Effect of climatic conditions on enzootic pneumonia of pigs

Enzootic pneumonia of pigs is a common worldwide problem affecting mainly growing pigs. It is caused byMycoplasma suipneumoniae (M. hyopneumoniae) but the pneumonia is usually complicated byM. hyorhinis and bacteria. The experimental evidence on the effect of temperature, UV light and drying on the survival ofM. suipneumoniae is reviewed and related to the data available onM. pneumoniae M. mycoides subsp.mycoides andM. gallisepticum which cause respiratory disease in man, cattle and chickens respectively. The external and internal climatic conditions which influence the severity of enzootic pneumonia in housed pigs are discussed. Possible further experiments withM. suipneumoniae are discussed in relation to the problem of cultivating one of the most fastidious of all known mycoplasmas.Presented at the Seventh International Biometeorological Congress, 18–25 August 1975, College Park, Maryland, USA.     

Large-Quantity Production of Chicken Embryo Tracheal Organ Cultures and Use in Virus and Mycoplasma Studies

Chicken tracheal organ cultures were made from embryos which were 19 to 20 days old. Transversely cut rings of trachea were placed in screw-capped tissue-culture tubes with Eagle's-N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) medium and incubated in roller drums. The method had advantages over other organ culture systems in that these cultures were prepared in numbers similar to conventional tissue cultures, ciliary activity was quickly and accurately evaluated, and contamination occurred less frequently than with organ cultures in petri dishes. Ciliary activity persisted for at least 1 month when the medium was changed at 5-to 7-day intervals and for 10 to 15 days without a change. Infectious bronchitis virus stopped ciliary movement, and this effect was used as a basis for titrating the virus and for determining the neutralizing capacity of immune mouse ascitic fluid. Twenty-four Mycoplasma strains were tested. Organisms of 17 strains, both avian and mammalian, multiplied in the organ cultures, and 7 strains, belonging to the species M. gallisepticum and M. mycoides var. capri, inhibited ciliary activity.     

Detection ofMycoplasmain Avian Live Virus Vaccines by Polymerase Chain Reaction

The polymerase chain reaction (PCR) was evaluated to detect mycoplasma contamination of avian live virus vaccines. The specificity of the primers showed that 34 strains belonging to nine species of avian mycoplasma DNA could be detected. The sensitivity of PCR to detect mycoplasma DNA was 10^0·2colony forming units (cfu) ofMycoplasma synoviaeand 10^0·7cfu ofMycoplasma gallisepticum. WhenM. synoviaeandM. gallisepticumwere spiked into several avian live virus vaccines, PCR gave a positive reaction except for the avian pox and the avian encephalomyelitis vaccines which were prepared from organ homogenates. Short-term incubation of avian encephalomyelitis vaccine improved the sensitivity of PCR to detect bothM. synoviaeandM. gallisepticum. Therefore, PCR, combined with the short-term incubation, were shown to be most effective in detecting mycoplasma contamination in all of avian live virus vaccines.     

Effect of cell membrane-active antimicrobial agents on membrane potential and viability of mycoplasmas

The membrane potential ofMycoplasma mycoides subsp.capri has been determined to beE _M=−48 mV±10%, inside negative. In this study we investigated the influence of cell membrane-active antimicrobial agents, viz., valinomycin, gramicidin, polymyxin, and clotrimazole, on membrane potential and viability ofM. mycoides subsp.capri. Valinomycin, an ionophore with extreme potassium selectivity, induced a membrane hyperpolarization,E _M=−110 mV. Valinomycin was not cidal, but static to mycoplasmas. Obviously the potassium drain induced by valinomycin can be compensated for by the organisms. Gramicidin is an antibiotic forming cation conduction channels across membranes. It induced a rapid depolarization,E _M=+23 mV, of mycoplasma membranes. At low concentrations, gramicidin had a static effect, whereas at high concentrations it was cidal to mycoplasmas. The rapid permeation of cations through the stationary ion channels formed by gramicidin obviously exerts an inhibitory or even lethal effect on mycoplasma metabolism and growth. Polymyxin B induced a depolarization,E _M=−35 mV, of mycoplasma membranes only when the organisms had been pretreated and hyperpolarized with valinomycin. After treatment with both valinomycin and polymyxin B, a slight inhibition of mycoplasma growth was observed. Clotrimazole, a synthetic imidazole antimycotic, hyperpolarized mycoplasma membranes (E _M=−80 mV). At high concentrations clotrimazole was cidal, whereas at low concentrations it was static to mycoplasmas.     

