intervalos semanais. O tratamento combinado P + G foi eficaz na eliminação de Mycoplasma spp. de quatro cultivos entre os dias 35 e 42, seguido pelo tratamento com Plasmocin® (3/4) e gentamicina (1/4). A linhagem CRIB não pôde ser descontaminada. Posteriormente, um protocolo de descontaminação de cepas virais foi realizado, consistindo em amplificação em cultivo celular, centrifugação, filtração, diluição limitante e amplificação em células livres de Mycoplasma spp. As quatorze cepas virais foram descontaminadas após 1 a 4 ciclos do protocolo. Em conclusão, detectou-se uma alta frequência de contaminação por Mycoplasma spp. em cultivos celulares e cepas virais e aplicou-se com sucesso drogas e protocolos de descontaminação. [ABSTRACT FROM AUTHOR]
Copyright of Ciência Rural is the property of Ciencia Rural and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
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  Data: Detection and decontamination of Mycoplasma in cultured cells and viral strains.
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  Data: Detecção e descontaminação de Mycoplasma em cultivos celulares e cepas virais.
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  Data: <searchLink fieldCode="AR" term="%22Prado+de+Andrade%2C+Glaucia%22">Prado de Andrade, Glaucia</searchLink><relatesTo>1,2</relatesTo><br /><searchLink fieldCode="AR" term="%22Hettwer+Pedroso%2C+Natália%22">Hettwer Pedroso, Natália</searchLink><relatesTo>1,2</relatesTo><br /><searchLink fieldCode="AR" term="%22Becker%2C+Alice%22">Becker, Alice</searchLink><relatesTo>1,2</relatesTo><br /><searchLink fieldCode="AR" term="%22Soares+dos+Santos%2C+Vinicius%22">Soares dos Santos, Vinicius</searchLink><relatesTo>2</relatesTo><br /><searchLink fieldCode="AR" term="%22Bonella%2C+Juciane%22">Bonella, Juciane</searchLink><relatesTo>3</relatesTo><br /><searchLink fieldCode="AR" term="%22Joaquim+Silva+Júnior%2C+José+Valter%22">Joaquim Silva Júnior, José Valter</searchLink><relatesTo>2,4,5,6,7</relatesTo><br /><searchLink fieldCode="AR" term="%22Weiblen%2C+Rudi%22">Weiblen, Rudi</searchLink><relatesTo>2</relatesTo><br /><searchLink fieldCode="AR" term="%22Furtado+Flores%2C+Eduardo%22">Furtado Flores, Eduardo</searchLink><relatesTo>2</relatesTo><i> eduardofurtadoflores@gmail.com</i>
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  Data: <searchLink fieldCode="JN" term="%22Ciência+Rural%22">Ciência Rural</searchLink>. 2025, Vol. 55 Issue 4, p1-9. 9p.
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  Data: *<searchLink fieldCode="DE" term="%22BOVINE+viral+diarrhea+virus%22">BOVINE viral diarrhea virus</searchLink><br />*<searchLink fieldCode="DE" term="%22PRIMARY+cell+culture%22">PRIMARY cell culture</searchLink><br />*<searchLink fieldCode="DE" term="%22BIOLOGICAL+products%22">BIOLOGICAL products</searchLink><br />*<searchLink fieldCode="DE" term="%22PARAINFLUENZA+viruses%22">PARAINFLUENZA viruses</searchLink><br />*<searchLink fieldCode="DE" term="%22RESPIRATORY+syncytial+virus%22">RESPIRATORY syncytial virus</searchLink>
– Name: Abstract
  Label: Abstract (English)
  Group: Ab
  Data: Contamination of biological products with Mycoplasma spp. represents a major problem for laboratories and vaccine industry. Herein, we investigated Mycoplasma spp. contamination in cell cultures and viral strains maintained at the Virology Section of the Universidade Federal de Santa Maria and evaluated drugs and protocols for decontamination. Among 25 cell lines and primary cultures tested by PCR, 16 (64%) were contaminated. Fourteen bovine viral strains were also found contaminated, including three bovine viral diarrhea virus 1 (BVDV-1), two BVDV-2, four bovine alphaherpesvirus 1 (BoHV-1), one each: HoBiPeV, BoHV-2, BoHV-5, bovine parainfluenza virus 3 (bPI-3V) and bovine respiratory syncytial virus (BRSV). Two drugs (Plasmocin® [P] and gentamicin [G]) were used individually or mixed for decontamination. Bovine (MDBK and CRIB cells), porcine (PK-15), rabbit (RK-13) and hamster (BHK-21) cell lineages were maintained in media containing the drugs and tested by PCR at weekly intervals. The combined P + G treatment was effective in eliminating Mycoplasma spp. from four cultures between days 35 and 42, followed by treatment with Plasmocin® (3/4) and gentamicin alone (1/4). The CRIB cell line could not be decontaminated. After, a decontamination protocol of viral seeds was carried out, consisting of amplification in cell culture, centrifugation of the viral suspension, filtration, limiting dilution and culture in mycoplasma-free cells. The fourteen viral strains were decontaminated after 1 to 4 cycles of the protocol. Overall, we detected a high frequency of Mycoplasma spp. contamination in cell cultures and viral strains and successfully applied drugs and protocols for decontamination. [ABSTRACT FROM AUTHOR]
– Name: Abstract
  Label: Abstract (Portuguese)
  Group: Ab
  Data: A contaminação de produtos biológicos com Mycoplasma spp. representa um grande problema para laboratórios e indústria de vacinas. Este trabalho investigou contaminação por Mycoplasma spp. em cultivos celulares e cepas virais do Setor de Virologia da Universidade Federal de Santa Maria e avaliou drogas e protocolos de descontaminação. Dentre 25 cultivos celulares testados por PCR, 16 (64%) estavam contaminados. Quatorze cepas virais também estavam contaminadas, incluindo três do vírus da diarreia viral bovina 1 (BVDV-1), dois BVDV-2, quatro do alfaherpesvírus bovino tipo 1 (BoHV-1) e um de HoBiPeV, BoHV-2, BoHV-5, vírus parainfluenza bovino 3 (bPI-3V) e vírus sincicial respiratório bovino (BRSV). Duas drogas (Plasmocin® [P] e gentamicina [G]) foram utilizadas individualmente ou combinadas para a descontaminação celular. Linhagens celulares bovinas (MDBK e CRIB), suínas (PK-15), de coelho (RK-13) e de hamster (BHK-21) foram mantidas em meio contendo as drogas e testadas por PCR a intervalos semanais. O tratamento combinado P + G foi eficaz na eliminação de Mycoplasma spp. de quatro cultivos entre os dias 35 e 42, seguido pelo tratamento com Plasmocin® (3/4) e gentamicina (1/4). A linhagem CRIB não pôde ser descontaminada. Posteriormente, um protocolo de descontaminação de cepas virais foi realizado, consistindo em amplificação em cultivo celular, centrifugação, filtração, diluição limitante e amplificação em células livres de Mycoplasma spp. As quatorze cepas virais foram descontaminadas após 1 a 4 ciclos do protocolo. Em conclusão, detectou-se uma alta frequência de contaminação por Mycoplasma spp. em cultivos celulares e cepas virais e aplicou-se com sucesso drogas e protocolos de descontaminação. [ABSTRACT FROM AUTHOR]
– Name: AbstractSuppliedCopyright
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  Data: <i>Copyright of Ciência Rural is the property of Ciencia Rural and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.)
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        Value: 10.1590/0103-8478cr20240168
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