Detecting Mycoplasma contamination in cell cultures by
polymerase chain reaction.
Uphoff CC, Drexler HG.
Contamination of cell cultures
with bacteria, fungi, and yeasts represents a major problem in cell culture.
Whereas these microorganisms are easily detected during routine cell culture
by the turbidity of the culture and observation under the inverted microscope,
one class of bacteria regularly evades detection. These bacteria belong to the
class of Mollicutes,
commonly known as mycoplasma.
Mycoplasma
may persist undetected in cell cultures for a long time without visibly
affecting the culture. Nevertheless, mycoplasma
can cause extensive alterations in the cell cultures. The frequency of
contamination is about 10-30% with pronounced variations in series regarding
the laboratory, type of cell culture examined, and the origin of the culture,
for example. The infecting mycoplasmas are limited to a few species of the
genera Mycoplasma and Acholeplasma with human, swine, and bovine as
predominant natural hosts. The ubiquitous use of cell cultures and their
transfer from one laboratory to another has lead to widespread dissemination
of such infections. Hence, adequate detection methods need to be established
and frequently employed in every laboratory applying cell cultures. Every
incoming cell culture should be kept in quarantine until mycoplasma detection
assays are completed and the infection status is determined. Positive cultures
should either be discarded and replaced by clean cultures or cured with
specific antibiotics. Only clean cultures should be used for research
experiments and for the production of biologically active pharmaceuticals.
Additionally, stringent rules for the prevention of further mycoplasma
contamination of cell cultures should be followed (2).
DSMZ German Collection of Microorganisms and Cell Cultures, Braunschweig,
Germany.
Comment: Although this and many other articles
on contamination with mycoplasma are directed to the effect it has on cells
for research purposes, mycoplasma has the potential to cause disease.
Here is what Joseph Tully wrote in Mycoplasma Infection of Cell Cultures
(1978): "Mycoplasmas were initially recognized as important
animal-pathogens. They were found to involve a variety of organs and
tissues, and to be be associated with acute and chronic respiratory disease,
mastitis, polyarthritis, and genitourinary tract infections (113). The
concept of a vast noormal flora of mycoplasmas in animals (and including man)
was not well appreciated until major advances occurred in the cultivation of
the more fastidious mycoplasmas, such as M. pneumoniae (3). From
that time in the early 1960s, we have seen a continual extension of the number
of mycoplasmas in each animal host selectively surveyed. The great
majority of these mycoplasmas appear to be strictly part of the normal
microbial flora. However, it is important to stress that limited
information is available on the interrelationships of normal mycoplasma flora
and other microbial agents, including virulent as well as avirulent bacteria,
viruses, etc. Thus, normal flora might still play some role in human or
animal diseases when acting in concert with other microbial agents or when the
host is compromised in some way."
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. Birgit.Makoschey@Intervet.com
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.
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.
Detection of bovine viral diarrhea virus by TaqMan reverse
transcription polymerase chain reaction.
Mahlum CE, Haugerud S, Shivers JL, Rossow KD, Goyal SM, Collins JE, Faaberg
KS.
Department of Veterinary Diagnostic Medicine, University of Minnesota, St.
Paul 55108, USA.
Detection and elimination of calves and cows persistently infected with bovine
viral diarrhea virus (BVDV) is important for the control of this pathogen.
Historically, BVDV detection involved cell culture isolation followed by virus
detection through immunofluorescence or immunoperoxidase monolayer assay (IPMA)
methods. More recently, immunohistochemistry (IHC) has been added as a routine
test for BVDV detection. The detection of BVDV by gel-based reverse
transcription polymerase chain reaction (RT-PCR) is more sensitive and rapid
than by cell culture isolation, but test results can be compromised by sample
contamination during nucleic acid amplification. This study was designed to
develop a closed-tube format of BVDV nucleic acid amplification and detection,
TaqMan RT-PCR. The results of this new technique were compared with those
obtained with virus isolation, IPMA, and IHC. With TaqMan RT-PCR, BVDV was
detected in many samples negative by IPMA, IHC, and virus isolation with the
exception of 1 sample that was positive by IHC. TaqMan RT-PCR in a closed-tube
format offers a rapid, economical, high volume, and sensitive method for BVDV
detection without the concerns of amplified cDNA product contamination
associated with open-tube gel-based PCR tests.
