Discrimination between some Mycoplasma spp. and Acholeplasma laidlawii in bovine milk using high resolution melting curve analysis.

OBJECTIVES: This study aimed to provide a rapid, accurate and cost-effective diagnostic real time polymerase chain reaction-high resolution melting curve assay (PCR-HRM) to identify and distinguish between four different mycoplasmas and Acholeplasma laidlawii isolated at cow-level from a single commercial dairy farm in South Australia. One set of genus-level universal primers was designed targeting the 16S ribosomal RNA gene. RESULTS: Real time PCR-HRM analysis was able to identify and distinguish between five different mollicutes, namely A. laidlawii, M. arginini, M. bovirhinis, M. bovis and uncultured Mycoplasma. Results were confirmed through sequencing. Our developed assay provides rapid and accurate screening for Mycoplasma mastitis detection.

Retention of Acholeplasma laidlawii by Sterile Filtration Membranes: Effect of Cultivation Medium and Filtration Temperature.

This experimental study compares cell size, zeta potential, and the ability to penetrate tailor-made size exclusion membrane filters of mycoplasma Acholeplasma laidlawii cultivated in five different cultivation media. The influence of relevant filtration process parameters, in particular transmembrane pressure and filtration temperature, on their respective retention was tested. The impact of the filtration temperature was further evaluated for the Gram-negative bacteria species Brevundimonas diminuta, the Gram-positive bacteria species Staphylococcus epidermidis, the Pseudomonas phage PP7, and the mycoplasma species Mycoplasma orale The findings were correlated to the different mechanical properties of the particles, especially also with respect to the different bacterial cell envelopes found in those species. This study suggests that mycoplasma, surrounded by a flexible lipid bilayer, are significantly susceptible to changes in temperature, altering the stiffness of the cell envelope. Mycoplasma retention could thus be increased significantly by a decreased filtration temperature. In contrast, Gram-negative and Gram-positive bacteria species, with a cell wall containing a cross-linked peptidoglycan layer, as well as bacteriophages PP7 exhibiting a rigid protein capsid, did not show a temperature-dependent retention within the applied filtration temperatures between 2 and 35 °C. The trends of the retention of A. laidlawii with increasing temperature and transmembrane pressure were independent of cultivation media. Data obtained with mycoplasma M. orale suggest that the trend of mycoplasma retention at different filtration temperatures is also independent of the membrane pore size and thus retention level.LAY ABSTRACT: Media in biopharmaceutical processes are sterile-filtered to prevent them from bacterial contamination. Mycoplasma represent a relevant class of bacteria. In this publication it is shown that mycoplasma cell size depends on the media they are cultivated in. Membranes used for sterile filtration retain bacteria predominantly by size exclusion. Thus, an altered cell size can result in different retention values. Another characteristic of mycoplasma is the flexible lipid bilayer and the absence of a rigid cell wall. The lipid bilayer can undergo a phase transition from a gel to a liquid-crystal phase at a certain temperature, which makes it stiffer at lower temperatures. A higher stiffness can result in higher retention values during filtration, as the deformability of the mycoplasma cell is lower and the cell does not squeeze through the membrane pores. ABBREVIATIONS: ALCM: A. laidlawii culture medium; ASTM: American Society for Testing and Materials; ATCC: American Type Culture Collection; CFU/mL: colony-forming units per milliliter; DLS: Dynamic light scattering; LRV: Log reduction value; PES: Polyethersulfone; PFU/mL: Plaque-forming units per milliliter; PSD: Particle size distribution; PVP: Polyvinylpyrrolidone; SDS: Sodium dodecyl sulfate; SEM: Scanning electron microscopy; SLB: Saline lactose broth; TMP: Transmembrane pressure; TSB: Tryptic soy broth.

Mycoplasma Clearance and Risk Analysis in a Model Bioprocess.

Mycoplasmas are a type of bacteria that lack cell walls and are occasional cell culture contaminants. In a biotechnology setting, because they can pass through 0.2 µm filters, mycoplasmas could pose a potential patient safety hazard if undetected contaminants from the production culture were not completely removed by downstream biotechnology manufacturing. In this study we investigated the ability of typical commercial monoclonal antibody purification operations to clear and kill mycoplasmas, using Acholeplasma laidlawii as a model organism. Our spike/removal studies have shown that protein A column chromatography clears about 4-5 log10 Column regeneration effectively prevents A. laidlawii column carryover between chromatography runs. Moreover, low-pH hold steps, typically implemented after protein A purification, effectively kill A. laidlawii using either pH 3.8 glycine or acetate solutions (LRV ≥5.30 and ≥4.57, respectively). Solvent/detergent treatment, used in some processes instead of low-pH hold, also completely kills highly concentrated A. laidlawii (LRV ≥5.95).LAY ABSTRACT: Biotechnology medicines need to be free from contaminating microorganisms such as mycoplasmas, a type of bacteria that can cause disease in humans (e.g., walking pneumonia). Here we show that some monoclonal antibody manufacturing steps can effectively clear and/or kill Acholeplasma laidlawii, a model mycoplasma species used in our study. This provides an additional level of safety assurance of biotechnology medicines for patients.