Surveillance of Mycoplasma agalactiae and Mycoplasma mycoides subsp. capri in dairy goat herds

This study was designed to monitor the presence of Mycoplasma agalactiae and Mycoplasma mycoides subsp. capri (Mmc) in 66 dairy goat herds of a genetic improvement programme in a region of Spain where contagious agalactia is endemic. Over a whole lactation period, 300 bulk tank milk and 381 milk samples from goats with clinical mastitis were subjected to polymerase chain reaction (PCR) to detect the two mycoplasma species. The presence of mycoplasmas (either species or both) was detected in 66.7% of the herds and M. agalactiae was identified in 95.45% of these positives herds. In a given infected herd, mycoplasmas were not continuously detected over the whole study period. Our findings indicate that in an endemic area, M. agalactiae and Mmc can be monitored through PCR analysis of mastitic milk and bulk tank milk (BTM) samples. Over a lactation period we recommend testing multiple BTM samples on a herd. No relationship was observed between the use of inactivated mycoplasma vaccines and the PCR detection of both mycoplasmas.     

Disruption of the S41 Peptidase Gene in Mycoplasma mycoides capri Impacts Proteome Profile,H2O2 Production,and Sensitivity to Heat Shock

Members of the Mycoplasma mycoides cluster are among the most virulent of the mycoplasmas, causing worldwide economically significant diseases of cattle and goats. A distinguishing phenotype among the members of the cluster is the ability to degrade casein. The MMCAP2_0241 gene, an S41 peptidase, confers the proteolytic phenotype in Mycoplasma mycoides subsp. capri GM12. In order to determine the impact of disruption of the gene, we used differential proteome profiling to compare the M. mycoides subsp. capri wild type with a mutant lacking the proteolytic phenotype. Disruption of MMCAP2_0241 resulted in altered phenotypes reminiscent of M. mycoides subsp. mycoides SC and had significant impacts on the proteome profile of the microbe. The mutant exhibited increased production of hydrogen peroxide, decreased lactate dehydrogenase activity, and increased sensitivity to heat shock.     

Preparation of reference strains for validation and comparison of mycoplasma testing methods

Aims: To optimize growth conditions for preparation of stocks of mycoplasma reference strains to obtain highly viable and disperse samples with low ratios of genomic copy (GC) number to that of colony forming units (CFU). These stocks are required for assessment of relative limits of detection (LOD) of alternative nucleic acid testing (NAT)‐based methods in comparison to the conventional microbiological methods. Methods and Results: A kinetics study was used to assess the changes in ratios between the numbers of GC and CFU at different growth phases of six different mycoplasma cultures Acholeplasma laidlawii, Mycoplasma gallisepticum, Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma orale and Mycoplasma pneumoniae. All tested mycoplasmas demonstrated low GC/CFU ratios (≤10) within the log and early stationary growth phases. A significant increase in GC/CFU ratios was observed at the very late stationary and death phases, when the titre of cultures has declined. Similar patterns of GC/CFU profiles were observed for A. laidlawii and Myc. gallisepticum co‐cultured with suspension of Chinese hamster ovary (CHO) cells. Conclusions: Tested mycoplasma strains harvested at the exponential‐early stationary phases of growth demonstrated the lowest GC/CFU ratios and low propensity to form filamentous structures or aggregates under proposed conditions and can be used for the preparation of a mycoplasma reference panel for methods comparability study. Significance and Impact of the Study: This study shows that the preparation and use of viable mycoplasma reference strains with low CG/CFU ratios is the most reliable way to adequately evaluate the LOD of alternative NAT‐based mycoplasma testing methods.     

Isolation and Characterization of Mycoplasma and Acholeplasma from Apparently Healthy and Diseased (Infectious Sinusitis) Turkeys

Investigation of 136 turkeys (24 manifesting infra-orbital sinusitis, 112 apparently healthy) resulted in isolation of 79 strains of Mycoplasma and 4 of Acholeplasma. By the disc growth inhibition test with 16 reference antisera of avian serogroups, 55 strains were identified serologically and 28 remained unidentified. Thirteen strains of Mycoplasma gallisepticum, 1 of M. meleagridis, and 2 of Acholeplasma laidlawii were isolated from turkey sinusitis whereas serogroups C (2), D (19), F (8), M. meleagridis (4), M. anatis (4), A. laidlawii (2), and 28 unidentified strains were isolated from apparently healthy turkeys. Three patterns were recognized on the basis of glucose, maltose, and sucrose, fermentation. The most frequent, pattern I, included 13 M. gallisepticum strains whereas 5 M. meleagridis strains belonged to fermentation pattern III. Isolates were also studied for reduction of tetrazolium, methylene blue, potassium tellurite, resistance to methylene blue and sodium taurocholate, and production of arginine deiminase and “film and spots.” Inoculation of selected isolates into developing chick embryos revealed that 2 A. laidlawii strains were nonpathogenic and 13 M. gallisepticum, 1 serogroup D and 2 serogroup F strains were pathogenic, causing 50–100% mortality. In vitro antibiotic disc sensitivity tests indicated that rovamycin (solubilized spiramycin) may be recommended for turkey mycoplasmosis. Isolation of 2 A. laidlawii strains from turkey sinusitis and 4 M. anatis strains from apparently healthy turkeys appears interesting.     

