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Return to: Scandals: On "mad cows" and sick monkeys: From the people who brought you SV40 in vaccines....


Click here for More links re: bovine contamination


J Microbiol. 2006 Feb;44(1):42-9. Related Articles, Links
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PCR-based detection of mycoplasma species.

Sung H, Kang SH, Bae YJ, Hong JT, Chung YB, Lee CK, Song S.

College of Pharmacy, Chungbuk National University, 12 Gaeshindong, Cheongju, Chungbuk, Republic of Korea.

In this study, we describe our newly-developed sensitive two-stage PCR procedure for the detection of 13 common mycoplasmal contaminants (M. arthritidis, M. bovis, M. fermentans, M. genitalium, M. hominis, M. hyorhinis, M. neurolyticum, M. orale, M. pirum, M. pneumoniae, M. pulmonis, M. salivarium, U. urealyticum). For primary amplification, the DNA regions encompassing the 16S and 23S rRNA genes of 13 species were targeted using general mycoplasma primers. The primary PCR products were then subjected to secondary nested PCR, using two different primer pair sets, designed via the multiple alignment of nucleotide sequences obtained from the 13 mycoplasmal species. The nested PCR, which generated DNA fragments of 165-353 bp, was found to be able to detect 1-2 copies of the target DNA, and evidenced no cross-reactivity with the genomic DNA of related microorganisms or of human cell lines, thereby confirming the sensitivity and specificity of the primers used. The identification of contaminated species was achieved via the performance of restriction fragment length polymorphism (RFLP) coupled with Sau3AI digestion. The results obtained in this study furnish evidence suggesting that the employed assay system constitutes an effective tool for the diagnosis of mycoplasmal contamination in cell culture systems.

PMID: 16554716 [PubMed - in process]


Biologicals. 2005 Jun;33(2):81-5. Epub 2005 Apr 26. Related Articles, Links
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Microbial contamination of cell cultures: a 2 years study.

Mirjalili A, Parmoor E, Moradi Bidhendi S, Sarkari B.

Cell and Gene Bank, Biotechnology Department of Razi Vaccine and Serum Research Institute (CGBRI), Hesarrak, Karaj, Iran.

Cell line contamination is a major drawback of main cell banks of the world and it has cost of losing important biological products or valuable research. The causative agents are different chemicals, invertebrates, bacteria, fungi, parasites, viral species and even other cell lines. In this retrospective study, cell lines from various species such as human, fish, insect, animals either offered or accessed through usual official accession in CGBRI were studied during 2 years (2002-2004) to detect their microbial contaminations and the causative organisms. Samples were taken for sterility test upon cell lines receipt and upon each cell line sub-culture. Samples were examined for bacterial (including mycoplasmas) and fungal contamination using conventional microbiological techniques. The study excluded parasites, viruses and other contaminating agents. This study revealed 39% of specimens were contaminated. The major contaminating agents were mycoplasmas (19%) followed by mixed infection (8%), fungi (8%) and bacteria (4%). Among various bacterial species (except mycoplasmas) Bacillus sp., Enterococcus sp. and Staphylococcus sp. are main bacterial agents and among various fungi Aspergillus sp. followed by Penicillium sp., Sepedonium sp. and Botrytis sp. were main fungal causative agents of CGBRI cell line contamination. Our study also delineates each cell line contamination rate and its causative agents. This is the first report of cell culture contamination from cell banks of Middle-East countries like Iran.

PMID: 15939285 [PubMed - indexed for MEDLINE]

Appl Microbiol Biotechnol. 2005 Sep;68(4):456-66. Epub 2005 Oct 26. Related Articles, Links
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Microbiological control in stem cell banks: approaches to standardisation.

Cobo F, Stacey GN, Hunt C, Cabrera C, Nieto A, Montes R, Cortes JL, Catalina P, Barnie A, Concha A.

Stem Cell Bank of Andalucia (Spanish Central Node), Hospital Universitario Virgen de las Nieves, Avda Fuerzas Armadas, 2, 18014, Granada, Spain.

The transplant of cells of human origin is an increasingly complex sector of medicine which entails great opportunities for the treatment of a range of diseases. Stem cell banks should assure the quality, traceability and safety of cultures for transplantation and must implement an effective programme to prevent contamination of the final product. In donors, the presence of infectious micro-organisms, like human immunodeficiency virus, hepatitis B virus, hepatitis C virus and human T cell lymphotrophic virus, should be evaluated in addition to the possibility of other new infectious agents (e.g. transmissible spongiform encephalopathies and severe acute respiratory syndrome). The introduction of the nucleic acid amplification can avoid the window period of these viral infections. Contamination from the laboratory environment can be achieved by routine screening for bacteria, fungi, yeast and mycoplasma by European pharmacopoeia tests. Fastidious micro-organisms, and an adventitious or endogenous virus, is a well-known fact that will also have to be considered for processes involving in vitro culture of stem cells. It is also a standard part of current good practice in stem cell banks to carry out routine environmental microbiological monitoring of the cleanrooms where the cell cultures and their products are prepared. The risk of viral contamination from products of animal origin, like bovine serum and mouse fibroblasts as a "feeder layer" for the development of embryonic cell lines, should also be considered. Stem cell lines should be tested for prion particles and a virus of animal origin that assure an acceptable quality.

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PMID: 16012832 [PubMed - indexed for MEDLINE]


In Vitro Cell Dev Biol Anim. 2005 Mar-Apr;41(3-4):65-70. Related Articles, Links
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Review: production, characterization, and testing of banked mammalian cell substrates used to produce biological products.

Schiff LJ.

Biopharmaceutical Services, Charles River Laboratories, Ijamsville, Maryland 21754, USA.

A critical component in controlling the production of biological products derived from human and animal cell lines is the characterization and testing of banked cell substrates. The objective is to confirm the identity, purity, and suitability of these cells for manufacturing use. Quality concerns for biological products derived from cell lines originate from the presence of cellular and adventitious contaminants. Well-characterized cell banks not only permit a consistent source of production cells throughout the life of a product but also decrease the likelihood of contamination by other cell lines and adventitious agents. An important part of qualifying a cell line is choosing the appropriate testing for the presence of adventitious contaminants. The qualification of cell banks includes tests for cell identity and endogenous and adventitious microbial contaminants (bacteria, fungi, mycoplasmas, and viruses). For cells producing recombinant deoxyribonucleic acid-derived products, analysis of the expression construct at the nucleic acid level (genetic stability) is also a primary concern. The strategy for designing a safety-testing program for banked cells should be based on sound scientific principles and current regulatory guidance.

Publication Types:

PMID: 16029074 [PubMed - indexed for MEDLINE]

Methods Mol Biol. 2005;290:13-23. Related Articles, Links
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Detection of mycoplasma contaminations.

Uphoff CC, Drexler HG.

DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany.

Mycoplasma contamination of cell lines is one of the major problems in cell culture technology. The specific, sensitive, and reliable detection of mycoplasma contamination is an important part of mycoplasma control and should be an established method in every cell culture laboratory. New cell lines as well as cell lines in continuous culture must be tested in regular intervals. The polymerase chain reaction (PCR) methodology offers a fast and sensitive technique to monitor all cultures in a laboratory. The technique can also be used to determine the contaminating mycoplasma species.The described assay can be performed within 3 h, including sample preparation, DNA extraction, performing the PCR reaction, and analysis of the PCR products. Special precautions necessary to avoid false-negative results resulting from inhibitors of the Taq polymerase present in the crude samples and the interpretation of the results are also described.

PMID: 15361652 [PubMed - indexed for MEDLINE]

Microbiol Immunol. 2005;49(9):859-63. Related Articles, Links
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Rapid detection and differentiation of the major mycoplasma contaminants in cell cultures using real-time PCR with SYBR Green I and melting curve analysis.

Harasawa R, Mizusawa H, Fujii M, Yamamoto J, Mukai H, Uemori T, Asada K, Kato I.

Veterinary Microbiology, Department of Veterinary Medicine, Iwate University, Morioka, Iwate 020-8550, Japan.

A quantitative real-time polymerase chain reaction (PCR) procedure followed by melting curve analysis, using the green fluorescence dye SYBR Green I, was developed for rapid detection and differentiation of mycoplasma contaminants in cell cultures. This method showed that the detection of the target sequence was linear over a range from 10(4) to 10 colony-forming units (CFU) of the mycoplasma cells. Analysis of the melting temperature of the PCR products allowed differentiation of the major mycoplasma contaminants. These results demonstrate that the protocol described in the present study can decrease the time to obtain reproducible results by simultaneous detection and differentiation of the Mycoplasma species contaminating cell cultures.

Publication Types:

PMID: 16172541 [PubMed - indexed for MEDLINE]


Appl Environ Microbiol. 2004 Mar;70(3):1483-6. Related Articles, Links
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Simultaneous detection and identification of common cell culture contaminant and pathogenic mollicutes strains by reverse line blot hybridization.

Wang H, Kong F, Jelfs P, James G, Gilbert GL.

Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research, Westmead, New South Wales, Australia.

We have developed a reverse line blot (RLB) hybridization assay to detect and identify the commonest mollicutes causing cell line contamination (Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale, and Acholeplasma laidlawii) and human infection (Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma parvum, and Ureaplasma urealyticum). We developed a nested PCR assay with "universal" primers targeting the mollicute 16S-23S rRNA intergenic spacer region. Amplified biotin-labeled PCR products were hybridized to membrane-bound species-specific oligonucleotide probes. The assay correctly identified reference strains of 10 mollicute species. Cell cultures submitted for detection of mollicute contamination, clinical specimens, and clinical isolates were initially tested by PCR assay targeting a presumed mollicute-specific sequence of the 16S rRNA gene. Any that were positive were assessed by the RLB assay, with species-specific PCR assay as the reference method. Initially, 100 clinical and 88 of 92 cell culture specimens gave concordant results, including 18 in which two or more mollicute species were detected by both methods. PCR and sequencing of the 16S-23S rRNA intergenic spacer region and subsequent retesting by species-specific PCR assay of the four cell culture specimens for which results were initially discrepant confirmed the original RLB results. Sequencing of amplicons from 12 cell culture specimens that were positive in the 16S rRNA PCR assay but negative by both the RLB and species-specific PCR assays failed to identify any mollicute species. The RLB hybridization assay is sensitive and specific and able to rapidly detect and identify mollicute species from clinical and cell line specimens.

PMID: 15006769 [PubMed - indexed for MEDLINE]

Methods Mol Med. 2004;88:309-17. Related Articles, Links
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Cell culture contamination: an overview.