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. cup@dsmz.de
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.
Evaluation of a new sandwich enzyme-linked immunosorbent
assay for detection of bovine viral diarrhea virus in unprocessed fetal
bovine serum.
Plavsic MZ, Prodafikas G.
Life Technologies, Grand Island Cell Culture R&D, NY 14072, USA.
A new sandwich enzyme-linked immunosorbent assay (S-ELISA) kit that uses raw
(unprocessed) fetal bovine serum (FBS) as the testing sample was evaluated
for upstream bovine viral diarrhea virus (BVDV) testing. Pooled FBS samples
(n = 84) were tested using the S-ELISA. Thirty serum samples originating
from persistently infected (PI) calves that had been confirmed by virus
isolation (VI) as BVDV positive and another 30 samples previously confirmed
by VI as BVDV negative were also evaluated. Of the 84 field samples, the
S-ELISA detected 13 (15.5%) BVDV-positive specimens. When these 13 positive
samples were tested by VI and immunofluorescent assay, 11 (84.6%) were
positive and 2 (15.4%) were negative. The S-ELISA was positive for all 30 PI
samples (100%) and negative for all 30 negative samples (100%). These data
indicate that the new kit is a relatively reliable diagnostic tool and can
be considered for upstream detection of BVDV-contaminated raw FBS pools.
The early detection of microbial
contamination is an important issue in the production of biopharmaceuticals
using animal cell culture systems. A new method, based on the ChemScanRDIanalyser,
was evaluated for the rapid detection and enumeration of low concentrations of
microorganisms within a large population of animal cells and in cell culture
media. The method was tested with suspension cell cultures and is
applicable to adherent cell cultures. In both cases, the method uses an
initial step to eliminate the animal cells followed by collection and
fluorescent labelling of the viable microbial contaminants on a filter
membrane. Total counts of the viable microorganisms were obtained after
analysis of the membrane by the ChemScan RDI analyser. The results showed that
the ChemScan RDI detected individual bacterial cells after filtration of the
pre-treated animal cell culture. The detection limit of the ChemScan RDI was
less than 10 bacteria/ml in cell culture containing 10(6) mammalian cells/ml
and one bacterium in 500 ml of cell culture medium. A strong correlation
between the standard plate count and the ChemScan RDI was observed, even at
low bacterial concentrations. The total time for each analysis was less than
two hours.
[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.
Mycoplasma detection in cell cultures: a comparison of four
methods.
Garner CM, Hubbold LM, Chakraborti PR.
Oncology Research Laboratory, Derby Cancer Centre, Derby City General
Hospital, Uttoxeter Road, Derby DE22 3NE, UK.
Mycoplasma is a common contaminant of
tissue culture samples. Infection is persistent, difficult to detect and
diagnose, and very difficult to cure. The concentration of mycoplasma
in infected cultures can be as high as 10(7) colony-forming units per mL, and
their presence can change many of the cell reactions, including altering cell
growth rate, inducing morphological changes or cell transformation, and
mimicking virus infection. Therefore, it should be assumed that a
mycoplasma-contaminated cell line may be significantly influenced in every
respect, and, thus, experimental data derived from such a cell line is likely
to be invalid. Contamination is not obvious, either macroscopically or
microscopically; thus, routine mycoplasma testing is essential for any cell
culture laboratory. Many of the testing procedures developed so far are
time-consuming, expensive, inconclusive and unsuitable for screening large
numbers of test specimens. This study compares DNA staining, enzyme-linked
immunosorbent assay (ELISA), polymerase chain reaction (PCR) and PCR ELISA, to
determine which is the best procedure for routine assessment of cell cultures.
All four methods gave reproducible results with both infected and non-infected
cell lines. Both ELISA methods were easy to perform, reproducible and easily
interpreted.
Detection of mycoplasma contaminations in cell cultures by
PCR analysis.
Uphoff CC, Drexler HG.