Reduction of Hydrogen Peroxide Accumulation and Toxicity by a Catalase from Mycoplasma iowae

Mycoplasma iowae is a well-established avian pathogen that can infect and damage many sites throughout the body. One potential mediator of cellular damage by mycoplasmas is the production of H₂O₂ via a glycerol catabolic pathway whose genes are widespread amongst many mycoplasma species. Previous sequencing of M. iowae serovar I strain 695 revealed the presence of not only genes for H₂O₂ production through glycerol catabolism but also the first documented mycoplasma gene for catalase, which degrades H₂O₂. To test the activity of M. iowae catalase in degrading H₂O₂, we studied catalase activity and H₂O₂ accumulation by both M. iowae serovar K strain DK-CPA, whose genome we sequenced, and strains of the H₂O₂-producing species Mycoplasma gallisepticum engineered to produce M. iowae catalase by transformation with the M. iowae putative catalase gene, katE. H₂O₂-mediated virulence by M. iowae serovar K and catalase-producing M. gallisepticum transformants were also analyzed using a Caenorhabditis elegans toxicity assay, which has never previously been used in conjunction with mycoplasmas. We found that M. iowae katE encodes an active catalase that, when expressed in M. gallisepticum, reduces both the amount of H₂O₂ produced and the amount of damage to C. elegans in the presence of glycerol. Therefore, the correlation between the presence of glycerol catabolism genes and the use of H₂O₂ as a virulence factor by mycoplasmas might not be absolute.     

Factors influencing microbiological assay of cell-culture mycoplasmas

Summary A total of 6432 cell cultures was assayed for mycoplasmas over a 6-year period by aerobic and anaerobic incubation of agar and broth media. Mycoplasmas were detected in 375 cultures (5.8%).M. orale andA. laidlawii accounted for 61.3% of the isolates. Anaerobic incubation detected 98.1% of the isolates; aerobic incubation detected 45.8%. Of factors studied to determine their effect on mycoplasma assay, only two, anaerobic incubation and presence of mycoplasmacidal/static antibiotics, were significant. In separate studies, 86 of 2656 cell cultures (3.2%) were infected with strains ofM. hyorhinis that did not grow on cell-free media. Recommendations are given for microbiological assay of cell-culture mycoplasmas. These studies were supported in part by Contracts N01-AG-4-2865 and N01-AG-8-2117 from the National Institute on Aging and N01-GM-6-2119 from the National Institute of General Medical Sciences.     

Host specificity of mollicutes oriC plasmids: functional analysis of replication origin

Recently, artificial oriC plasmids containing the chromosomal dnaA gene and surrounding DnaA box sequences were obtained for the mollicutes Spiroplasma citri and Mycoplasma pulmonis. In order to study the specificity of these plasmids among mollicutes, a set of similar oriC plasmids was developed for three mycoplasmas belonging to the mycoides cluster, Mycoplasma mycoides subsp. mycoides LC (MmmLC), M.mycoides subsp. mycoides SC (MmmSC) and Mycoplasma capricolum subsp. capricolum. Mycoplasmas from the mycoides cluster, S.citri and M.pulmonis were used as recipients for transformation experiments by homologous and heterologous oriC plasmids. All five mollicutes were successfully transformed by homologous plasmids, suggesting that the dnaA gene region represents the functional replication origin of the mollicute chromosomes. However, the ability of mollicutes to replicate heterologous oriC plasmids was found to vary noticeably with the species. For example, the oriC plasmid from M.capricolum did not replicate in the closely related species MmmSC and MmmLC. In contrast, plasmids harbouring the oriC from MmmSC, MmmLC and the more distant species S.citri were all found to replicate in M.capricolum. Our results suggest that the cis-elements present in oriC sequences are not the only determinants of this host specificity.     