Langdon SP.

Cancer Research UK Oncology Unit, Western General Hospital, Edinburgh, UK.
For the cell culturist, two types of contamination require careful monitoring and constant vigilance: the contamination of cell cultures with microbiological organisms and the contamination of one cell line with another. Both forms of contamination are extremely prevalent and cannot be underestimated. Neither type can be eliminated, only controlled and managed to minimize the possibility of occurrence. Contamination consequences can range from minor inconvenience (a flask of cells becoming contaminated with bacteria) to a major disaster (published results that may be invalid owing to cross-contamination of one cell line with another). Other types of contaminants, such as chemical contamination, may also cause problems (e.g., deposits of disinfectants or detergents on glassware; residues, impurities, and toxins in water, media or sera), but the common recurring problems are likely to be biological in origin.
Comment:  Is the reason there is no mention of possible disease transmission that it is assumed that such contamination cannot cause disease, or has it been thoroughly tested and been shown to (almost) never occur?  (Examples of cross-species disease transmission, and in particular that of SV40, suggest that the former may be the case.)

PMID: 14634243 [PubMed - in process]

Nat Med. 2004 Dec;10(12 Suppl):S70-6. Related Articles, Links
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Social and environmental risk factors in the emergence of infectious diseases.

Weiss RA, McMichael AJ.

Wohl Virion Centre, Division of Infection and Immunity, University College London, W1T 4JF, UK.

Fifty years ago, the age-old scourge of infectious disease was receding in the developed world in response to improved public health measures, while the advent of antibiotics, better vaccines, insecticides and improved surveillance held the promise of eradicating residual problems. By the late twentieth century, however, an increase in the emergence and re-emergence of infectious diseases was evident in many parts of the world. This upturn looms as the fourth major transition in human-microbe relationships since the advent of agriculture around 10,000 years ago. About 30 new diseases have been identified, including Legionnaires' disease, human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS), hepatitis C, bovine spongiform encephalopathy (BSE)/variant Creutzfeldt-Jakob disease (vCJD), Nipah virus, several viral hemorrhagic fevers and, most recently, severe acute respiratory syndrome (SARS) and avian influenza. The emergence of these diseases, and resurgence of old ones like tuberculosis and cholera, reflects various changes in human ecology: rural-to-urban migration resulting in high-density peri-urban slums; increasing long-distance mobility and trade; the social disruption of war and conflict; changes in personal behavior; and, increasingly, human-induced global changes, including widespread forest clearance and climate change. Political ignorance, denial and obduracy (as with HIV/AIDS) further compound the risks. The use and misuse of medical technology also pose risks, such as drug-resistant microbes and contaminated equipment or biological medicines. A better understanding of the evolving social dynamics of emerging infectious diseases ought to help us to anticipate and hopefully ameliorate current and future risks.

PMID: 15577934 [PubMed - indexed for MEDLINE]
Vet Microbiol. 2004 Sep 8;102(3-4):131-40. Related Articles, Links
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Comparison of the sensitivity of in vitro and in vivo tests for detection of the presence of a bovine viral diarrhoea virus type 1 strain.

Antonis AF, Bouma A, de Bree J, de Jong MC.

Division of Infectious Diseases and Food Chain Quality, Animal Sciences Group, Wageningen University and Research Centre (WUR), P.O. Box 65, Lelystad AB8200, The Netherlands.

Veterinary vaccines are usually tested for the absence of contaminants. However, the quality control does not always imply that vaccines are not contaminated as, for example, illustrated by the bovine herpes virus 1 (BHV1) vaccine used in The Netherlands in 1999 that contained a small amount of bovine viral diarrhoea virus (BVDV1). Thousands of cows were vaccinated with BHV1 vaccine batches, and the question arose as to whether these small amounts of BVDV1, most likely not detected with in vitro tests, could have infected cattle. More in general, the question was whether the outcome of the in vitro tests, i.e. the in vitro infectivity, was indicative for the infectivity for cattle, i.e. the in vivo infectivity. We therefore carried out in vitro experiments to determine the sensitivity of a BVDV1 isolation assay. In addition, we performed two animal experiments, in which we estimated the lowest dose needed to infect calves with BVDV1. We extrapolated the experimental in vitro and in vivo results from a tissue culture infectious dose (TCID50) to a cattle infectious dose (CID50). We observed a partial response in the calves inoculated with this dose: four out of six calves turned out to be infected. In the tissue culture test, all 20 samples tested negative. The response in vivo, however, was not significantly higher than the in vitro response, which implies that no difference in susceptibility was observed between the animal test and the tissue culture test. Based on the results in our experiments, some cattle may have been infected with BVDV1 after the application of the contaminated BHV1 vaccine during the vaccination campaign. The question remains that how many cattle received contaminated vaccine, and became infected with BVDV1.

PMID: 15327789 [PubMed - indexed for MEDLINE]
Vet Rec. 2004 Oct 30;155(18):563-4. Related Articles, Links
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Assessment of the risk of transmission of vaccine viruses by using insufficiently cleaned injection devices.

Makoschey B, Beer M.

Virological R & D Department, Intervet International, Wim de Korverstraat 35, NL-5831 AN Boxmeer, The Netherlands.

PMID: 15559423 [PubMed - indexed for MEDLINE]



Immunol Lett. 2004 Apr 15;92(3):215-6. Related Articles, Links
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Rapid test for early detection of mycoplasma contamination of the continuous cell line J774.2.

Loudova M, Novosad J.

Department of Clinical Immunology and Allergology, Faculty of Medicine in Hradec Kralove, University Hospital, Charles University in Prague, Sokolska tr. 408, 500 05 Hradec Kralove, Czech Republic.

The authors describe a rapid, simple and inexpensive method for the routine testing of mycoplasma contamination of the continuous mouse macrophage-like cell line J774.2 using specific anti-mouse monoclonal antibodies (antiCD14, antiCD80) and flow cytometry.

PMID: 15081614 [PubMed - indexed for MEDLINE]
Sci Cult (Lond). 2004 Mar;13(1):75-88. Related Articles, Links

Intersecting discourses: MMR vaccine and BSE.

Wilson C.

School of Humanities, Faculty of Arts, Education, and Social Sciences, University of Western Sydney, Locked Bag 1797, South Penrith Distribution Centre, NSW 1797, Australia.

PMID: 15971371 [PubMed - indexed for MEDLINE]
Biologicals. 2003 Dec;31(4):303-6. Related Articles, Links
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Detection of infectious Bovine polyomavirus.

Nairn C, Lovatt A, Galbraith DN.

Q-One Biotech Limited, Todd Campus, West of Scotland Science Park, Glasgow, G20 0XA, UK.

Bovine polyomavirus (BPyV) is a member of the Polyomaviridae, a virus that was originally thought to be of simian origin but was later shown to be of bovine origin, the primate cultures having been contaminated through the use of foetal bovine serum. The significance of this agent to the biotechnology industry cannot be underestimated. The presence of BPyV in serum batches poses a serious risk for the contamination of human therapeutic products. The current PCR based assays provide a means of detecting virus sequences but give no indication as to the infectious nature of the virus. The communication reports the successful development of an assay to detect infectious BPyV using an in vitro amplification system followed by PCR. A lengthy culture period on bovine cells was required before replicating BPyV could be detected and distinguished from non-replicating virus in the cell culture supernatant. A mock-test assay using foetal bovine serum positive for BPyV showed that there was no evidence of replicating BPyV in the serum sample. The BPyV spiked serum control showed that replicating virus was present thus confirming that the serum itself did not inhibit replication of the virus. Cells harvested during the culture period were subjected to fixation, embedding and sectioning and examined by electron microscopy. Intact virus-like particles of approximately 40-50nm were observed in the nucleus of the bovine kidney cells, the site of polyomavirus replication.

PMID: 14624801 [PubMed - in process]

Biologicals. 2003 Sep;31(3):203-8. Related Articles, Links
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Bovine viral diarrhoea virus antigen in foetal calf serum batches and consequences of such contamination for vaccine production.

Makoschey B, van Gelder PT, Keijsers V, Goovaerts D.

Virological R&D Department, Intervet International b.v., Wim de Korverstraat 35, NL-5831 AN, Boxmeer, The Netherlands.

A protocol to test foetal calf serum (FCS) for contamination with bovine viral diarrhoea virus (BVDV) is described. Following this protocol, which combines cell culture methods and detection of pestivirus RNA, seven batches of FCS were tested. Infectious BVDV was detected in four of those batches. One of the remaining batches contained a relatively high number of non-infectious BVDV particles. A sample of this batch was formulated with aluminium hydroxide and aluminium phosphate as adjuvant into an experimental vaccine preparation. This product was injected twice into BVDV seronegative cattle with a 4 week interval. Blood samples taken 4 weeks after the second application were negative for BVDV specific antibodies. Our data stress that detection of BVDV RNA is not sufficient for a complete risk assessment on FCS. Discrimination between infectious and non-infectious BVDV is essential. This can only be achieved by cell culture methods.

PMID: 12935809 [PubMed - in process]

Chang Gung Med J. 2003 Apr;26(4):250-8. Related Articles, Links

Detection and treatment of mycoplasma contamination in cultured cells.

Jung H, Wang SY, Yang IW, Hsueh DW, Yang WJ, Wang TH, Wang HS.

Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital, Taipei, ROC.

BACKGROUND: Mycoplasmas, the smallest and simplest prokaryotes that reside in endosomes of mammalian cells, are widespread contaminants found in cell cultures. About 30% of all cell cultures, varying from 15 to 80%, are reportedly contaminated with mycoplasmas. Here, we present our experience in successfully detecting and treating mycoplasmal infection in various cell lines. METHODS: The nested polymerase chain reaction (PCR) detection and microscopic examination, including phase-contrast, fluorescent, as well as differential interference contrast, were used for detecting potential mycoplasma contamination of cell lines used in our laboratory. As soon as mycoplasma was identified, antibiotic treatment was initiated. RESULTS: Mycoplasmal contamination was detected in six of 15 cell lines using the nested PCR amplification of mycoplasma DNA, which was further demonstrated using 4, 6-Diamidino-2-phenylindole (DAPI) staining and fluorescent microscopy. Alternate treatment with two antibiotics, macrolide (tiamulin) and tetracycline (minocycline), effectively eliminated mycoplasma, which was validated by both PCR and microscopic studies. CONCLUSIONS: The nested PCR using genomic DNA extracted from cultured cells as templates is a rapid and sensitive method for detecting mycoplasma contamination. Treatment with combined antibiotics can completely eradicate mycoplasmal infection from cultured cells. For the ease of use, PCR and/or DAPI staining appear suitable for detecting potential mycoplasmal contamination in laboratories that rely heavily on the cell culture system.