The Development of a Microbial Challenge Test with Acholeplasma laidlawii To Rate Mycoplasma-Retentive Filters by Filter Manufacturers.

Mycoplasma are bacteria that can penetrate 0.2 and 0.22 µm rated sterilizing-grade filters and even some 0.1 µm rated filters. Primary applications for mycoplasma filtration include large scale mammalian and bacterial cell culture media and serum filtration. The Parenteral Drug Association recognized the absence of standard industry test parameters for testing and classifying 0.1 µm rated filters for mycoplasma clearance and formed a task force to formulate consensus test parameters. The task force established some test parameters by common agreement, based upon general industry practices, without the need for additional testing. However, the culture medium and incubation conditions, for generating test mycoplasma cells, varied from filter company to filter company and was recognized as a serious gap by the task force. Standardization of the culture medium and incubation conditions required collaborative testing in both commercial filter company laboratories and in an Independent laboratory (Table I). The use of consensus test parameters will facilitate the ultimate cross-industry goal of standardization of 0.1 µm filter claims for mycoplasma clearance. However, it is still important to recognize filter performance will depend on the actual conditions of use. Therefore end users should consider, using a risk-based approach, whether process-specific evaluation of filter performance may be warranted for their application. LAY ABSTRACT: Mycoplasma are small bacteria that have the ability to penetrate sterilizing-grade filters. Filtration of large-scale mammalian and bacterial cell culture media is an example of an industry process where effective filtration of mycoplasma is required. The Parenteral Drug Association recognized the absence of industry standard test parameters for evaluating mycoplasma clearance filters by filter manufacturers and formed a task force to formulate such a consensus among manufacturers. The use of standardized test parameters by filter manufacturers, including the preparation of the culture broth, will facilitate the end user's evaluation of the mycoplasma clearance claims provided by filter vendors. However, it is still important to recognize filter performance will depend on the actual conditions of use; therefore end users should consider, using a risk-based approach, whether process-specific evaluation of filter performance may be warranted for their application.

Sensitive and selective detection of mycoplasma in cell culture samples using cantilever sensors.

In this article we report a new biosensor-based method that is more sensitive and rapid than the current approach for detecting mycoplasma in cell culture samples. Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors respond to mass change via resonant frequency change. They are sensitive at femtogram level and can be used directly in liquid for label-free detection. Common cell culture contaminant, Acholeplasma laidlawii was detected in both buffer and cell culture medium. Two different sources (positive control from a commercial kit and ATCC 23206) were analyzed using antibody-immobilized PEMC sensor. Resonant frequency decrease caused by binding of A. laidlawii was monitored in real-time using an impedance analyzer. Positive detection was confirmed by a second antibody binding. The limit of detection (LOD) was lower than 10(3) CFU/mL in cell culture medium using PEMC sensor while parallel ELISA assays showed LOD as 10(7) CFU/mL. This study shows that PEMC sensor can be used for sensitive and rapid mycoplasma detection in cell culture samples.

Taxonomic status of the agent of cereals pale-green dwarf and proposition of founding in the class of Mollicutes, of the order III Acholeplasmatales, family I Acholeplasmataceae, genus II Pluraplasma gen. nov., and its first species Pluraplasma granulum sp. nov.

