Detection of mycoplasma in contaminated mammalian cell culture using FTIR microspectroscopy.

Mycoplasma contamination represents a significant problem to the culture of mammalian cells used for research as it can cause disastrous effects on eukaryotic cells by altering cellular parameters leading to unreliable experimental results. Mycoplasma cells are very small bacteria therefore they cannot be detected by visual inspection using a visible light microscope and, thus, can remain unnoticed in the cell cultures for long periods. The detection techniques used nowadays to reveal mycoplasma contamination are time consuming and expensive with each having significant drawbacks. The ideal detection should be simple to perform with minimal preparation time, rapid, inexpensive, and sensitive. To our knowledge, for the first time, we employed Fourier transform infrared (FTIR) microspectroscopy to investigate whether we can differentiate between control cells and the same cells which have been infected with mycoplasmas during the culturing process. Chemometric methods such as HCA and PCA were used for the data analysis in order to detect spectral differences between control and intentionally infected cells, and spectral markers were revealed even at low contamination level. The preliminary results showed that FTIR has the potential to be used in the future as a reliable complementary detection technique for mycoplasma-infected cells. Graphical abstract FTIR microspectroscopy is able to differentiate between mycoplasma infected cells (LC for low contamination and HC for high contamination) and control non-infected cells (CN).

Management practices associated with the bulk tank milk prevalence of Mycoplasma spp. in dairy herds in Northwestern Portugal.

The objective of this study was to evaluate the effect of some management practices on the prevalence of Mycoplasma spp. in Northwestern Portuguese dairy farms from bulk tank milk (BTM) samples. Additionally, the within-herd prevalence of Mycoplasma spp. was also determined, but only in BTM positive herds. From May 2007 to November 2008, 492 BTM samples from 164 dairies randomly chosen in a population of 1234 dairy farms were analyzed. Five herds (3.0%) had positive mycoplasmal culture results, from which 4 out of 164 (2.4%) were Mycoplasma bovis, with simultaneous presence of Mycoplasma bovigenitalium or Mycoplasma canadense in two of those samples. In one out of 164 (0.6%) herds Mycoplasma capricolum subsp. capricolum was also found. In BTM positive Mycoplasma spp. herds, the apparent intra-herd prevalence was low and varied between 2.5% and 4.5%. Multiple locus variable-number of tandem-repeat analysis was conducted in order to compare the genetic relationship between the isolates. Mycoplasma spp. was found to be present in cows with subclinical mastitis with or without California Mastitis Test positive results, hence all cows should be tested when the agent is isolated from bulk tank rather than selecting suspected cows. A multivariable logistic regression using the Firth's penalized maximum likelihood estimation was performed showing that increasing number of lactating cows (OR=1.05; P<0.01) was associated with a higher probability of isolating Mycoplasma spp. On the other hand, identifying problem cows was associated with a lower probability (OR=0.06; P<0.05). Particular importance was given to the prevalence of M. bovis, and the results obtained highlight the need to include this agent in mastitis control protocols in national dairies and in sanitary controls of transitioned animals between European countries.

The scope of mycoplasma contamination within the biopharmaceutical industry.

Mycoplasma is well recognized as one of the most prevalent and serious microbial contaminants encountered within the manufacturing of biopharmaceuticals from the research phase to clinical development and production. The potential for mycoplasma contamination within cell culture systems was first identified by Robinson et al. in 1956. Presently, contamination rates in established cell cultures have been reported between 15 and 35% with considerably higher occurrence cited in certain selected populations. In the last few years, there has been an expansion of diagnostic approaches for mycoplasma detection with the development and validation of rapid microbiological methods. The objective of this study was to determine current levels of mycoplasma infection of cell cultures, cell substrates and biologicals within a client based population. Retrospective comparison of 40,000 sample results was done to determine total contaminations rates amongst four (4) individual analytical assays. The establishment of reference data, such as existing contamination rates, becomes important in the critical appraisal of rapid microbiological methods for the detection of mycoplasma.

From bench to cageside: Risk assessment for rodent pathogen contamination of cells and biologics.

Many newly developed animal models involve the transfer of cells, serum, or other tissue-derived products into live rodents. These biologics can serve as repositories for adventitious rodent pathogens that, when used in animal studies, can alter research outcomes and result in endemic outbreaks. This review includes a description of some of the biologics that have inadvertently introduced infectious agents into in vivo studies and/or resulted in endemic outbreaks. I also discuss the points of potential exposure of specific biologics to adventitious rodent pathogens as well as the importance of acquiring a complete developmental and testing history of each biologic introduced into a barrier facility. There are descriptions of specific cases of mycoplasma and lactate dehydrogenase-elevating virus (LDHV), two of the most common organisms that contaminate cells and cell byproducts. The information in this article should help investigators and animal resource program personnel to perform an appropriate risk assessment of biologics before their use in in vivo studies that involve rodents.