DSMZ-German Collection of Microorganisms & Cell Cultures, Department of Human
and Animal Cell Cultures, Braunschweig, Germany. cup@dsmz.de
Mycoplasma contamination is still one
of the main problems in using cell cultures in biological and medical research
and in the production of bioactive substances, because mycoplasma can alter
nearly all parameters and products of the cell. They can persist
undetected in the culture if no special detection methods are applied. In
recent years, the PCR technology has become a commonly used method to analyze
genomic DNA and the expression of genes, with both high specificity and
sensitivity. This technique can be effectively employed for the detection and
even the identification of mycoplasma contaminations in cell cultures applying
primers complementary to the 16S rDNA region. Although this technique, once
established, is characterized by simplicity and speed, PCR is still a complex
process and its sensitivity and specificity can be influenced by a number of
different parameters, e.g. inhibiting compounds originating from the
preparation process of the DNA, RNA or cDNA, contamination of the solutions
with PCR products, and the selection of a primer pair which does not cover all
the mycoplasma species occurring in cell cultures. Thus, adequate controls
have to be included to obtain reliable results. The present review examines
the use of different primers of the 16S rDNA region including their
specificity, the sensitivity applying various DNA or RNA preparation
procedures, and the methods to detect finally the amplicons. In conclusion,
basic nucleic acid preparation and PCR product detection methods offer a
simple, fast and reliable technique for the examination of mycoplasma
contaminations in cell cultures, provided that the indispensable control
assays are implemented.
Cell culture contamination: sources, consequences,
prevention, and elimination.
Lincoln CK, Gabridge MG.
Bionique Testing Laboratories, Inc., Saranac Lake, New York 12983, USA.
The subject of the chapter is cell culture contamination. Contamination may
enter the cell culture system as a physical, chemical, and/or biological
component of the environment. The potential sources and consequences of cell
culture contamination are unique to the cell culture system and the
contaminant. A basic understanding of cell culture contamination is
necessary to appreciate the need to develop and practice standardized cell
culture procedures. General sources, consequences, and preventative measures
are discussed for physical and chemical contamination based on current
technology. Mycoplasmal contamination is the focus of the discussion on
biological contamination and its impact on cell cultures. The introduction
of other biological contaminants should be controlled by the institution of
cell culture management procedures needed to minimize the incidence of
mycoplasmal contamination. The need
to eliminate the routine use of antibiotics in cell culture systems and
institute routine testing to detect contamination is emphasized. More rapid
detection of contamination should reduce the incidence of
cross-contamination and minimize the consequences of any contamination
event.
Use of polymerase chain reaction to simultaneously detect
and type bovine viral diarrhoea viruses isolated from clinical specimens.
el-Kholy AA, Bolin SR, Ridpath JF, Arab RM, Abou-Zeid AA, Hammam HM,
Platt KB.
Veterinary Sera and Vaccines Researches Institute, Genetic Engineering
Research Unit, Cairo, Egypt.
The techniques of indirect immunofluorescence (IF), immuno-peroxidase (IP)
staining and the one-step reverse transcriptase polymerase chain reaction (RT-PCR)
were compared for detection of 102 isolates of bovine viral diarrhoea virus
(BVDV) in infected cell cultures. The BVDV was obtained from bovine clinical
specimens, including sera, buffy coats and tissues, submitted from farms
located in the States of Iowa and Wisconsin, United States of America. The
IF technique detected 88/102 (86.3%) of the viral isolates, whereas IP
staining detected an additional 4 isolates (92/102; 90%). The one-step
RT-PCR using primers derived from the 5' untranslated region of the BVDV
genome detected 102/102 (100%) of the BVDV isolates. A second-round PCR
utilising another pair of PCR primers from the 5' untranslated region,
allowed rapid genotyping of BVDV. The procedure used showed that the PCR
assay based on the 5' untranslated region of the virus genome is the most
sensitive indicator for BVDV detection in cell culture, and is also of
considerable epidemiological importance since it allowed rapid genotyping of
BVDV isolated from clinical specimens. In addition to detection and
genotyping of BVDV isolated from clinical specimens, the RT-PCR procedure
can be used for routine screening of locally produced and imported
biologicals for BVDV contamination. However, the procedure requires further
refinement to enable direct application on the clinical specimen.
European Collection of Cell Cultures, Centre for Applied Microbiology and
Research, Porton Down, Salisbury, UK.