PMID: 12846524 [PubMed - indexed for MEDLINE]

Biologicals. 2002 Dec;30(4):289-96. Related Articles, Links
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Detection and characterization of pestivirus contaminations in human live viral vaccines.

Studer E, Bertoni G, Candrian U.

Official Medicines Control Laboratory Biologika and R&D Unit, Division of Biologicals, Swiss Federal Office of Public Health, P.O. Box 3003, Bern, Switzerland.

In view of the use of potentially contaminated foetal calf serum (FCS) in cell cultures pestiviruses may be present in live viral vaccines. Thirty-six lots of human live viral vaccines produced by three manufacturers were tested for the presence of pestiviruses. Bovine viral diarrhoea virus (BVDV) RNA was detected in 33% of the vaccine lots. All positive results were caused by the mumps component of a single manufacturer. Partial sequences of the 5' untranslated region of BVD viral RNA were determined. The sequences were closely related to that of the NADL strain of BVDV. The amount of BVDV RNA in the vaccines was determined by real-time RT-PCR using the LightCycler. Between 3.3*10(2) and 6.2*10(5) RNA copies per dose were found to be present in the vaccine samples. Additionally, culture tests were done with FCS and human diploid cells used in the vaccine production of the manufacturer whose vaccines were positive by PCR. All attempts to detect virus antigen in MRC-5 human diploid cells or to infect these cells with BVDV failed. This suggests that BVDV RNA detected in human live viral vaccines represents passive carry over of BVDV from contaminated FCS rather than active virus replication in human diploid cells. Our results indicate that contamination with BVDV of FCS used in vaccine production does not appear to be of immediate concern to human health. Furthermore, our results indicate that gamma-irradiation of FCS destroys BVDV particles and is also effective in preventing the presence of BVDV RNA in the vaccines. Copyright 2002 The International Association for Biologicals. Published by Elsevier Science Ltd. All rights reserved.
Comment: Let's hope they are right about the consequences re: human health..

PMID: 12421586 [PubMed - indexed for MEDLINE]

In Vitro Cell Dev Biol Anim. 2002 Feb;38(2):79-85. Related Articles, Links
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Comparative PCR analysis for detection of mycoplasma infections in continuous cell lines.

Uphoff CC, Drexler HG.

Department of Human and Animal Cell Cultures, DSMZ-German Collection of Microorganisms & Cell Cultures, Braunschweig.

Mycoplasma contamination of cell lines is one of the major problems in cell culturing. About 15-35% of all cell lines are infected with a limited number of mycoplasma species of predominantly human, swine, or bovine origin. We examined the mycoplasma contamination status in 495 cell cultures by polymerase chain reaction (PCR) assay, microbiological culture method, and deoxyribonucleic acid-ribonucleic acid (DNA-RNA) hybridization, and in 103 cell cultures by PCR and DNA-RNA hybridization, in order to determine the sensitivity and specificity of the PCR assay in routine cell culture. For those two cohorts, results for the three or two assays were concordant in 92 and 91% of the cases, respectively. The sensitivity (detection of true positives) of this PCR detection assay was 86%, and the specificity (detection of true negatives) was 93%, with positive and negative predictive values (probability of correct results) of 73 and 97%, respectively. PCR defined the mycoplasma status with 92% accuracy (detection of true positives and true negatives). The mycoplasma contaminants were speciated by analyzing the PCR amplification fragment using several restriction enzymes. Most of the cultures (47%) were infected with Mycoplasma fermentans, followed by M. hyorhinis (19%), M. orale (10%), M. arginini (9%), Acholeplasma laidlawii (6%), and M. hominis (3%). To sum up, PCR represents a sensitive, specific, accurate, inexpensive, and quick mycoplasma detection assay that is suitable for the routine screening of cell cultures.

PMID: 11928999 [PubMed - indexed for MEDLINE]

J Clin Microbiol. 2001 Feb;39(2):675-84. Related Articles, Links
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No evidence of infectious retroviruses in measles virus vaccines produced in chicken embryo cell cultures.

Shahabuddin M, Sears JF, Khan AS.

Laboratory of Retrovirus Research, Division of Viral Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Bethesda, Maryland 20892, USA.

All vaccines that are prepared in chicken embryo fibroblasts (CEFs) contain a low level of particle-associated reverse transcriptase (RT) activity, which is produced from the avian cell substrate. The RNAs present in the particles have sequence homology to viral DNAs belonging to the ancient endogenous avian virus (EAV) family or to the avian sarcoma-leukosis virus (ALV)-related subgroup E endogenous virus loci. Although no replication-competent retrovirus has been associated with the RT activity produced from CEFs, there have been some theoretical safety concerns regarding potential consequences of integration of EAV and ALV sequences in human DNA, which may result from nonproductive infection with replication-defective particles or infection with EAV and ALV pseudotypes bearing measles virus envelopes. To address these possibilities, we have analyzed EAV and ALV particles in a measles virus vaccine equivalent (MVVE) preparation, obtained from a U.S. manufacturer, for integration and for replication in human peripheral blood mononuclear cells (PBMCs). The results show the absence of EAV and ALV integrants in DNA prepared from MVVE-inoculated human cells by direct DNA PCR and Alu PCR assays and no propagation of retrovirus in 18-day cultures of MVVE-inoculated human PBMCs by a highly sensitive PCR-based RT assay. These results provide further confidence regarding the safety of chicken RT activity in live viral vaccines and support the continued use of chick-cell-derived vaccines in humans.

        Comment:  Again, let's hope they are right.

PMID: 11158127 [PubMed - indexed for MEDLINE]

Philos Trans R Soc Lond B Biol Sci. 2001 Jun 29;356(1410):849-51. Related Articles, Links
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Responsibility for truth in research.

Nelson-Rees WA.

For over half a century, cell cultures derived from animals and humans have served researchers in various fields. To this day, cross-contamination of cultures has plagued many researchers, often leading to mistaken results, retractions of results, cover-ups and some out-and-out falsification of data and results following inadvertent use of the wrong cells. Also, during years of examining cultures for purity we learned that many virologists were not too concerned about the specificity of the cultures they used to propagate the particular virus under study as long as the substrate (whatever it might have been) gave optimal virus yield. Polio virus propagates in primate cells, and much research has involved cells from man and various species of primates. In the 1950s a large number of chimpanzees were held in captivity in Africa for extensive studies of the efficacy of polio vaccine in production at the Wistar Institute in Philadelphia and elsewhere. Chimpanzee tissues, particularly kidneys, were thus readily available and could have also provided substrates for polio virus production, since little was known about the purity of substrates and little attention was paid to their specificity at that time.

Publication Types:
  • Review
  • Review, Tutorial

Comment:  How much concern has there been about possible cross-species transfer of disease?

PMID: 11405932 [PubMed - indexed for MEDLINE]

Vet Res Commun. 2003 Oct;27(7):577-89. Related Articles, Links

Experimental infection of calves with bovine viral diarrhoea virus type-2 (BVDV-2) isolated from a contaminated vaccine.

Falcone E, Cordioli P, Tarantino M, Muscillo M, Sala G, La Rosa G, Archetti IL, Marianelli C, Lombardi G, Tollis M.

Istituto Superiore di Sanita, Laboratorio di Medicina Veterinaria, Viale Regina Elena 299, 00161 Rome, Italy.

A non-cytopathic strain of BVDV-2 was isolated from a batch of live infectious bovine rhinotracheitis (IBR) vaccine, and inoculated intranasally into four 3-month-old calves. Severe signs of disease developed by days 4 and 6 in three of the calves, free of BVDV and antibodies to BVDV, that had been exposed to the virus. These calves survived the acute phase of the infection and progressively recovered. BVDV was consistently isolated, or the respective viral RNA was detected, in the buffy coats from blood samples collected starting from days 2 or 4 up to days 11 or 14 after the experimental infection. Viral RNA was also detected in sera from these infected calves until the presence in the serum of virus neutralizing antibodies was demonstrated. By contrast, the only calf having pre-existing neutralizing antibodies to BVDV at the start of the study was protected from the disease. No virus was detected at any time after experimental inoculation of this calf. Genomic characterization of the BVDV-2 isolated in cell cultures, or detected in sera from the experimentally infected animals, revealed 100%, homology in the nucleotide sequence with the BVDV-2 detected as a contaminant of the live IBR virus vaccine. These findings provided evidence of the infective nature of the contaminant BVDV-2 and of its potential to generate disease outbreaks when inoculated into susceptible animals.
Comment:  Are humans susceptible? 

PMID: 14609269 [PubMed - indexed for MEDLINE]
Br J Haematol. 2003 Jul;122(1):3-9. Related Articles, Links

The pathophysiology of variant Creutzfeldt-Jacob disease: the hypotheses behind concerns for blood components and products.

Burthem J, Roberts DJ.

Department of Biomedical Sciences, University of Manchester Institute of Science and Technology, Manchester, UK.

Publication Types:

PMID: 12823340 [PubMed - indexed for MEDLINE]

Biologicals. 2002 Dec;30(4):289-96. Related Articles, Links
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Detection and characterization of pestivirus contaminations in human live viral vaccines.

Studer E, Bertoni G, Candrian U.

Official Medicines Control Laboratory Biologika and R&D Unit, Division of Biologicals, Swiss Federal Office of Public Health, P.O. Box 3003, Bern, Switzerland.

In view of the use of potentially contaminated foetal calf serum (FCS) in cell cultures pestiviruses may be present in live viral vaccines. Thirty-six lots of human live viral vaccines produced by three manufacturers were tested for the presence of pestiviruses. Bovine viral diarrhoea virus (BVDV) RNA was detected in 33% of the vaccine lots. All positive results were caused by the mumps component of a single manufacturer. Partial sequences of the 5' untranslated region of BVD viral RNA were determined. The sequences were closely related to that of the NADL strain of BVDV. The amount of BVDV RNA in the vaccines was determined by real-time RT-PCR using the LightCycler. Between 3.3*10(2) and 6.2*10(5) RNA copies per dose were found to be present in the vaccine samples.Additionally, culture tests were done with FCS and human diploid cells used in the vaccine production of the manufacturer whose vaccines were positive by PCR. All attempts to detect virus antigen in MRC-5 human diploid cells or to infect these cells with BVDV failed. This suggests that BVDV RNA detected in human live viral vaccines represents passive carry over of BVDV from contaminated FCS rather than active virus replication in human diploid cells. Our results indicate that contamination with BVDV of FCS used in vaccine production does not appear to be of immediate concern to human health. Furthermore, our results indicate that gamma-irradiation of FCS destroys BVDV particles and is also effective in preventing the presence of BVDV RNA in the vaccines. Copyright 2002 The International Association for Biologicals. Published by Elsevier Science Ltd. All rights reserved.