Tetrazolium reduction methods for assessment of substrate oxidation and strain differentiation among mycoplasmas, with particular reference to Mycoplasma bovigenitalium and some members of the Mycoplasma mycoides cluster.

AIMS: To apply a rapid nitroblue tetrazolium (NBT) reduction assay of substrate metabolism by mycoplasmas that would help to differentiate Mycoplasmas. METHODS AND RESULTS: Growth, substrate preferences and tetrazolium reduction were assessed for 18 strains of Mycoplasma bovigenitalium and Mycoplasma ovine serogroup 11. NBT reduction was detectable in 1 h with 10(8) CFU ml(-1). Use of alpha-ketobutyrate, lactate and pyruvate to support growth and NBT reduction were correlated: pyruvate was preferred and lactate was used by only four of the 18 strains. Selected members of the Mycoplasma mycoides cluster were also assessed and monotetrazoles tested as alternatives to NBT. The NBT method was applied to a further 19 species. CONCLUSIONS: This simple and reproducible method requires only small amounts of cells, enabling routine assessment of substrate use within 1 h, and the rapid assignment of numerous mycoplasmas to one of six physiological groups. The four physiological groups of M. bovigenitalium and Mycoplasma serogroup 11 strains were indistinguishable from each other, which supports the view that these belong to the same species. SIGNIFICANCE AND IMPACT OF THE STUDY: Strain-specific substrate-utilization patterns by mycoplasmas can be obtained rapidly and reliably. The method has potential as a large-scale semi-automated procedure to monitor numerous strains and substrates simultaneously.

Assessment of infectious organisms associated with chronic rhinosinusitis in cats.

OBJECTIVE: To determine detection rates for feline herpesvirus type 1 (FHV-1), Mycoplasma spp, fungi, and bacteria in flush samples and biopsy specimens from the nasal cavities of cats with and without chronic rhinosinusitis (CRS). DESIGN: Prospective study. ANIMALS: 10 CRS-affected cats and 7 cats without signs of respiratory tract disease. PROCEDURES: Nasal flush samples and biopsy specimens were collected from all cats for bacterial (aerobic and anaerobic), fungal, and mycoplasmal cultures; additional biopsy specimens were collected for virus isolation and polymerase chain reaction (PCR) assay (to detect FHV-1 DNA). RESULTS: Aerobic bacteria were detected in flush samples from 5 of 7 control cats; culture of flush samples from CRS-affected cats yielded aerobic bacteria (9/10 cats), anaerobic bacteria (3/10), and Mycoplasma spp (2/10). No fungal organisms were isolated from any cat. Potential pathogens were isolated significantly more often from CRS-affected cats than from control cats. Bacterial culture of biopsy specimens yielded aerobic bacteria (2/7 control cats and 4/10 CRS-affected cats) and anaerobic bacteria (2/10 CRS-affected cats). Although FHV-1 was not detected in nasal biopsy specimens from control or CRS-affected cats, FHV-1 DNA was detected via PCR assay in specimens from 4 of 7 control cats and 3 of 10 CRS-affected cats. CONCLUSIONS AND CLINICAL RELEVANCE: Compared with findings in control cats, anaerobic bacteria, Mycoplasma spp, and a variety of potentially pathogenic organisms were detected more commonly in samples from cats with CRS. In both groups, FHV-1 was detected via PCR assay as a nonviable organism or in noncultivable amounts.

Assessment of PCR for routine identification of species of the Mycoplasma mycoides cluster in ruminants.

DNA amplification techniques offer considerable promise for the identification of Mycoplasma mycoides cluster members. They avoid antigenic cross-reactivity and variability that hamper serological methods. Many sets of primers, specific of these different members and of Mycoplasma putrefaciens, have been proposed. To assess the reliability of some of these PCR tests in routine laboratory diagnostic use, 230 field strains supposed to belong to this group were simultaneously identified by PCR and an antigenic method. The results were well correlated to antigenic identification for M. putrefaciens, but PCR failed to identify respectively 74% and 52% of M. mycoides subsp. mycoides Large Colony type and M. capricolum subsp. capricolum strains. Any identification of M. mycoides subsp. mycoides Small Colony type must be confirmed by two different tests. Difficulties in defining the M. species bovine serogroup 7 were also encountered with both the PCR and immunological methods. The occurrence of putative variable antigen(s) on the mycoplasma surface may explain part of the identification difficulties encountered with the immunological methods.

Bovine mastitis pathogens in New York and Pennsylvania: prevalence and effects on somatic cell count and milk production.