Continuous cell lines offer a level of reproducibility, and thus
standardization, which cannot normally be achieved using primary cells.
However, even with continuous cell lines adoption of correct cell banking
and appropriate quality control procedures are critical to the provision of
reliable, reproducible and safe cell stocks. These procedures enable
establishment of cryopreserved stocks of pure cultures of correct identity
and phenotype which are free from adventitious agents. In addition to
quality control techniques, culture conditions and growth medium used often
require standardization. In particular different sources of serum, growth
factors and cell attachment substrates may lead to significant variation in
the 'performance' of cell lines. To ensure a high degree of reliability it
is essential to obtain cells from authenticated and quality controlled
sources. In culture collections, the principles of correct cell banking
should be applied with appropriate quality control for which the minimum
standard should be confirmation of viability and mycoplasma testing. Such
approaches will afford increased confidence in research data and avoid the
waste of time and resources which result from the use of cross-contaminated
or infected cells.
Detection of bovine polyomavirus contamination in fetal
bovine sera and modified live viral vaccines using polymerase chain
reaction.
Kappeler A, Lutz-Wallace C, Sapp T, Sidhu M.
Biologics Evaluation Laboratory, Animal Diseases Research Institute,
Agriculture and Agri-Food Canada, Nepean, Ontario, Canada.
A nested polymerase chain reaction (PCR) assay has been developed for the
detection of bovine polyomavirus (BPyV) DNA. The assay has been used to
screen commercial lots of fetal bovine serum and modified live veterinary
vaccines for the presence of the agent. A PCR product of the expected size
was detected after the first round of PCR for eight out of 20 serum lots,
but in none of the 14 vaccines tested. The subsequent nested assay
revealed that four more serum lots were positive for BPyV DNA, as well as
two vaccine lots. When hybridized with a labelled probe, blots of the PCR
products from vaccines revealed that in one of the two positive samples a
specific product was present after the first PCR at a level not detectable
in gel electrophoresis. Nested PCR appears to be a useful tool for the
detection of low level contamination with BPyV DNA of products used in,
and derived from cell culture.
A comparison of methods for the estimation of retroviral
burden.
Bierley ST, Raineri R, Poiley JA, Morgan EM.
IEM Section, Pathology Associates International, Durham NC, USA.
The presence of retroviral contamination is of vital concern in the
manufacture of cell culture-derived biopharmaceuticals. These cell lines
usually have A- or C-type retrovirus-like particles which are visible by
transmission electron microscopy (TEM) even when infectivity (IF) or reverse
transcriptase activity (RTA) cannot be demonstrated. The supernatant of the
post-production cell cultures, therefore, also needs to be evaluated by TEM
for viral burden. A major question, however, is how to establish a
quantitative viral load estimate for the evaluation of a purification
process. The FDA recommends that a purification process for viral
contaminants remove or inactivate 3-5 logs over the estimated viral burden.
Viral particles are difficult to identify and quantify, however, by
conventional negative staining. We present a comparison of infectivity
assay, reverse transcriptase assay, negative staining, and thin sectioned
TEM. These assays were performed on four samples. Ultracentrifuged sediments
of cleared cell-culture media were measured, fixed and processed. Thin
sections were evaluated by TEM and the number of viral particles estimated
by morphometric derived quantification. Retrovirus particles were easily
identified and quantified when examined by TEM as compared to negative
staining and correlated with the other viral assays (IF, RTA). These results
demonstrate that the TEM thin section method was a superior technique to
negative staining for estimating viral particle load in cell-culture
supernatant. To validate further the plastic embedding with thin sectioning,
we evaluated cell culture supernatants (pellets) for retroviral burden at
various dilutions, from two cell lines. Morphometric determinations were
made of the number of viral particles present per unit volume and compared
to results obtained by infectivity assay. Since the morphometric calculation
for viral density assumes even distribution of viral particles, we also
evaluated and calculated viral counts on multiple thin sections taken
throughout selected pellets.
Detection by polymerase chain reaction of all common
Mycoplasma in a cell culture facility.
Pruckler JM, Pruckler JM, Ades EW.
Biological Products Branch, Centers for Disease Control and Prevention,
Atlanta, Ga 30333, USA.