PMID: 12421586 [PubMed - indexed for MEDLINE]
Curr Opin Neurol. 2002 Jun;15(3):333-8. Related Articles, Links
Click here to read 
Neurological adverse events associated with vaccination.

Piyasirisilp S, Hemachudha T.

Division of Neurology, Department of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand.

Public tolerance to adverse reactions is minimal. Several reporting systems have been established to monitor adverse events following immunization. The present review summarizes data on neurologic complications following vaccination, and provides evidence that indicates whether they were directly associated with the vaccines. These complications include autism (measles vaccine), multiple sclerosis (hepatitis B vaccine), meningoencephalitis (Japanese encephalitis vaccine), Guillain-Barre syndrome and giant cell arteritis (influenza vaccine), and reactions after exposure to animal rabies vaccine. Seizures and hypotonic/hyporesponsive episodes following pertussis vaccination and potential risks associated with varicella vaccination, as well as vaccine-associated paralytic poliomyelitis following oral poliovirus vaccination, are also described. In addition, claims that complications are caused by adjuvants, preservatives and contaminants [i.e. macrophagic myofasciitis (aluminium), neurotoxicity (thimerosal), and new variant Creutzfeldt-Jakob disease (bovine-derived materials)] are discussed.

Publication Types:

PMID: 12045734 [PubMed - indexed for MEDLINE]

Am J Health Syst Pharm. 2002 Feb 1;59(3):254-60; quiz 261-3. Related Articles, Links
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Implications of prion-induced diseases for animal-derived pharmaceutical products.

Erstad BL.

Department of Pharmacy Practice and Science, College of Pharmacy, University of Arizona, 1703 E. Mabel Street, Tucson, AZ 85721-0207, USA.

The implications of prion-induced diseases for the use of medications that theoretically could harbor the infectious pathogens are discussed. Prions have been identified as protein particles that lack nucleic acids. There is evidence that prions cause the transmissible neurodegenerative diseases known as transmissible spongiform encephalopathies. Of these diseases, bovine spongiform encephalopathy (BSE) and the human spongiform encephalopathy to which it has been linked, new variant Creutzfeldt-Jakob disease (CJD), have generated the most attention. The first cases of new variant CJD appeared in Britain in the mid-1990s. Ingestion of prion-infected beef remains the only known cause of new variant CJD. No cases of BSE or new variant CJD have been documented in the United States. The time from exposure to the development of clinical sequelae appears to be about 10 years. The median duration of illness is 14 months, and the outcome is invariably death. There is no treatment; currently the only available approach is prevention. There is no reliable method of predicting the number of new cases that might occur because of lack of definitive information on the efficiency of transmission from animals to humans and the number of people currently infected and at risk for infection. The infectivity of medications and plasma fractionation products containing material from cattle with BSE is unknown, but the risk is believed to be very low. No cases of such transmission have been identified. Guidelines to keep the risk of transmission via medications low have been promulgated by FDA, and further research is warranted. There have been no reports of medications or plasma fractionation products being contaminated with the prions that cause new variant CJD. Ongoing vigilance and research are appropriate, however.

Publication Types:

PMID: 11862637 [PubMed - indexed for MEDLINE]

Methods Cell Sci. 2000 Mar;22(1):33-41. Related Articles, Links
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Ten commandments for preventing contamination of primary cell cultures.

Vierck JL, Byrne K, Mir PS, Dodson MV.

Department of Animal Sciences, Washington State University, Pullman, Washington, USA.

Procedures for preventing contamination in primary cell cultures must be carefully defined and strictly followed in order to obtain healthy cells. Protocols have been developed and refined in our laboratory for establishing primary cultures of muscle and fat stem cells without contamination from a variety of animals. Contamination of cell cultures is not only frustrating, but is also very expensive both in time and loss of materials. Through the consistent use of proper aseptic techniques, most instances of contamination may be avoided. We suggest that the basic principles detailed here will find wide applicability in the culturing of primary cells without contamination from many different types of animals and tissues.
Comment:  Is it merely "frustrating" and "expensive", or are there negative health consequences associated with their use as well?

PMID: 10650333 [PubMed - indexed for MEDLINE]

Rev Argent Microbiol. 2000 Jan-Mar;32(1):27-32. Related Articles, Links

[Contamination of bovine fetal serum with bovine viral diarrhea virus]

[Article in Spanish]

Zabal O, Kobrak AL, Lager IA, Schudel AA, Weber EL.

Instituto de Virologia, CICVyA, INTA Castelar, Buenos Aires, Argentina.

Fetal bovine serum (FBS) used in cell culture may be contaminated with viruses, among them bovine viral diarrhea virus (BVDV) affecting the production of biological reagents and the results of diagnosis. The filtration process used in the preparation of commercial FBS abrogates most viral agents that may be present in raw FBS, but BVDV may pass through the filters because of its small size and its pleomorphism. While detection of bovine herpes virus-1 and parainfluenza-3 (PI-3) is determined by observation of the cytopathic effect, and also by hemadsorption in the case of PI-3, the most frequently isolated BVDV is non cytopathic, and infects cells without morphological alterations, inducing problems that arise after several cell generations. Batches of raw and processed FBS were analyzed. Frequencies of BVDV detection in raw serum in Argentina were similar to those published for USA. By conventional methods for BVDV detection, only 2 of 20 commercial batches of FBS had BVDV. Using cell cultures maintained with high concentrations of the serum under study for at least 2 weeks, with detection of viral antigen by indirect immunofluorescence, the percentage of BVDV detection was 80%. This method shows that most lots of commercial FBS contain BVDV. RT-PCR allows faster detection of the viral genome, but it must be validated, as it does not show viral replication. To eliminate the problem of BVDV contamination in FBS, only gamma irradiated FBS is used in our laboratory.

PMID: 10785940 [PubMed - indexed for MEDLINE]

Dev Biol Stand. 1999;99:3-8. Related Articles, Links

Benefits and risks due to animal serum used in cell culture production.

Wessman SJ, Levings RL.

USDA, APHIS, VS, Center for Veterinary Biologics-Laboratory, Ames, Iowa, USA.

Infection with bovine viral diarrhoea virus (BVDV) and other viruses is frequent in the bovine population. In utero infection leads to virus and antibody contamination of foetal and other serum used in cell culture production. The use of contaminated cells for vaccine production may result in contaminated vaccines, which may lead to seroconversion or disease in the vaccinated animal. Contaminated serum or cell cultures may also interfere with the diagnosis of viral infections. Methods for the detection of BVDV and other viruses in serum, cell cultures, seed viruses and vaccines at the CVB-L, and the frequency of detection are described. Reasons for continued use of serum in cell culture production, and the risks of using serum, are discussed.

PMID: 10404869 [PubMed - indexed for MEDLINE]

Anticancer Res. 1999 May-Jun;19(3B):2173-80. Related Articles, Links

Cancer risk associated with simian virus 40 contaminated polio vaccine.

Fisher SG, Weber L, Carbone M.

Cancer Cause and Prevention Program, Loyola University Medical Center, Maywood, Illinois 60153, USA.

BACKGROUND: The presence of SV40 in monkey cell cultures used in the preparation of the polio vaccine from 1955 through 1961 is well documented. Investigations have consistently demonstrated the oncogenic behavior of SV40 in animal models. Early epidemiologic studies were inadequate in demonstrating an increase in cancer incidence associated with contaminated vaccine. Recently, investigators have provided persuasive evidence that SV40 is present in human ependymomas, choroid plexus tumors, bone tumors, and mesotheliomas, however, the etiologic role of the virus in tumorigenesis has not been established. MATERIALS AND METHODS: Using data from SEER, we analyzed the incidence of brain tumors, bone tumors, and mesotheliomas from 1973-1993 and the possible relationship of these tumors with the administration of the SV40 contaminated vaccine. RESULTS: Our analysis indicates increased rates of ependymomas (37%), osteogenic sarcomas (26%), other bone tumors (34%) and mesothelioma (90%) among those in the exposed as compared to the unexposed birth cohort. CONCLUSIONS: These data suggest that there may be an increased incidence of certain cancers among the 98 million persons exposed to contaminated polio vaccine in the U.S.; further investigations are clearly justified.

PMID: 10472327 [PubMed - indexed for MEDLINE]

Dev Biol Stand. 1998;93:133-4. Related Articles, Links

Safety of biological products prepared from mammalian cell culture. Bovine spongiform encephalopathy and other non-viral transmissible agents.

[No authors listed]

Publication Types:
  • Review
  • Review, Tutorial

PMID: 9737391 [PubMed - indexed for MEDLINE]

In Vitro Cell Dev Biol Anim. 1998 Jan;34(1):1-8. Related Articles, Links

Cell cross-contamination in cell cultures: the silent and neglected danger.

Markovic O, Markovic N.

BioSciCon, Inc., Rockville, MD 20852, USA.

Cell cross-contamination in cell cultures is a common problem during cell culturing and use. Contamination invalidates research results, compromises the comparison of results between laboratories, reduces reproducibility required in industrial production of cell lines, and may lead to unusable therapeutic products. The problem can be solved by increasing the awareness of its seriousness and by introducing regular quality control of cell cross-contamination in every laboratory where cells are grown and used.

Publication Types:
  • Review
  • Review, Academic

PMID: 9542623 [PubMed - indexed for MEDLINE]

Rev Argent Microbiol. 1998 Jul-Sep;30(3):147-53. Related Articles, Links

[Presence of Mycoplasma in laboratory cell cultures from Cordoba, Argentina]

[Article in Spanish]

Cumino AC, Cordoba P, Zapata TM.

Instituto de Virologia J.M. Vanella, Facultad de Medicina, Universidad Nacional de Cordoba, Argentina.

In this paper we determined the prevalence of mycoplasma contamination in 17 cell lines. Eighty per cent of the laboratories that currently use cell culture techniques participated in this study. Hoechst 33258 dye was used to detect mycoplasma contamination. The relationship between culture maintenance conditions and the presence of mycoplasma were analyzed, considering the use of antibiotics in the culture media, fetal calf serum (FCS) quality, culture media processing, use of disponsable labware, type of laminar flow cabinet, quantity of operators, and cell culture system. Thirty-five per cent of the analyzed cell lines showed mycoplasma contamination. Those lines belonged to 2 of the 8 surveyed laboratories. When confronting the working conditions versus mycoplasma contamination, 66% of the laboratories that employ non-certified FCS or reuse their labware, show mycoplasma contamination. Mycoplasma presence was found in 50% of the laboratories that use closed culture system, or more than one operator. Laboratories that process their culture media or that include antibiotic in the growing media, show a 40% contamination. The results obtained help to establish working conditions necessary to avoid introducing or spreading the microorganism.