Milk samples were collected from 108,312 dairy cows during 1601 farm visits made between January 1991 and June 1995. The herd visits were made by personnel from the Central Laboratory of the Quality Milk Promotion Services at Cornell University (Ithaca, NY) to farms located in central New York and northern Pennsylvania. Dairy Herd Improvement Association records were available for 32,978 cows in 327 herds. Intramammary infections, as defined by positive milk cultures, were present in 48.5% of all cows and in 36.3% of cows in herds enrolled in the Dairy Herd Improvement Association. Over 75% of the intramammary infections were caused by Streptococcus agalactiae, Streptococcus spp. other than Strep. agalactiae, Staphylococcus aureus, and coagulase-negative staphylococci. Mean days in milk at the time of diagnosis, linear score of the somatic cell count, cost of milk loss per lactation, and milk production effects were calculated for 24 etiologic agents of bovine mastitis.

Detection and identification of avian mycoplasmas by polymerase chain reaction and restriction fragment length polymorphism assay.

The polymerase chain reaction (PCR) with primers complementary to the 16S rRNA genes was used to detect avian mycoplasmas. A primer pair designed for the detection of human and rodent mycoplasmal species was examined for its ability to detect the most important avian mycoplasmas. After testing the respective reference strains, we found that Mycoplasma iowae, Mycoplasma meleagridis and Mycoplasma synoviae could be detected by PCR with this primer pair, and distinction could be made among them by restriction fragment length polymorphism (RFLP) assay with two restriction enzymes (BamHI and RsaI). For the detection of Mycoplasma gallisepticum by PCR, we needed species-specific primers. The results of the PCR- and RFLP, based identification procedures of 17 different field isolates agreed with those obtained by conventional methods.

Mycoplasmal infections of cerebrospinal fluid in newborn infants from a community hospital population.

Mycoplasma hominis or Ureaplasma urealyticum have previously been isolated from cerebrospinal fluid (CSF) in 13 of 100 newborn infants tested from a high risk university hospital population where the mothers were of predominantly lower income and socioeconomic status and had often received little or no prenatal care. We sought to determine whether such infections occur in neonates born to women cared for mainly through private obstetric practices and who delivered in 4 suburban community hospitals. CSF cultures were done in 318 infants during an 8-month period. M. hominis was isolated from 9 and U. urealyticum from 5 CSF cultures. Four infants infected with U. urealyticum and 3 infected with M. hominis were born at term. One infant infected with U. urealyticum had a birth weight of less than 1000 g. In 5 infants clearance of the infecting organism was documented without specific treatment. Twelve infants had good perinatal outcomes regardless of treatment and 2 died. One death in a 2240-g infant infected with M. hominis was associated with Haemophilus influenzae sepsis and pneumonia. The other death occurred 3 days after birth in a 630-g infant infected with U. urealyticum who had evidence of meningitis and intraventricular hemorrhage. Results of this study suggest that mycoplasmas are common causes of neonatal CSF infections, not only in high risk populations, but also in the general population.

Vaginal colonization with mycoplasma hominis and ureaplasma urealyticum.

Vaginal cultures obtained from unselected young women who consulted the gynecologist in a student health service were examined for Ureaplasma urealyticum and Mycoplasma hominis. Each participant completed a confidential questionnaire. Multiple logistic regression analysis was used to determine which variables, of a large number ascertained, were associated with mycoplasmal colonization. U. urealyticum was isolated from 273 (56.8%) of 481 participants. The following variables were significantly predictive of colonization with U. urealyticum: black race, absence of antibiotic use, cigarette smoking, and number of sexual partners during the last year. Lifetime number of sexual partners was significantly predictive only in women who used nonbarrier methods of contraception. M. hominis was isolated from 85 (17.7%) of the 481 participants. Independent variables that were significantly predictive of colonization with M. hominis included black race, young age, and, for users of nonbarrier methods of contraception, lifetime number of sexual partners.

Porcine respiratory disease in the western Cape Province.

The aetiology and pathogenesis of respiratory syndromes in pigs are reviewed with emphasis on the role of environmental factors and diagnosis. Therapeutic and control measures are suggested and the cost of various regimens is dealt with in detail.

Prevalence of Mycoplasma hominis and Ureaplasma urealyticum (T strains) in urine of adolescents.

Adolescent children were surveyed for colonization with Mycoplasma hominis and Ureaplasma urealyticum by culturing urine specimens. Rates were compared between three study groups: (i) 397 children attending parochial schools, (ii) 293 children attending an adolescent clinic specializing in adjustment problems, and (iii) 86 children attending a renal clinic. The recovery rate was higher among postpubertal girls attending the renal clinic (33%) and the adolescent clinic (26%) than among students attending parochial high school (males 2%, females 8%). Girls had approximately eightfold higher rates than boys of the same age. Isolation of Mycoplasmataceae was associated with certain sociological determinants, such as dating, cigarette smoking, and coming from a broken home, but also with abnormal findings (protein, leucocytes) in urine.