The identification of cell cultures contaminated with organisms from the
class Mollicutes has led us to examine the effectiveness of polymerase chain
reaction (PCR) for detecting these organisms in genomic DNA. We developed a
previously identified nested PCR primer set and compared its ability to
detect Mycoplasma with that of a commercially available PCR kit for
detecting Mycoplasma. We found that although the commercial system detected
and identified a few of the most common Mycoplasma species, the primer set
(GPO-1, GPO-2, MGSO) detected the presence of all the common Mycoplasma
species and many of the rare mycoplasma species previously encountered in
tissue culture.
Rapid detection of mycoplasma contamination in cell
cultures by enzymatic detection of polymerase chain reaction (PCR) products.
Kobayashi H, Yamamoto K, Eguchi M, Kubo M, Nakagami S, Wakisaka S,
Kaizuka M, Ishii H.
National Institute of Animal Health, Ibaraki, Japan.
Enzymatic detection of polymerase chain reaction (ED-PCR) was applied for
rapid and easy identification of mycoplasmas from contaminated cell culture.
This method was based on the capture of amplified products via biotin-streptavidine
affinity and the detection of an incorporated hapten in amplified products
with enzyme-linked antibody. Primers corresponding to common sequence of
Mollicutes in 16S ribosomal RNA dominated gene was used. Nineteen of twenty
Mollicutes so far reported as cell contaminants appeared positive by ED-PCR,
whereas remaining one, Acholeplasma axanthum, appeared negative. Samples
from sixty-two cell culture were tested for contamination of mycoplasmas by
means of ED-PCR, cultivation, and electronmicroscopy. The results of ED-PCR
were the same as those of cultivating method. The time required for all the
detection process in ED-PCR was about 5 hr for 20 samples. We suggest that
ED-PCR can be used in the rapid detection of mycoplasms from cell culture.
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)
Detection of mycoplasma contamination in cell cultures by
a mycoplasma group-specific PCR.
van Kuppeveld FJ, Johansson KE, Galama JM, Kissing J, Bolske G, van der
Logt JT, Melchers WJ.
Department of Medical Microbiology, University of Nijmegen, The Netherlands.
The suitability of a 16S rRNA-based mycoplasma group-specific PCR for the
detection of mycoplasma contamination in cell cultures was investigated. A
total of 104 cell cultures were tested by using microbiological culture, DNA
fluorochrome staining, DNA-rRNA hybridization, and PCR techniques. A
comparison of the results obtained with these techniques revealed agreement
for 95 cell cultures. Discrepant
results, which were interpreted as false negative or false positive on the
basis of a comparison with the results obtained with other methods, were
observed with nine cell cultures. The microbiological culture technique
produced false-negative results for four cell cultures. The hybridization
technique produced false-negative results for two cell cultures, and for one
of these cell cultures the DNA staining technique also produced a
false-negative result. The
PCR may have produced
false-positive results for one cell culture. Ambiguous results were obtained
with the remaining two cell cultures. Furthermore, the presence of
contaminating bacteria interfered with the interpretation of the DNA
staining results for 16 cell cultures. For the same reason the hybridization
signals of nine cell cultures could not be interpreted. Our results
demonstrate the drawbacks of each of the detection methods and the
suitability of the PCR
for the detection of mycoplasmas in cell cultures.
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?
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.
Detection of bacterial and mycoplasma contamination in
cell cultures by polymerase chain reaction.
Spaepen M, Angulo AF, Marynen P, Cassiman JJ.
Center for Human Genetics, University of Leuven, Belgium.
A fast and simple method to detect bacterial and especially mycoplasma
contamination in tissue culture by means of polymerase chain reaction (PCR)
amplification is described. In a first step the universal primer pairs P1/P2
(190-bp fragment) and P3/P4 (120-bp fragment) directed to different
conserved parts of the prokaryotic 16S rRNA gene are used. A positive signal
after amplification on cell culture DNA with these primers provides an
indication of bacterial infection. Using the internal primers IP1, IP3 and
IP'3 complementary to a part of the V4 and V8 variable regions of the 16S
rRNA gene, in combination with a universal primer, cultures contaminated
with mycoplasma could be identified. Six mycoplasma species, typical
contaminants in tissue cultures, were investigated: Mycoplasma orale, M.
fermentans, M. arginini, M. hyorhinis, M. hominis and Aeromonas laidlawii.