The paper includes the data concerning the taxonomic status of the agent of cereals pale-green dwarf (ACPGD) which has been defined as the phytopathogenic variant of the mollicute Acholeplasma laidlawii and called A. laidlawii var. granulum. Since besides phytopathogenicity ACPGD has such fundamental differences from A.laidlawii as: a very large genome of 2200 thousand pairs of nucleotides (t.p.n.) to 2310 t.p.n. that practically equals a sum of genomes of A. laidlawii (1600 t.p.n.) and phytoplasmas (710 t.p.n.); two forms of DNA-dependent RNA-polymerase (one form functions in A. laidlawii); a capacity to form extracellular fructosobisphos-phatase which looks like its hypothetical phylogenetic precursor a bacteria Bacillus subtilis; availability of numerous fermentative activities which are absent in acholeplasmas; peculiar relation to sterols availability in nutrient media that is not characteristic of all the known acholeplasmas; extremely rich, as to quantity and quality, composition of antigens to react almost homologously with antibodies to representatives of Acholeplasma genus and separate species of Mycoplasma genus; great similarity (above 88 %) of sequences of 16S rRNA of ACPGD and representatives of Phytoplasma genus and other properties described in the paper, so it is concluded, that proceeding from its characteristics, ACPGD cannot be referred to either of the existing genera of the Mollicules class, because according to all its features this mollicute is a transition form of the microorganism, it is the hitherto unknown chain between these genera of mollicutes and sporiferous bacteria, ACPGD is a probable representative of mollicute precursor with genome of about 1600 t.p.n. and, first of all, of genera Acholeplasma and Phytoplasma which arised as a result of the evolutionary splitting of its genome. On this basis, it is recommended to found for ACPGD in the Mollicutes class, order III Acholeplasmatales, family I Acholeplasmataceae, a new genus II Pluraplasma gen. nov. and its first species Pluraplasma granulum sp. nov., the strain 118 being its typical representative.

[On the nature of the pathogen of pale-green dwarfism in cereals].

The paper presents more precise data concerning optimal temperature demands to growth of white-yellow dwarfness of cereals (WYDC) identified before as Acholeplasma laidlawii var. granulum, its relation to sterols and genome properties was determined using pulse-electrophoresis. It was established that the agent strains 84 and 118, characterized by phytopathogenicity, grew most intensively at 32 degrees C; they behaved as mesoplasmas but, as it had been found, they were capable to synthesis of carotenoids and displayed close serologic affinity for A. laidlawii PG8. That is, the above strains are typical acholeplasmas capable to live in leafhoppers which carry a disease and in cereals plants and cause a disease with typical symptoms of "yellows" in the latter. Molecular weight of the strain 84 genome was 2200 t.p.n. (GC = 33 mol %); in strains 118 it was 2310 t.p.n. (GC = 34.2 mol %). Allowing for the fact that molecular weight of genome of A. laidlavii var. granulum is almost by 1/3 (1600 + 710 t.p.n.) more than that of A. laidlawii PG8 genome, the authors think that the agent of WYDC is the evolution precursor (or one of precursors) which initiated the Acholeplasma and Phytoplasma genera as a results of splitting of their genomes.

[Change in lectin specificity of winter wheat seedlings in the course of infection with mycoplasms]. / Izmenenie lektinovoi aktivnosti prorostkov ozimoi pshenitsy pri infitsirovanii mikoplazmami.

The activity of soluble lectins in leaves and roots of seedlings of winter wheat (Triticum aestivum L.) cultivar Mironovskaya 808 increased 1 day and 2 days, respectively, after infection with the mycoplasma Acholeplasma laidlawii 118. Analysis of acid-soluble proteins of wheat leaves by PAGE revealed the appearance of 22- and 20-kDa polypeptides, the disappearance of a 14-kDa polypeptide, and an increase in the content of polypeptides with molecular weights of 76, 48, 25, and 18 kDa. The 18-kDa polypeptide is a subunit of wheat germ agglutinin. A change in the activity of lectins may be a nonspecific response of plants to infection with the pathogen.

Colistin sulfate as a suitable substitute of thallium acetate in culture media intended for mycoplasma detection and culture.

Thallium acetate in concentrations of 500 to 1000 mg/l is tolerated in the culture by the most mollicutes of the orders Mycoplasmatales and Acholeplasmatales and by this reason it is added in the culture media as a selective element for the detection and propagation of mycoplasmas and acholeplasmas. Because of the high toxicity of thallium acetate and its accumulation in the environment, thallium acetate is not biodegradable, an alternative was searched. The results and analysis of tests with nine mollicute species are presented here. It is recommended to replace thallium acetate in the formulations where it is used and colistin sulfate is proposed as its substitute.

Development and qualification of a novel virus removal filter for cell culture applications.

Commercial bioreactors employing mammalian cell cultures to express biological or pharmaceutical products can become contaminated with adventitious viruses. The high expense of such a contamination can be reduced by passing all gases and fluids feeding the bioreactor through virus inactivation or removal steps, which act as viral barriers around the bioreactor. A novel virus barrier filter has been developed for removing viruses from serum-free cell culture media. This filter removes the 20 nm minute virus of mice by >3 log reduction value (LRV), the 28 nm bacteriophage PhiX174 by >4.5 LRV, the mycoplasma Acholeplasma laidlawii by > or =8.8 LRV, and the bacteria Brevundimonas diminuta by > or =9.2 LRV. Robust removal occurs primarily by size exclusion as demonstrated over a wide range of feedstocks and operating conditions. The filtered media are indistinguishable from unfiltered media in growth of cells to high densities, maintenance of cell viability, and productivity in expressing protein product. Insulin and transferrin show high passage through the filter. The virus barrier filter can be autoclaved. The relatively high membrane permeability enables the use of a moderate filtration area.