PMID: 9793145 [PubMed - indexed for MEDLINE]

Dev Biol Stand. 1998;93:31-6. Related Articles, Links

Zoonoses and haemorrhagic fever.

Mahy BW.

Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.

Virus zoonoses causing haemorrhagic fever have been recognized in three major families: Arenaviridae, Bunyaviridae and Filoviridae. All are negative-stranded RNA viruses, with genomes in two segments, three segments, or non-segmented, respectively. Acquisition of haemorrhagic fever in man generally requires close contact with a vertebrate vector species, usually rodents, for the arenaviruses and bunyaviruses. In the case of filoviruses, the vector is currently unknown, but these viruses may infect monkeys, and may contaminate cell cultures prepared from them. Both bunyavirus and arenavirus haemorrhagic fevers have arisen in humans following exposure to rodents, and in the case of Hantaan, a virus causing haemorrhagic fever with renal syndrome (HFRS), there have been numerous laboratory-acquired infections among animal care workers. As the technology to differentiate virus species has improved, it has become clear that there are numerous potentially hazardous viruses capable of causing HFRS or hantavirus pulmonary syndrome (HPS) within the feral rodent population. In many cases it would be desirable to introduce screening methods for such viruses before preparing cell cultures from these rodent or simian species that will be used to prepare biological products for human use.

Publication Types:
  • Review
  • Review, Tutorial

Comment:  That seems like an understatement.

PMID: 9737374 [PubMed - indexed for MEDLINE]

J Infect Dis. 1996 Dec;174(6):1324-7. Related Articles, Links

Contamination of commercially available fetal bovine sera with bovine viral diarrhea virus genomes: implications for the study of hepatitis C virus in cell cultures.

Yanagi M, Bukh J, Emerson SU, Purcell RH.

Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-0740, USA.

The establishment of cell cultures for hepatitis C virus (HCV) is important for its study as a human pathogen. However, in reported cell lines, HCV demonstrates low levels of replication detected primarily by reverse transcription-polymerase chain reaction (RT-PCR) assays. In attempts to culture HCV, an additional complication was observed. From mock-infected cultures, cDNA of appropriate size was obtained by RT-PCR with primers deduced from conserved domains of the 5' noncoding region of HCV. However, sequence analysis revealed that the cDNA was amplified from bovine viral diarrhea virus (BVDV). All of 7 bovine sera tested were contaminated with BVDV. In conclusion, most commercially available bovine sera are contaminated with BVDV and, although there is no evidence that the virus is infectious, bovine sera should be screened for this virus by RT-PCR when used in conjunction with HCV or for the development or production of vaccine.

PMID: 8940226 [PubMed - indexed for MEDLINE]

Dev Biol Stand. 1996;88:49-56. Related Articles, Links

Experience with viral contamination in cell culture.

Garnick RL.

Genentech, Inc., South San Francisco, CA, USA.

Genentech has had direct experience with two contaminations of large-scale cell cultures by Minute Virus of Mice (MVM). No definitive source of either contamination has been identified, although the conclusions of the investigation were consistent with a raw material used in cell culture as the source. Effective analytical barriers were developed following the first contamination using polymerase chain reaction (PCR) and cell culture-based methodology for MVM, and these were employed on a routine basis. This approach was effectively used to detect and contain the second contamination, thus dramatically minimizing its impact. Close interactions with regulatory authorities were critical in the positive management of these situations.

Publication Types:
  • Historical Article

PMID: 9119162 [PubMed - indexed for MEDLINE]

J Reprod Fertil. 1995 Sep;105(1):17-24. Related Articles, Links

Detection of bovine viral diarrhoea virus antigen and RNA in oviduct and granulosa cells of persistently infected cattle.

Booth PJ, Stevens DA, Collins ME, Brownlie J.

Institute for Animal Health, Compton, Newbury, Berkshire, UK.

Large-scale in vitro bovine embryo production systems commonly use genital tracts obtained from an abattoir as a source of both cumulus-oocyte complexes and co-culture feeder cells. Tissues derived from this source may be contaminated with non-cytopathogenic bovine viral diarrhoea virus (BVDV) since, in several countries surveyed, approximately 1% of animals tested are persistently infected with this pathogen. Therefore, the use of such material in in vitro fertilization systems presents a potential risk for the transmission of BVDV to bovine embryos and via embryo transfer. This potential was investigated by obtaining oviduct epithelial cells and granulosa cells, which are commonly used as feeder cells, from cattle persistently infected with BVDV and examining them for the presence of BVD viral antigen (p80 non-structural protein and gp53 envelope glycoprotein) by indirect immunofluorescent histochemistry, and also viral RNA (encoding the p80 region) by in situ hybridization. In addition, titres of virus present in oviduct, ovary and blood were assayed by immunodetection on calf testis cell cultures. Luminal epithelial cells from the oviduct and primary cultures of granulosa cells and oviduct epithelial cells from such cattle were shown to contain both viral antigen and RNA. The susceptibility of both cell types to BVDV infection was further established by inoculating primary cell cultures of cells derived from cattle not infected with BVDV with a cloned isolate of non-cytopathogenic BVDV (Pe515). RNA encoding BVDV and the antigen were detected 12 h after inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 7490710 [PubMed - indexed for MEDLINE]

Vopr Virusol. 1995 Sep-Oct;40(5):225-7. Related Articles, Links

[Cross contamination of continuous cell cultures]

[Article in Russian]

Iurkov SG.

The possibility of air-droplet cell transmission during cell transfer was experimentally confirmed, this appearing to be the most probable route of cross contamination of cell cultures. The consequences of such contamination are assessed and measures reducing the risk of intercellular contamination proposed.

PMID: 8659179 [PubMed - indexed for MEDLINE]

Am Biotechnol Lab. 1994 Jul;12(8):42. Related Articles, Links

Mycoplasmal contamination of cell cultures.

Lundin DJ.

Bionique Testing Laboratories, Inc., Saranac Lake, NY 12983.

PMID: 7765012 [PubMed - indexed for MEDLINE]

Trends Biotechnol. 1993 Apr;11(4):143-51. Related Articles, Links

Beware of mycoplasmas.

Rottem S, Barile MF.

Department of Membrane and Ultrastructure Research, Hebrew University-Hadassah Medical School, Jerusalem, Israel.

Mycoplasma infection of cell cultures is widespread and has major detrimental effects on cellular physiology and metabolism. Since cell culture is used extensively, both in research and in industrial production processes, questions of primary concern arise, such as: how can mycoplasma contamination be detected; what are the effects of such contamination on cellular functions; what methods are available for eliminating contamination?

Publication Types:
  • Review
  • Review, Tutorial

PMID: 7763647 [PubMed - indexed for MEDLINE]

Appl Microbiol Biotechnol. 1993 May;39(2):141-7. Related Articles, Links

Safe biotechnology (5). Recommendations for safe work with animal and human cell cultures concerning potential human pathogens.

Frommer W, Archer L, Boon B, Brunius G, Collins CH, Crooy P, Doblhoff-Dier O, Donikian R, Economidis J, Frontali C, et al.

DECHEMA, Frankfurt/Main.

The benefits of using animal or human cell cultures have been clearly demonstrated in diagnostic and therapeutic research and in their application for manufacturing. Cell cultures serve as a tools for the production of vaccines, receptors, enzymes, monoclonal antibodies and recombinant DNA-derived proteins. They represent an integral part of drug development for which corresponding facilities, equipment and manufacturing processes are required. Although the cells themselves offer no particular risk to workers in laboratories and production areas or to the environment, the cell cultures may be contaminated with viruses, mycoplasma, bacteria, yeast and fungi or might contain endogenous viruses. The containment level for animal and human cells is therefore determined by the risk class of these agents. The history of animal and human cell cultures has proved that they can be handled safely. The recommendations in this publication concern the safe handling of cell cultures (tissue explants, primary cell cultures) and permanent cell lines of animal and human origin. A classification system of safety precautions has been elaborated according to the potential for contamination with the pathogenic agents involved.

PMID: 7763726 [PubMed - indexed for MEDLINE]

Folia Biol (Praha). 1993;39(5):270-6. Related Articles, Links

Comparison of methods used for detection of mycoplasma contamination in cell cultures, sera, and live-virus vaccines.

Benisheva T, Sovova V, Ivanov I, Opalchenova G.

National Drug Institute, Sofia.

Two methods for detection of mycoplasma contamination in cell cultures, sera, and live-virus vaccines were compared: the direct culture test and the DNA staining method employing bisBenzimide (Hoechst No. 33258). Contamination by different species of mycoplasma was found in 39% samples tested. It is recommended to use both techniques for a reliable detection of mycoplasma contamination.

PMID: 8206173 [PubMed - indexed for MEDLINE]

J Gen Virol. 1992 Nov;73 ( Pt 11):2871-8. Related Articles, Links

Bovine polyomavirus, a cell-transforming virus with tumorigenic potential.

Schuurman R, van Strien A, van Steenis B, van der Noordaa J, Sol C.

Department of Virology, University of Amsterdam, Academic Medical Centre, The Netherlands.

The early region of bovine polyomavirus (BPyV) was tested for its cell transformation potential employing an assay of dense focus formation. Dense foci of morphologically transformed cells were observed upon transfection of primary rodent cells with a plasmid construct encoding the complete early region of BPyV under the transcriptional control of the long terminal repeat of Rous sarcoma virus. No transformation of primary rodent cells was observed upon transfection of these cells with a plasmid encoding the complete early region of BPyV under the control of its own transcriptional regulatory sequences. In BPyV-transformed cells, the viral sequences had become integrated into the cellular genome, and expression of large T antigen could be detected in a high percentage of cells. The transformed cells were demonstrated to be capable of anchorage-independent growth and to be oncogenic in immunocompromised newborn rats. Therefore BPyV should be considered as a potentially tumorigenic polyomavirus. Since many commercial batches of calf serum have been shown to be contaminated with BPyV, our observations may have implications for the use of calf serum in cell culture.

PMID: 1331291 [PubMed - indexed for MEDLINE]

Zentralbl Bakteriol. 1992 Jun;277(1):49-53. Related Articles, Links

Detection of Mycoplasma contamination in primary calf kidney cell cultures.