This mycoplasma test is an easy, specific and sensitive assay which should
be extremely useful in any tissue culture setting.
The possibility for viral contamination exists in established cultures as
well as in primary cultures. The use of established genetically engineered
cultures in the production of biologicals for human use requires that these
cultures be monitored for adventitious viral agents. Among the methods used
for this purpose are animal inoculation and in vitro assays which provide a
broad-spectrum screen for viral agents.
The possibility for viral contamination exists in established cultures as
well as in primary cultures. The use of established genetically engineered
cultures in the production of biologicals for human use requires that these
cultures be monitored for adventitious viral agents. Among the methods used
for this purpose are animal inoculation and in vitro assays which provide a
broad-spectrum screen for viral agents.
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.
Development of in vitro tests for detection of extraneous
agents.
Thornton DH, Nicholas RA, Wood GW.
The use of animals in tests to detect extraneous agents is not only
undesirable from the ethical viewpoint but also because of the expense and
length of time involved in such tests. We have carried out tests on a
variety of potential contaminating avian pathogens to determine whether
tests in chicks offer any advantage over tests in embryos or cell cultures.
In many cases, but not all, in vitro tests were shown to be more sensitive.
The use fluorescent antibody or enzyme-linked assays serves to enhance the
sensitivity of the tests. In the future it may be possible to adapt
techniques such as nucleic acid hybridisation to the detection of extraneous
agents.
An enzyme-linked immunosorbent assay, ELISA, for SV40
antigen detection.
Edevag G, Grandien M, Mares I.
The exclusion of SV40 contamination in poliovaccine produced in Cynomolgus
monkey kidney cell cultures is routinely done by inoculation of the
inactivated vaccine into Cercopithecus monkey kidney cell cultures where a
cytopathic effect reveals the presence of the virus. An ELISA is described
for the detection of SV40 antigen and the efficiency of antigen detection
was compared with the development of cytopathic effect in Cercopithecus
tissue cultures. The assay shortened considerably the time for production
control and was in full agreement with the conventional test method.
Detection of avian leukosis virus: comparison of five
techniques.
Nicholas RA, Thornton DH.
Five techniques were compared for their ability to detect decreasing
dilutions of RAV-I, an avian leukosis sarcoma virus, in serially passaged
chick embryo fibroblast cell cultures. The indirect fluorescent antibody
test, sandwich enzyme-linked immunosorbent assay (ELISA) and reverse
transcriptase assay were equally sensitive in detecting the virus. The
indirect immunoperoxidase and complement fixation avian leukosis tests were
less sensitive. It is recommended that the sandwich ELISA be used for
routine detection of the avian leukosis sarcoma virus group as possible
vaccine contaminants because it is rapid and simple to perform and may be
carried out on a large number of samples conveniently.
[Use of a cocultivation method for detecting
cytomegalovirus contamination of cell cultures of simian origin]
[Article in Russian]
Karetnyi IuV, Dzagurov SG, Shalunova NV, Elekoev KA.
At present, nonanthropoid
primates are widely used as sources of cell cultures for manufacture of live
viral vaccines. Simian cell cultures, particularly kidney cell cultures are
also known to be frequently contaminated with cytomegaloviruses.
The isolation of the latter is rather difficult due to the late appearance
of the cytopathic
effect in cell cultures of natural hosts. In the present study, the
sensitivity of 4 methods virus isolation from the test cells was compared:
the method of long-term cultivation of cells; the method of long-term
cultivation with one subpassage of the cells; the method of cocultivation of
the test cells by mixing with sensitive cells; and the method of
co-cultivation by overlaying the test cells on an incomplete monolayer of
sensitive cells. The latter method shortened the observation period and
yielded a higher percentage of isolation of contaminating viruses from
African green monkey kidney cell cultures. This method is supposed to be
used in future for the detection of viral contamination of African group
monkey kidney cell cultures utilized in manufacture of live viral vaccines.