A polymerase chain reaction based method for detecting Mycoplasma/Acholeplasma contaminants in cell culture.

A detection system that utilizes a primer mixture in a nested polymerase chain reaction for detecting Mycoplasma contaminants in cell cultures is described. Primers were designed to amplify the spacer regions between the 16S and 23S ribosomal RNA genes of Mycoplasma and Acholeplasma. This detection system was able to detect 20-180 colony forming units per milliliter of sample. Eight commonly encountered Mycoplasma and Acholeplasma contaminants, which include Mycoplasma (M.) arginini, M. fermentans, M. hominis, M. hyorhinis, M. orale, M. pirum, M. salivarium, and Acholeplasma laidlawii, were consistently amplified. Mycoplasma contaminants generated a single DNA band of 236-365 base pairs (bp), whereas A. laidlawii produced a characteristic two-band pattern of 426 and 219 bp amplicons. Species identification could be achieved by size determination and restriction enzyme digestion. Minor cross-reactions were noted with a few closely related gram positive bacteria and DNA from rat cell lines. A Mycoplasma Detection Kit for detecting Mycoplasma contaminants in cell cultures has been developed based on this approach.

[Effect of mycoplasma contamination of the human uterine leiomyosarcoma cell line SK-UT-1B on karyotype structure]. / Vliianie mikoplazmennoi kontaminatsii kletochnoi linii leiomiosarkomy cheloveka SK-UT-1B na kariotipicheskuiu struktury.

The karyotypic variability has been investigated for human uterine leiomyosarcoma cell line SK-UT-1B, cultivated for 30-90 days after contamination with Acholeplasma laidlawii, strain PG-8. The character of cell distribution for chromosome number gradually changes in contaminated cells, comparatively to the control, with the lengthening of the term of contamination. So, in 30 days the analysed distributions do not differ in the experimental and in the control variants, the modal number of chromosomes being equal to 46. In 60 days the frequency of cells with modal number of chromosomes have a tendency to decrease, and the range of variability in the number of chromosomes tend to increase. In 90 days, the frequency of cells with modal number of chromosomes decreases significantly, and the range of variability on the number of chromosomes increases significantly. The number of chromosomal aberrations gradually increases in contaminated cells, as compared to the control, with the lengthening of the term of contamination. So, in 30 days the number of chromosomal aberrations does not increase, only the number of dicentrics (telomeric associations) has the tendency to increase. In 60 days, the number of chromosomal aberrations, mainly dicentrics, increases significantly. In 90 days, the number of chromosomal aberrations increases significantly, including both dicentrics and chromatid breaks. The possible reasons of the observed character of karyotypic variability is discussed. Our previous results make it possible to suppose that the increase in the number of dicentrics in "markerless" line SK-UT-1B with long term contamination may be an additional evidence on the role of dicentrics in cell adaptation to in vitro conditions in such lines.

Evaluation of current sterility tests for human live viral vaccines.

Current sterility tests for human viral vaccines were evaluated. A total of 43 lots of bulk suspension of live viral vaccines (measles, mumps, rubella and oral poliomyelitis) produced by six manufacturers in Japan were evaluated for bacteriostatic and mycoplasmastatic activities. Some of them showed fairly high bacteriostatic and mycoplasmastatic activities, due to antibiotics added during vaccine production. It was concluded that the current sterility test for mycoplasmas is not reliable to detect viable mycoplasmas in live viral vaccines.

Breeder turkey hens seropositive and culture-negative for Mycoplasma synoviae.

Four flocks of clinically normal turkey breeder hens were shown to have suspect and positive Mycoplasma synoviae (MS) hemagglutination-inhibition (HI), enzyme-linked immunosorbent assay, and, in some cases, serum plate agglutination serology in the absence of MS isolation. In all cases, HI serology for Mycoplasma gallisepticum (MG) and M. meleagridis was negative. Acholeplasma laidlawii was isolated from some hens in each of these MS-seropositive culture-negative flocks. Immunoblotting was used to help determine if this positive MS serology was a result of cross-reactive antibodies to A. laidlawii or to some other Mycoplasma species. When sera from two of the flocks were reacted with MS antigen in immunoblotting, a strong and characteristic MS immunoblot profile was seen. Immunoblotting gave no evidence of a strong antibody response to A. laidlawii, M. iowae, or MG. This suggests the presence (or earlier presence) of MS in these flocks that is difficult to isolate by routine methods. Furthermore, this work shows that immunoblotting can be an important tool in the diagnosis of poultry diseases.