Pfutzner H, Otto P.

Research Institute for Bacterial Animal Diseases, Jena, Germany.

A total of 31 primary cell culture preparations of calf kidney including their processing stages (kidney, washing fluid, cell suspension, cell culture monolayer) were investigated for mycoplasmas by cultural methods. Mycoplasma (M.) arginini, was isolated from 8 out of 27 investigated samples of calf kidney from 3 out of 26 washing fluids, 5 out of 20 cell suspensions, and from 9 out of 21 cell culture monolayers. Furthermore, M. arginini was repeatedly found in throat swabs of the cell laboratory technicians. The results give indications as to the source and route of mycoplasma infections of primary cell cultures.

PMID: 1520969 [PubMed - indexed for MEDLINE]

Dev Biol Stand. 1992;76:267-74. Related Articles, Links

Filovirus contamination of cell cultures.

Peters CJ, Jahrling PB, Ksiazek TG, Johnson ED, Lupton HW.

Disease Assessment Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21701-5011.

The filoviruses Marburg and Ebola comprise a newly recognized family of viruses. The first filovirus to be isolated was Marburg virus in 1967. This virus was imported in shipments of African green monkeys from Uganda and infected several cell-culture technicians, with serious illness resulting. The rarity of Marburg and Ebola virus transmission, decreasing use of imported African monkeys, and quarantine efforts have presumably been responsible for the lack of additional episodes until 1989, when a new filovirus related to Ebola was isolated from quarantined monkeys in Reston, Virginia. This virus was imported on multiple occasions from a Philippine supplier of cynomolgus macaques as a consequence of an epidemic of acute infections in the foreign holding facility. While quarantine procedures prevented the use of any of these animals in research and the three human infections that occurred were asymptomatic, this episode emphasizes that these little understood viruses have considerable potential for mischief. The finding of antibodies reacting with Ebola viruses in many biomedically important Old World primates, including colonized monkeys in the U.S., emphasizes the need for more research to understand the specificity of the antibodies, spectrum of filovirus strains in nature, potential hosts, and true distribution of the family. The filoviruses grow well in primary and established cell strains and cell lines, and cytopathogenic effects may be absent or require several days to be manifest, leading to the possibility of occult contamination. The known viruses are readily detected by polyclonal and monoclonal antibody staining of cells and by electron microscopy; nucleic acid probes exist to develop more sensitive techniques if warranted.

PMID: 1478345 [PubMed - indexed for MEDLINE]

Dev Biol Stand. 1992;76:285-93. Related Articles, Links

Latent parvoviral infection of continuous cell lines.

Fikrig MK, Tattersall P.

Department of Medicine, Yale University School of Medicine, New Haven, CT 06510.

The parvoviruses are a family of single-stranded DNA-containing viruses which are known to establish inapparent infections of continuous, and in some cases, primary cell cultures. Their small size and great stability suggest that they would be difficult to eliminate from a biological component purified from a contaminated cell line. Thus, precautions should be taken to exclude such agents from initial cell cultures, and from the reagents used to maintain them.

Publication Types:
  • Review
  • Review, Tutorial

PMID: 1335934 [PubMed - indexed for MEDLINE]

Dev Biol Stand. 1992;76:5-11. Related Articles, Links

Cell line issues: historical and future perspectives.

Petricciani JC.

Pharmaceutical Manufacturers Association, Washington, DC 20005.

The initial decision to use only primary cell cultures for the production of human biological products was challenged in the late 1960s by the introduction of human diploid cells (HDCs), and again in the 1980s by continuous cell lines (CCLs). The history of the HDC controversy is reviewed and lessons from that era that are relevant to the use of CCLs are pointed out. With the introduction of recombinant DNA technology in the 1980s, and the potential usefulness of CCLs in product development, the issue of cell acceptability became more urgent, and several attempts were made to reach a consensus on regulatory issues. In 1986, the World Health Organization convened a Study Group to review the safety issues related to products derived from CCLs. The Study Group made a clear recommendation to pursue CCLs in product development because of the demonstrated capability of modern manufacturing processes to cope with contaminants. Issues such as acceptable levels of cellular DNA in products, the relationship of purity to safety, and the relevance of the genetic stability of recombinant cells to product consistency are current examples of areas in need of discussion and agreement. A system in which regulatory authorities, industry, and the general biomedical community cooperate in finding solutions is ultimately in everyone's best interest.

Publication Types:
  • Historical Article

PMID: 1478355 [PubMed - indexed for MEDLINE]

J Gen Virol. 1991 Nov;72 ( Pt 11):2739-45. Related Articles, Links

Frequent detection of bovine polyomavirus in commercial batches of calf serum by using the polymerase chain reaction.

Schuurman R, van Steenis B, van Strien A, van der Noordaa J, Sol C.

Department of Virology, University of Amsterdam, The Netherlands.

Twenty commercial batches of calf serum, obtained from several suppliers, were tested for the presence of bovine polyomavirus (BPyV) DNA and antibodies against the virus. Using polymerase chain reaction (PCR) technology, BPyV DNA was detected in 70% of the batches; no BPyV was detected in any of the negative control samples. The specificity of the amplification reactions was proven by hybridization. PCR results were confirmed by virus isolation experiments performed with five PCR-positive and five PCR-negative serum batches. The results indicate that the use of calf serum to supplement tissue culture media involves a serious risk of contaminating cell cultures with BPyV. No correlation was observed between the presence or absence of anti-BPyV immunoglobulins and the detection of BPyV-specific DNA sequences in the serum batches.

PMID: 1658200 [PubMed - indexed for MEDLINE]

Dev Biol Stand. 1991;75:183-9. Related Articles, Links

Virus zoonoses and their potential for contamination of cell cultures.

Mahy BW, Dykewicz C, Fisher-Hoch S, Ostroff S, Tipple M, Sanchez A.

Division of Viral and Rickettsial Diseases, Centers for Disease Control, Atlanta, GA 30333.

Silent virus infections of laboratory animals present a human health hazard, from direct exposure and from contamination of biological products for human use. Here we report two recent examples. In 1989, an outbreak of lymphocytic choriomeningitis virus (LCMV) infections was recognized among workers at a cancer research center after an animal caretaker developed viral meningitis. Investigation revealed that multiple tumor cell lines at the facility were infected with LCMV, as were research animals injected with these cell lines. Of 82 workers tested, eight (10%) were found to have been infected. The infected workers were more likely than other animal handlers to report handling athymic (nude) mice (p less than .0.007). The number of nude mice used in this facilty had increased five-fold in the previous year, possibly explaining the timing of the outbreak. This is the first reported LCMV outbreak since 1975, and the first to implicate nude mice as a source of human LCMV infections. In November 1989 and January 1990, infections caused by two distinct Ebola-like filoviruses were discovered in non-human primates at quarantine facilities in Virginia and Pennsylvania. Although 22 persons were considered to have high- or medium-risk exposures for Ebola infection, no Ebola-compatible illnesses occurred. One of the medium-risk persons had Ebola IgG antibodies confirmed by IFA and Western blot. Rigorous use of barrier precautions may have limited exposure and infection with these filoviruses. In February 1990, new groups of filovirus-infected monkeys were identified in Virginia and in Texas. Seroconversion occurred in four animal handlers, including one to very high titer, but again no illness was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 1794619 [PubMed - indexed for MEDLINE]

Dev Biol Stand. 1991;75:211-7. Related Articles, Links

Correlations between virus infection and pathogenicity in humans.

Agut H, Huraux JM.

Laboratoire de Virologie, CERVI, Groupe Hospitalier Pitie-Salpetriere, Paris.

Infection is not synonymous with disease. Infection refers to the multiplication or the persistence of a virus in tissues while pathogenicity refers to the emergence of disease in the infected host. Pathogenicity is the result of a competition between the growth of the virus and the host response to infection, and its genesis involves many intricate factors. Consequently, most viruses of medical interest exhibit a wide spectrum of pathogenicity ranging from asymptomatic infection to lethal disease. The study of pathogenicity is far more complex than the recognition of infection. Cell cultures, animal models and molecular biology investigations have provided substantial insights both into the virulence of viruses and the susceptibility of the host. However, the prediction of disease often remains hazardous whereas the detection of a virus can now be obtained in most cases by the combination of classical methods with recent molecular techniques. Therefore the prevention of virus diseases transmitted by biologicals is logically founded on the prevention of virus infections, which implies a constant adaptation of safety control procedures to the rapid evolution of knowledge in medical virology.

Publication Types:
  • Review
  • Review, Tutorial

PMID: 1794623 [PubMed - indexed for MEDLINE]


Dev Biol Stand. 1991;75:177-81. Related Articles, Links

Bovine viral diarrhea virus contamination of nutrient serum, cell cultures and viral vaccines.

Levings RL, Wessman SJ.

National Veterinary Services Laboratories, Animal and Plant Health Inspection Service, USDA, Ames, IA 50010.

Bovine viral diarrhea virus (BVDV) infection is common in the bovine population. Infection in utero leads to virus and antibody contamination of the fetal bovine serum used in cell cultures. These contaminants can interfere with diagnosis of viral infection. The high frequency of virus and antibody detection in individual animal or small pool samples suggests that any large pool of unscreened sera will be contaminated. Infection of cell cultures with BVDV can lead to interference with the growth of other viruses. Vaccine produced on contaminated cells may in turn be contaminated, leading to seroconversion or disease in the vaccine. The safety, purity, and efficacy of viral vaccines require BVDV testing of ingredients, cell substrates and final product. Methods for detection of BVDV in nutrient serum, cell cultures, seed viruses, and viral vaccines, and the frequency of their detection at the National Veterinary Services Laboratories are discussed.

PMID: 1665461 [PubMed - indexed for MEDLINE]


Dev Biol Stand. 1989;70:59-66. Related Articles, Links

Detection and elimination of adventitious agents in continuous cell lines.

Erickson GA, Landgraf JG, Wessman SJ, Koski TA, Moss LM.

National Veterinary Services Laboratories, Ames, IA 50010.