[Problems in detection of mycoplasma contamination in
cell cultures (author's transl)]
[Article in French]
Bonissol C, Gilbert M, Ivanova LM.
A total of 135 cell lines was
examined for mycoplasma contamination using two techniques: isolation and
specific DNA-staining with
DAPI or "Hoechst 33258". The
two techniques showed similar results in 64% of the cases of cell
contamination while the remainder was detected only by one or the other
techniques: 12,82% by the staining technique and 23% by the isolation
technique, which shows that the 2 techniques are complementary. The
staining technique is very quick, easy to execute, very sensitive, and
should be the method of choice to detect contamination when the mycoplasma
do not grow in acellular media.
Comparison of methods for the detection of Mycoplasmal
contamination of cell cultures: a review.
Schneider EL, Stanbridge EJ.
Several reviews in recent years have emphasized the problems created by
mycoplasmal contamination of cultured cells (1-5). Because of the hazards of
interpreting data derived from mycoplasma contaminated cells, most cell
biologists routinely screen their cultures for the presence of these
organisms. In recent years, the limitations of standard microbiological
testing for mycoplasmas have become increasingly apparent and have led to
the development of several new biochemical techniques for detection of these
organisms. The aim of this review is to describe and compare available
detection techniques and to evaluate their relative efficacy. Those
properties of mycoplasmas that are relevant to their role as cell culture
contaminants will be briefly discussed.
Mycoplasma in African green monkey kidney cell cultures:
biochemical detection and effects in virus-infected cells.
Van Roy F, Fiers W.
Among a number of techniques for the detection of mycoplasmal contamination
in African green monkey kidney (AGMK) cell lines, the assay of uridine
phosphorylase activity is unsuitable because of the presence of high levels
of endogenous enzymatic activity. A thymidine phosphorylase test, however,
based on the chromatographic analysis of radiolabeled thymidine breakdown,
turned out to be a simple and sensitive mycoplasma detection method. We
found, using the latter technique, that 0.22-micrometer-filtered virus
inocula could still transfer mycoplasma unless treated with diethyl ether.
The effect of mycoplasmal contamination on the synthesis of simian virus 40
and adenovirus in AGMK cells was negligible under the conditions used (no
depletion of arginine). Incorporation of radioactive thymidine in viral
macromolecules, however, was inhibited severely by the presence of
mycoplasma.
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.
Monitoring bovine viral diarrhea virus vaccines for
adventitious virus, using T1 ribonuclease viral RNA oligonucleotide
fingerprinting.
Kelling CL, Kennedy JE, Rump KK, Stine LC, Paul PS, Partridge JE.
Department of Veterinary Science, University of Nebraska, Lincoln 68583-0905.
Viral RNA oligonucleotide fingerprinting was used to discriminate 3 cytopathic
vaccine bovine viral diarrhea viruses (BVDV) grown in medium supplemented with
serum contaminated with noncytopathic BVDV from the same 3 viruses grown in
cell culture free of BVDV. Oligonucleotide fingerprinting also effectively
discriminated between reference Singer BVDV, NADL BVDV, and New York-1 BVDV
grown in BVDV-free noncontaminated or BVDV-contaminated cell cultures.
Oligonucleotide fingerprint mapping of viral RNA maybe used to determine the
purity of virus stocks, as well as that of BVDV vaccines.
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.
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.
DISCLAIMER: All
information, data, and material contained, presented, or provided here is for
general information purposes only and is not to be construed as reflecting the
knowledge or opinions of the publisher, and is not to be construed or intended
as providing medical or legal advice. The decision whether or not to vaccinate
is an important and complex issue and should be made by you, and you alone, in
consultation with your health care provider.
"A foolish faith in authority is the worst enemy of truth."
-- Albert Einstein, letter to a friend, 1901
"I know of no safe depository of the ultimate powers of the society but the people themselves, and if we think them not enlightened enough to exercise control with a wholesome discretion, the remedy is not to take it from them, but to inform their discretion by education."
-- Thomas Jefferson, letter to William C. Jarvis, September 28, 1820
"What's the point of vaccination if it doesn't protect you from the unvaccinated?"
-- Sandy Gottstein
"Who gets to decide what the greater good is and how many will be sacrificed to it?"