[Contamination of fish tissue cell cultures by Mycoplasma]. / Mikoplazmennaia kontaminatsiia kul'tur kletok iz tkanei ryb.

The inoculated and primary cell cultures of fish (carp, salmon, and sturgeon) have been studied. Acholeplasma typed as A. laidlawii in terms of its biochemical properties shown in inhibited metabolism has been isolated from 19 samples. The authors consider the source of Acholeplasma-induced contamination of the cell cultures under study.

Effects of age, environmental temperature and relative humidity on the colonization of the nose and trachea of calves by Mycoplasma spp.

Nasal and tracheal swabs sequentially collected from three groups of eight calves between the ages of 1 and 98 days indicated that the nose and trachea were colonized by Mycoplasma spp. during the first weeks of life. Over 92% of all calves harboured Mycoplasma spp. in their noses when they were 2 weeks old, the rate of recovery falling gradually thereafter. The peak period of recovering mycoplasmas from the noses and tracheas of calves was at 6 weeks old. M. bovirhinis, M. arginini and Acholeplasma laidlawii predominated in the nose while M. dispar and M. bovirhinis predominated in the trachea. There was no association between rates of isolation and clinical signs of respiratory disease. There were no significant differences between the frequencies of isolation of Mycoplasma spp. from groups of calves kept under different environmental temperatures and relative humidities.

Evaluation of a digitonin disk assay to discriminate between acholeplasma and mycoplasma isolates from bovine milk.

A study was undertaken to evaluate effectiveness of a digitonin disk inhibition test to discriminate between Acholeplasma laidlawii and Mycoplasma sp. isolated from bovine milk. The test measured zone diameters of growth inhibition surrounding a digitonin-containing disk on solid medium. Zones of inhibition for 20 isolates of A. laidlawii, ranging from 8-14 mm, did not overlap those of 261 isolates of Mycoplasma sp., ranging from 16 to 38 mm. Examination of variation in zone diameters for M. bovis found that inhibition was not appreciably affected by agar dehydration. Zones of inhibition increased with increasing dilutions of stock culture and decreased with increasing incubation time. Analysis of variance and Fisher's least significant difference test of logn zone diameters revealed that differences in mean logn zone diameters were different at the 0.01 level of significance between some of the six species of mycoplasma examined, indicating that growth among some species of mycoplasma was effected differently by digitonin. The digitonin test was found to clearly discriminate between A. laidlawii and Mycoplasma sp. indicating that the test would be useful as a practical screening test of individual-cow and bulk tank milk for mycoplasmas.

[Detection certainty of the contamination of bull sperm with mycoplasma]. / Untersuchungen zur Nachweissicherheit der Kontamination von Bullensperma mit Mykoplasmen.

Tests for presence of mycoplasmas were conducted on 20 insemination bulls known as mycoplasma spreaders, with 5 sperm pellet batches of 20 pellets each being investigated for each animal. Mycoplasma contamination was positively recorded from 83 of the above 100 batches. Mycoplasma (M.) bovigenitalium, M. californicum. M. arginini, M. bovirhinis, and Acholeplasma laidlawii were typed by means of indirect immunofluorescent technique, which confirmed the presence also in the GDR of mycoplasma species described in the literature and detected in sperm samples. The solid culturing media used in the above tests, medium-B agar and cattle blood agar with staphylococcal nutrix, proved to be equally suitable for isolation of mycoplasma from sperm samples. Mycoplasma was positively identified in about one third of all pellets/batches tested. 3 pellets to one batch should be sufficient a random sample size from which to obtain information at least very close to real contamination.

Comparison of two methods for detection of mollicutes (Mycoplasmatales and Acholeplasmatales) in cell cultures in the Netherlands.

A total of 1949 cell cultures was tested for contamination with mollicutes by cultivation on and in mycoplasma media, 25.7% of the cell cultures was positive, 243 strains of Mycoplasma hyorhinis were isolated. Furthermore, mainly M. arginini and M. orale were detected, less often Acholeplasma laidlawii, M. fermentans and M. pneumoniae. Optimal conditions for isolation were discussed. About one third of 217 hybridoma cultures and two third of 57 myeloma cultures proved to be contaminated, all with M. hyorhinis. A DNA fluorochrome staining method (DAPI-test) was compared to cultivation for testing 1039 cell cultures. The efficiency of the DAPI-test could be estimated to be about 96% that of cultivation about 89%, but cultivation is more specific. The highest assurance is obtained when both methods are applied.