The National Veterinary Services Laboratories (NVSL) routinely monitors continuous cell lines (CCL's) used for veterinary biologicals and diagnostic virology. All veterinary biologicals produced in CCL's must follow the master seed concept which limits the use of the master seed CCL to up to 20 passages beyond the passage level characterized and deposited at NVSL. All CLL's are evaluated for the presence of adventitious agents such as mycoplasma, bovine viral diarrhea virus, and other bacteria and viruses. Previously, CCLs were evaluated for tumorigenicity by the Syrian hamster cheek pouch method; however, this procedure has now been eliminated. The adventitious agents most frequently detected in CCL's have been bovine viral diarrhea virus and mycoplasma. Our laboratory has consistently found that the source of bovine viral diarrhea contamination of CCLs has been the use of contaminated fetal bovine cell culture enrichment serum. Gamma irradiation at 2.5-3.5 megarads at -40 degrees C of carefully screened fetal bovine serum has been used in the Diagnostic Virology Laboratory for over 10 years. If the irradiated serum is used at a final concentration of 10 percent, there is no untoward effect on cell susceptibility for virus propagation or cell culture growth. Gamma irradiation has also been demonstrated to be a very efficient inactivator of mycoplasma. Specific conditions utilized by our laboratory to preserve fetal bovine serum cell culture growth factors while eliminating adventitious bovine viral diarrhea virus will be presented.

PMID: 2759356 [PubMed - indexed for MEDLINE]

Dev Biol Stand. 1989;70:3-10. Related Articles, Links

Cells, science and health.

Petricciani JC.

National AIDS Program, U.S. Public Health Service, Washington, D.C.

The initial discussions and decisions in 1954 on the use of various types of mammalian cell substrates for the production of human biological products set the stage for controversy in the late 1960's and again in the 1980's when "abnormal" cell substrates were proposed as alternates for primary cell cultures. In the 1960's the issue was human diploid cells, and in the 1980's it has been the use of continuous cell lines. The parallels between the two issues have been obvious to all who have been interested in the subject. The history of the human diploid cell controversy is reviewed from a personal point of view, and lessons from that era that are relevant to the use of continuous cell lines are pointed out. Pragmatism based on the perceived need for human interferon in the late 1970's led to the exploration of human lymphoblastoid cells as substrates. That bold venture into a prohibited zone set the stage for a reconsideration of the acceptability of continuous cell lines as substrates for the production of a wide range of human biologicals. With the introduction of recombinant DNA technology in the 1980's and the potential usefulness of continuous cell lines in product development, the issue of acceptability took on a more acute aspect and several attempts were made to reach a consensus and a resolution of regulatory issues. In 1986 the World Health Organization identified the use of continuous cell lines as of sufficient global importance to convene a Study Group to review the safety issues and to make recommendations on their use, especially for vaccines. The result of that international effort was a clear recommendation to pursue the use of continuous cell lines in product development because of the demonstrated capability of modern manufacturing processes to cope with even theoretically worrying contaminants.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication Types:
  • Historical Article

PMID: 2668074 [PubMed - indexed for MEDLINE]

Rev Saude Publica. 1989 Feb;23(1):39-44. Related Articles, Links

[Mycoplasma as a contaminant of cell cultures maintained in laboratories of private and official institutions]

[Article in Portuguese]

Miyaki C, Pral MM, Gallina NM, de Rizzo E.

Mycoplasma is one of the most serious contaminants of cell cultures. Its detection is very important in virology, as well as its eradication. The aim of this study was to verify the incidence of mycoplasma in cell lines maintained in seven laboratories of private, government and college institutions of the State of Sao Paulo, Brazil, for the purposes of research, production of reagents for diagnosis and production of biologicals for human and animal use. Of the 29 cell lines, eight were derived from human tissues and 21 from other animal species (dog, rabbit, mouse, hamster, monkey, pig, chicken and ox). Using the direct method with specific liquid and solid media for detection of mycoplasma, 48 out of the 106 cell samples tested were positive, corresponding to a contamination index of 45.28%. The incidence of contamination among the 35 cell samples of human origin was 51.43% (18 positive). Of the 71 samples originated from other species, 30 were positive (42.25%). The high incidence of contamination found calls for the adoption of measures for the prevention of this hazard: the elimination of mouth pipetting, the use of aseptic techniques and a rigid control of trypsin, serum and other components of cell culture media. The substitution of mycoplasma-free cultures for all contaminated ones and the performance of periodical tests for mycoplasma detection must also be carried out to prevent and avoid the dissemination of these organisms. Data obtained showed that contamination appeared in the 2nd (72.92%), in the 3rd (20.83%) and in the 4th passage (6.25%).(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 2814309 [PubMed - indexed for MEDLINE]

Res Vet Sci. 1981 Mar;30(2):251-2. Related Articles, Links

Virus contamination of bovine testis cell cultures.

Luther PD, Stott EJ, Jebbett J.

PMID: 7255918 [PubMed - indexed for MEDLINE]

Vopr Virusol. 1980 May-Jun;(3):327-30. Related Articles, Links

[Spontaneous contamination of primary green monkey kidney cell cultures by foamy virus]

[Article in Russian]

Mironova LL, Preobrazhenskaia NK, Kniaginskaia IuG, Belova AG.

Examinations of 1653 batches of green monkey kidney cell cultures revealed contamination with foamy virus (FV) in 243 (16%) batches. From some of these cultures 65 strains of virus belonging to two serotypes were isolated. Tests on sera from 1122 monkeys revealed antibody to FV in 985 (87.8%) animals. No correlation between the presence of antibody in the serum and virus recovery from monkey kidney cell cultures was observed, however. This might be due either to the lack of the virus in the kidneys or to the failure in its isolation from primary cultures. Passages of cell cultures were shown to facilitate additional recovery of FV. These experimental results may be used for screening and selection of FV-free cultures and for control purposes in the course of vaccine manufacture.

PMID: 6254265 [PubMed - indexed for MEDLINE]

J Clin Microbiol. 1978 Feb;7(2):214-8. Related Articles, Links

Contamination of primary embryonic bovine kidney cell cultures with parainfluenza type 2 simian virus 5 and infectious bovine rhinotracheitis virus.

Crandell RA, Hierholzer JC, Krebs JW Jr, Drysdale SS.

Two different viruses were isolated from bovine embryonic cell cultures after two subcultures from the primary cells. One virus was identified as parainfluenza type 2 simian virus 5 (SV-5), and the other was identified as infectious bovine rhinotracheitis virus. Six months later, stock cultures of pig kidney (PK-15) cells were found to be contaminated with SV-5 virus. We believe that the source of the SV-5 virus in the bovine cells was a cross-contamination from monkey kidneys during preparation of the cell cultures. The infectious bovine rhinotracheitis contamination was probably of endogenous origin. The bovine embryonic cell cultures were the probable source of contamination of the PK-15 cells with SV-5 virus.

PMID: 75889 [PubMed - indexed for MEDLINE]

Science. 1977 Mar 25;195(4284):1343-4. Related Articles, Links

Inter- and intraspecies contamination of human breast tumor cell lines HBC and BrCa5 and other cell cultures.

Nelson-Rees WA, Flandermeyer RR.

It is shown that the two most recently reported cell lines derived from malignant human breast tissue, HBC and BrCa5 are, respectively, rat and HeLa cell contaminants. The incidence of inter- and intraspecies contamination among 279 cell cultures from 45 laboratories in an 18-month survey is also presented.

PMID: 557237 [PubMed - indexed for MEDLINE]

Nature. 1977 Apr 28;266(5605):835-7. Related Articles, Links

Viral contamination of bovine foetal serum and cell cultures.

Nuttall PA, Luther PD, Stott EJ.
From the article: The presence of adventitious viruses in cell cultures is well recognized, and when the cultures are of primate origin there are serious hazards for the production of human viral vaccines.  This is one reason for the increasing use of bovine cell cultures.  These cultures, however, are not free from viral contamination.  We found that calf kidney (CK) and calt testis (CT) cells were often infected by non-cytopathic mucosal disease virus (MDV): the cells seemed morphologically healthy but nearly all showed fluorescence with MDV antiserum and rabbit-anti-bovine conjugate.  We report here that both the cells and foetal calf serum, an essential growth factor of cell culture medium, are sources of the virus...These results indicate a much higher incidence of contamination than reported previously, and illustrate the inadequacy of commercial screening methods...Infection of bovine cell cultures by non-cytopathic MDV has particular significance for the production of viral vaccines...The importance of contamination by MDV with regard to human viral vaccines is unknown, but measles virus vaccine and a potential respiratory syncytial virus vaccine are produce in bovine kidney cells grown in the presence of unheated commercial FCS...Regular screening of bovine cell cultures is essential if stocks are to be kept free from MDV.

PMID: 194159 [PubMed - indexed for MEDLINE]

Am J Hematol. 1976;1(2):237-42. Related Articles, Links

Identification of cells in culture.

Stulberg CS, Peterson WD Jr, Simpson WF.

Most laboratories using cells cultured in vitro maintain multiple cell lines. Such lines should be monitored for species and intraspecies characteristics to prevent invalidation of research work due to incidents of cell line cross-contamination. This report describes the results obtained when 246 cell cultures were examined for evidence of cross-contamination or mislabeling. Using species-specific antigens, isoenzyme electrophoresis, and chromosomes as markers of identity, 14% of the cultures submitted were found to be contaminated by cells of another species. Of human cell lines submitted 25% were of HeLa cell origin, as determined by 2 intraspecies markers, glucose-6-phosphate dehydrogenase and chromosome analyses. The fact that, overall, nearly 30% of the cell lines examined were incorrectly designated makes the importance of cell line monitoring self-evident.

PMID: 63243 [PubMed - indexed for MEDLINE]

Vopr Virusol. 1976 May-Jun;(3):371-9. Related Articles, Links

[Several methodologic problems in the control of cell cultures]

[Article in Russian]

Demidova SA, Tsareva AA, mikhailova GR, Perekrest VV, Gushchin BV.

Some human and animal continuous cell lines as well as primary cell cultures were examined by karyological, electron microscopial, virological and molecular biological methods and also by the electrophoretic motility of glucose-6-phosphate dehydrogenase (G-6-PDG) in polyacrylamide gel. All human and animal continuous cell lines were shown to contain mycoplasma, 17-to contain intracytoplasmic particles of type A oncornaviruses, 5 -- type B oncornaviruses similar to Mason-Pfizer virus, 8 -- paramyxoviruses, 2 --oncornaviruses type C. A high molecular RNA with sedimentation constant 64--70 S was found in oncornaviruses isolated from cell cultures. Intracellular virus or subviral structures were detected by association of the reverse transcriptase activity with high molecular RNA. The presence in the cell cultures of marker chromosomes of HeLa cells, the absence in these cultures of Y chromosomes, the presence of the G-6-PDG enzyme with type A motility indicate the possibility of contamination of human continuous cell lines with HeLa cells.

