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.

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.

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.

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.

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.

[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.

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.

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.

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.

Experimentally produced calf pneumonia.

Experimental pneumonia was produced in calves by the endobronchial inoculation of pneumonic lung homogenates. Irradiated homogenates produced minimal pneumonia. Ampicillin treatment of the homogenates and the experimental calves reduced the extent of pneumonia. Treatment with tylosin tartrate prevented experimental pneumonia. These results suggest that the total pneumonia was due to organisms susceptible to tylosin tartrate and that the residual pneumonia remaining after ampicillin treatment was due to organisms susceptible to tylosin tartrate but not to ampicillin. Of the organisms isolated from the lungs, the ones in this latter category most likely to be responsible are Mycoplasma dispar and ureaplasmas (T-mycoplasmas).

Isolation of Mycoplasma arginini from swine and from a swine waste disposal system.

Mycoplasma arginini was isolated from the pharynges and tonsils of 4 of 6 swine necropsied during an epizootic of respiratory tract diseases and lameness. The organism was also isolated from 2 of 16 asymptomatic swine examined in the same herd approximately 10 months after the epizootic. In addition, M arginini, Acholeplasma laidlawii, and other acholeplasmas were isolated from recycled effluent of an anaerobic lagoon used for disposal of waste from the swine herd. This observation represents the 1st report of a Mycoplasma species isolated from a lagoon or sewage system. The pathogenicity of a strain of M arginini isolated during the epizootic was examined by inoculating young swine intranasally, intratracheally, intravenously, and intraperitoneally. The agent colonized in the pharynges of 2 pigs inoculated intranasally, but did not produce overt signs of clinical disease. Although neutralizing antibodies to M arginini were not detected, 1 animal that had been infected naturally and 2 that had been inoculated intravenously did develop complement-fixing antibodies. The findings indicate that M arginini can colonize in the nasal and pharyngeal mucosae of swine, but that it is not highly pathogenic for these animals.

Infectious bovine keratoconjunctivitis: experimental induction of infection in calves with mycoplasmas and Moraxella bovis.

Eyes of 14 calves were exposed by conjunctival instillation to cultures of either Mycoplasma conjunctivae (6 calves) or Acholeplasma laidlawii (8 calves). Calves were observed for clinical signs of infectious bovine keratoconjunctivitis (IBK), and eyes were examined for the test organisms by bacteriologic cultural technique for 60 days. Acholeplasma laidlawii became established in the eyes of 5 of 8 calves; M conjunctivae became established in the eyes of 4 of 6 calves. On day 28, eyes of 9 of the 14 calves were exposed by conjunctival instillation to Moraxella bovis, and all developed IBK. Five calves exposed to Moraxenjunctivae or A laidlawii, but not to Mor bovis, did not develop IBK. Four calves not exposed to M conjunctivae or A laidlawii, but exposed to Mor bovis, developed IBK. Mycoplasmas do not have a major role in IBK, but might produce ancillary effects similar to those of infectious bovine rhinotracheitis virus, wind, ultraviolet radiation, dust, and other irritants.

A severe outbreak of bovine mastitis assoicated with Mycoplasma bovigenitalium and Acholeplasma laidlawii.

An outbreak of clinical mastitis is described in which 75 cows of a herd of 200 Friesian cows were affected in one or more quarters over a period of three months. Sixty animals failed to return to normal despite various intramammary and systemic antibiotic treatments. Heifers, dry cows and lactating animals were affected. The milk of 15 out of 53 animals tested yielded mixed cultures of Mycoplasma bovigenitalium and Acholeplasma laidlawii. Thirteen of 34 sera showed evidence of antibodies to A laidlawii.

Mycoplasmas and cuffing pneumonia in a group of calves.

The mycoplasmas found in the lungs of 20 calves, housed together for six months, and the related pulmonary pathology are reported. Twelve calves had cuffing pneumonia and in this group there was a significantly higher isolation frequency of Mycoplasma dispar and Ureaplasma spp compared with the non-pneumonic group. Mycoplasma bovirhinis and Acholeplasma laidlawii were isolated from the lungs of calves in both groups. Mycoplasma arginini was not recovered from the lungs of any calf. The significance of the peribronchiolar lymphocytic accumulation in the lungs of the non-pneumonic animals and their differentiation from peribronchiolar lymphocytic cuffs is discussed.

Bovine genital mycoplasmosis.

Infection, lesions and clinical significance of Acheloplasmas, Mycoplasma bovis and Mycoplasma bovigenitalium in genital disease of cattle are described. A more detailed account is given of ureaplasma infections. Acute and chronic forms of granular vulvitis in both field and experimental disease are described as well as the role of the organism in abortion. Recovery rates of ureaplasma and mycoplasma from semen and preputial washings in bulls are outlined and their significance in disease is discussed. There are problems in differentiating pathogenic from nonpathogenic isolates. Methods are being developed to treat semen for these organisms. This paper provides a concise summary of clinical and microbiological aspects of bovine genital mycoplasmosis.

[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.

[Biochemical and serologic study of Acholeplasmae isolated from monkeys]. / Biokhimicheskoe i serologicheskoe izuchenie akholeplazm, vydelennykh ot obez'ian.

Biochemical and serological properties of mycoplasmas isolated from the blood, feces and parenchymatous organs of monkeys have been studied to determine their species. It was established that the isolated strains belong to the family Acholeplasmatoceae. The study of their biochemical properties in different tests has revealed the presence of 5 biochemically heterogeneous groups. Their serological properties suggest that 13 out of 45 strains are identical to the reference strain of A. laidlawii A, and all other strains have been classified as new Acholeplasma species which have never been isolated from monkeys before.

[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.

[Identification of mycoplasmas isolated from monkeys]. / Identifikatsiia mikoplazm, vydelennykh ot obez'ian

The authors identified 15 strains of mycoplasmae isolated from Papio hamadryas suffering from leukemia and from healthy Macacus rhesus, a green monkey and saimiri. A study was made of their biochemical properties, antigenic properties in the reaction of growth depression with immune sera to a number of standard strains, and also the protein composition by electrophoresis in polyacrylamide gel (EPAG). A number of mycoplasmae were identified as A. laidlawii, M. arthritidis--Campo and M. cynomolgus KI, affilated to M. orale II. Four strains were apparently a mixture of three mycoplasmae (M. cynomolgus KI, M. arthritidis--Campo, and M. hyorhinis). Six strains of mycoplasmae (three enzymatically active, belonging according to EPAG data to a single serological type, and three--enzymatically inert, differing by EPAG) could not be identified.

[Use of the growth inhibition test, the CFT, and the agar gel diffusion precipitation test to identify mycoplasmae isolated from monkeys]. / Ispol'zovanie reaktsii ingibitsii rosta, RSK i reaktsii diffuznoi pretsipitatsii v agarovom gele dlia identifikatsii mikoplazm, vydelennykh ot obez'ian.

The data are presented on comparative evaluation of the growth inhibition test (GIT), the diffuse precipitation test in agar gel (DPTAG) and the complement fixation test (CFT) in the identification of Mycoplasma isolated from monkeys. The results obtained in the study of 4 Mycoplasma strains from the internal organs of monkeys in the CFT and the DPTAG with hyperimmune rabbit sera provided by Microbiological Associates, Bethesda, Maryland, USA, allowed these Mycoplasma to be identified as M. laidlawii (l 6916, p 6240 and M 6802) and M. hominis (C 7034), which was confirmed by the study of the biological properties of these strains and in the GIT. The CFT and the DP-TAG, equally to the GIt and other serological tests, can be used for identification of newly isolated Mycoplasma strains.