PMID: 983006 [PubMed - indexed for MEDLINE]


Dev Biol Stand. 1976 Dec 13-15;37:191-200. Related Articles, Links

The need for non-cultural methods for the detection of mycoplasma contaminants.

Stanbridge EJ, Schneider EL.

Mycoplasmas are common contaminants of cell cultures and are of great importance because of the deleterious effects they have upon the infected host cells. Routine detection testing usually relies upon cultural methods and demonstration of characteristic colonies on agar. Questions as to the efficacy of this method have been raised because of increasing realization of the presence of "non-cultivable" mycoplasmas. Several non-cultural detection methods have been developed to aid in the detection of these fastidious microorganisms. These methods include morphology by light and electron microscopy, immunofluorescence, enzyme assays, autoradiography, sucrose gradient separation, altered transport of nucleic acid precursors and altered nucleic acid profiles. A comparison of the relative sensitivities of these methods will be outlined. The need for, and the feasibility of non-cultural detection methods as quality control tests in human virus vaccine production will be discussed.

PMID: 1031685 [PubMed - indexed for MEDLINE]

In Vitro. 1976 Sep;12(9):643-8. Related Articles, Links

Spread and control of mycoplasmal infection of cell cultures.

McGarrity GJ.

Environmental sampling was performed during trypsinization and passage of 3T-6 cell cultures that contained a mean of 4.3 X 10(7) colony forming units (CFU) per ml supernatant of A. laidlawii. The lip of the culture flask and the outside of the used pipet were always heavily contaminated. The outside of the culture flask (3/7), the work surface (8/12) and the outside of a pan of disinfectant (4/5) were regularly contaminated with mycoplasmas. Airborne mycoplasmas were detected eight of 32 times (25%) by settling plates; simultaneous forced-air samplers by two different methods were always negative. The technician's hands were contaminated two of 15 samples. When hands were contaminated, more contamination was detected in the environment. Droplets of A. laidlawii and M. orale inoculated onto work surfaces survived drying for a minimum of 3 days, even in laminar airflow cabinets. Twenty-five of 31 (80.6%) cell culture technicians carried M. salivarium in their throats; only two carried M. orale. It is concluded that mycoplasma-infected clltures are the most common source of further infection. Recommendations for prevention and control of mycoplasmal infection are listed.

PMID: 1034618 [PubMed - indexed for MEDLINE]
In Vitro. 1975 Nov-Dec;11(6):400-3. Related Articles, Links

Detection of bovine viruses in fetal bovine serum used in cell culture.

Kniazeff AJ, Wopschall LJ, Hopps HE, Morris CS.

This investigation employed a viral screening method detect endogenous bovine virus contaminants in commercially supplied fetal bovine serum. Fifty-one lots of fetal bovine serum from 14 suppliers were examined. Over 30% of the lots tested were found to contain bovine viruses; they included bovine virus diarrhea virus, parainfluenza type3-like virus, bovine herpesvirus-1, bovine enterovirus type 4, and an unidentified cytopathogenic agent. Of the 51 lots, 20 had been pretested by the suppliers and were considered to be free of known viral contaminants. Our viral screening methods revealed that five of these pretested lots, or 25%, contained endogenous bovine viruses.

PMID: 172434 [PubMed - indexed for MEDLINE]


J Infect Dis. 1975 May;131(5):588-91. Related Articles, Links

Bacteriophages and endotoxin in licensed live-virus vaccines.

Moody EE, Trousdale MD, Jorgensen JH, Shelokov A.

In confirmation of recent reports, coliphages were found in seven of 19 unselected samples of the currently licensed live-virus vaccines. Coliphages and pseudomonas phage were found in 11 and 14, respectively, of the 20 bovine sera commonly used in the cell culture phase of virus vaccine production. The same lots of vaccine and serum were examined by the limulus assay for endotoxin, another product of bacterial contamination. Eighteen of 20 sera had detectable endotoxin-like activity. Our preliminary results suggest that endotoxin activity may serve as a sensitive indicator of residual products of previous bacterial contamination, including bacteriophages.

PMID: 805187 [PubMed - indexed for MEDLINE]

Am J Vet Res. 1975 Jan;36(1):41-4. Related Articles, Links

Porcine parvovirus: frequency of naturally occurring transplacental infection and viral contamination of fetal porcine kidney cell cultures.

Mengeling WL.

The frequency of naturally occurring transplacental infection of swine with porcine parvovirus (PPV) and one of the possible consequences of such infection--the presence of PPV in cell cultures prepared from fetal tissues--were investigated. Transplacental infection was indicated by the presence of high titers of hemagglutination inhibiting (HI) antibody for PPV in serums of 0-day-old, hysterectomy-derived, colostrum-deprived pigs of 3 of 82 litters. All letters were farm-raised dams. Moreover, cell cultures prepared from 3 of 49 lots of fetal porcine kidneys (FPK) collected from an abattoir during an interval of 14 months were found contaminated with PPV. Because each lot was usually comprised of kidneys from 2 litters, the latter finding suggests that 3 of approximately 98 litters were infected. Prior infection of FPK cell cultures with PPV resulted in only slight interference of replication of other selected viruses; i.e., porcine enterovirus (PEV), pseudorabies virus (PRV), vesicular stomatitis virus (VSV), and hemagglutinating encephalomyelitis virus (HEV). Moreover, PPV and HEV were propagated in the same cell cultures during 5 serial passages of the viruses. In contrast, when copropagation of PPV and VSV was attempted, PPV was not detected after the 2nd serial passage.

PMID: 163603 [PubMed - indexed for MEDLINE]

Pathol Biol (Paris). 1975 Feb;23(2):151-9. Related Articles, Links

[Viral contamination in laboratories and hospital units]

[Article in French]

Chastel C.

Viral contamination is at least as important in hospital laboratories and wards as contamination by bacteria or microscopic fungi, but it is much more insidious and sometimes unrecognized. There are two main types: The first has a purely technical effect and only interest the virologist. This is contamination of reagents, reference strains, cell cultures, etc.., by foreign viral agents. It may be the cause of errors on diagnosis or regrettable errors of interpretation of certain experimental data. It is most difficult to detect, if not to avoid. The second is much more worrying as it is liable to cause disease in man, which may induce severe, and even fatal infections in patients or in the medical, para-medical and technical personnel. This is the case with type B hepatitis virus which tends to invade surgical units using extra-corporeal circulation, hemodialysis units and transplantation units, blood transfusion centres, dental units and even causes victims in routine laboratories. However, type B hepatitis is not the only virus which may lead to severe infections; other viruses include: poxvirus, cytomegalovirus, arbovirus, etc. Finally, other often severe accidents may occur in research laboratories and in the pharmaceutical industry, owing to manipulation of dangerous viruses or by contact with experimental animals, e.g. rodents, or monkeys, which contain the virus in a latent state, e.g. lymphocytic choriomeningitis, Sabin virus, Marburg virus, type A hepatitis virus, etc. With regard to such accidents, we are almost completely powerless from the therapeutic point of view and, even poorly equipped, from the point of view of prophylaxis.

PMID: 165448 [PubMed - indexed for MEDLINE]

Zentralbl Bakteriol [Orig A]. 1975 Jul;232(2-3):131-40. Related Articles, Links

[Studies on simian viruses as possible contaminants of inactivated virus vaccines. I. Direct and serologic detection of simian adenovirus SV20 (author's transl)]

[Article in German]

von Mettenheim AE.

In the Federal Republic of Germany inactivated vaccines against poliomyelitis and measles are still produced in monkey kidney cell cultures which may be contaminated by simian viruses. One of these viruses is the oncogenic adenovirus SV20. A control of monkey sera from the institute's monkey house showed a high incidence of hemagglutination-inhibiting and neutralizing antibodies against this virus (table 1). Experimentally could be demonstrated that inactivated virus vaccines may be contaminated with SV20 antigen. Vero cells were infected at the time of their seeding with small doses of SV20. After 7 and 14 days CPE and hemagglutinin were frequently undetectable although infectivity could be shown by passages (table 2). Vaccines experimentally contaminated with SV20 were injected into guinea-pigs or rabbits (tables 3-6). In this way small amounts of the contaminating virus antigen could be detected by demonstrating neutralizing and hemagglutination-inhibiting antibodies. It is suggested to include passages of control cultures and of the virus harvest, after neutralization of the vaccine virus, for the control of virulent extraneous viruses. Alternatively to the last suggestion high amounts already inactivated virus pools could be inoculated into animals for the detection of antibodies against extraneous virus antigens. These controls are necessary until inactivated virus vaccines will be produced in safer substrates.

PMID: 1179876 [PubMed - indexed for MEDLINE]

Vopr Virusol. 1974 Sep-Oct;(5):521-7. Related Articles, Links

[Contaminamts of cell cultures]

[Article in Russian]

Demidova SA, Ritova NM.

Publication Types:
  • Review

PMID: 4216160 [PubMed - indexed for MEDLINE]

Vopr Virusol. 1974 Mar-Apr;(2):253-4. Related Articles, Links

[Editorial: Oncornaviruses--contaminants of cell cultures]

[Article in Russian]

[No authors listed]

PMID: 4374830 [PubMed - indexed for MEDLINE]

In Vitro. 1974 Sep-Oct;10(3-4):238-42. Related Articles, Links

On the contamination of cell cultures by Leptospira biflexa.

Tumilowicz JJ, Alexander AD, Stafford K.

PMID: 4476725 [PubMed - indexed for MEDLINE]

Zentralbl Bakteriol [Orig A]. 1972 Apr;219(4):550-4. Related Articles, Links

On the incidence of mycoplasma contamination in cell cultures.

Sethi KK.

PMID: 4145491 [PubMed - indexed for MEDLINE]

Appl Microbiol. 1970 Feb;19(2):381-2. Related Articles, Links

Type 2 bovine adenovirus as an adventitious contaminant in primary bovine embryonic kidney cell cultures.

Mohanty SB, Lillie MG.

PMID: 5437309 [PubMed - indexed for MEDLINE]

J Natl Cancer Inst. 1969 Mar;42(3):489-96. Related Articles, Links

Cytomegaloviruses as common adventitious contaminants in primary African green monkey kidney cell cultures.

Smith KO, Thiel JF, Newman JT, Harvey E, Trousdale MD, Gehle WD, Clark G.

PMID: 4305018 [PubMed - indexed for MEDLINE]

Natl Cancer Inst Monogr. 1968 Dec;29:123-32. Related Articles, Links

Viruses infecting cattle and their role as endogenous contaminants of cell cultures.

Kniazeff AJ.

Publication Types:
  • Review

PMID: 4305954 [PubMed - indexed for MEDLINE]


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