Mycoplasma adaptation to stress factors: Nucleotide sequences absent in vegetative forms of <Emphasis Type="Italic">Mycoplasma gallisepticum</Emphasis> S6 cells are found in nonculturable forms

The adaptation of Mycoplasma gallisepticum S6 to adverse environmental conditions is associated with the transformation of vegetative cell forms to viable but nonculturable (VBNC) forms. The vegetative and VBNC forms proved to differ in the spectrum of PCR products amplified from pvpA-gene, which codes for the phase-variable cytoadhesion protein. The vegetative forms displayed only one amplicon, which contained one open reading frame (1086 bp) with a high homology (97%) to pvpA-gene of M. gallisepticum R and Pendik. The VBNC forms of M. gallisepticum S6 had additional amplicons, whose open reading frames were absent from the complete database sequence of the mycoplasma genome. A high nucleotide sequence homology (54–55%) between pvpA-gene and the additional pvpA-gene amplicons made it possible to suggest that pvpA-gene provided a source for the formation of new regions within the mycoplasma genome during adaptation to adverse environmental conditions.     

Adsorption of Mycoplasma pneumoniae to Neuraminic Acid Receptors of Various Cells and Possible Role in Virulence

Monkey, rat, and chicken tracheal epithelial cells, as well as monkey, rat, guinea pig, and chicken erythrocytes, adsorbed firmly to colonies of Mycoplasma pneumoniae and M. gallisepticum. Colonies of M. pulmonis also adsorbed erythrocytes but with less avidity than M. pneumoniae or M. gallisepticum; unlike the latter organisms, M. pulmonis did not adsorb tracheal epithelial cells. Colonies of M. orale type 1 and M. orale type 3 adsorbed only chicken red cells. Other mycoplasma species tested, including four of human origin and one of animal origin, did not adsorb red cells or epithelial cells. M. pneumoniae and M. gallisepticum appeared to attach to erythrocytes or tracheal epithelial cells by neuraminic acid receptors on these cells, whereas M. orale types 1 and 3 and M. pulmonis seemed to utilize another type or other types of receptors. Pretreatment of red cells or tracheal epithelial cells with receptor-destroying enzyme, neuraminidase, or influenza B virus removed the adsorption receptors for M. pneumoniae. Similarly, pretreatment of M. pneumoniae colonies with neuraminic acid-containing materials prevented adsorption of erythrocytes or respiratory tract cells. The adsorption sites on M. pneumoniae were specifically blocked by homologous but not heterologous antisera. This property made it possible to study the nature of the mycoplasma adsorption sites by testing the capacity of different fractions of the organism to block the action of adsorption-inhibiting antibodies. Such studies suggested that the mycoplasma binding sites were probably lipid or lipoprotein in nature. The glycerophospholipid hapten was implicated as one such site, since this serologically active hapten blocked the action of hemadsorption-inhibiting antibodies in M. pneumoniae rabbit antiserum. The affinity of M. pneumoniae for respiratory tract epithelium, unique among the mycoplasmas that infect man, may play a role in virulence, since this type of attachment provides an unusual opportunity for peroxide, secreted by the organism, to attack the tissue cell membrane without being rapidly destroyed by catalase or peroxidase present in extracellular body fluids.     

The complete genome sequence of the murine respiratory pathogen Mycoplasma pulmonis

Mycoplasma pulmonis is a wall-less eubacterium belonging to the Mollicutes (trivial name, mycoplasmas) and responsible for murine respiratory diseases. The genome of strain UAB CTIP is composed of a single circular 963 879 bp chromosome with a G + C content of 26.6 mol%, i.e. the lowest reported among bacteria, Ureaplasma urealyticum apart. This genome contains 782 putative coding sequences (CDSs) covering 91.4% of its length and a function could be assigned to 486 CDSs whilst 92 matched the gene sequences of hypothetical proteins, leaving 204 CDSs without significant database match. The genome contains a single set of rRNA genes and only 29 tRNAs genes. The replication origin oriC was localized by sequence analysis and by using the G + C skew method. Sequence polymorphisms within stretches of repeated nucleotides generate phase-variable protein antigens whilst a recombinase gene is likely to catalyse the site-specific DNA inversions in major M.pulmonis surface antigens. Furthermore, a hemolysin, secreted nucleases and a glyco-protease are predicted virulence factors. Surprisingly, several of the genes previously reported to be essential for a self-replicating minimal cell are missing in the M.pulmonis genome although this one is larger than the other mycoplasma genomes fully sequenced until now.     

Biological Enrichment of Mycoplasma Agents by Cocultivation with Permissive Cell Cultures

In this study, we describe our results on the evaluation of the ability of different permissive mammalian cell lines to support the biological enrichment of mycoplasma species known to be bacterial contaminants of cell substrates. The study showed that this approach is able to significantly improve the efficiency of mycoplasma detection based on nucleic acid testing or biochemical technologies (e.g., MycoAlert mycoplasma detection). Of 10 different cell lines (Vero, MDBK, HEK-293, Hep-G2, CV-1, EBTr, WI-38, R9ab, MDCK, and High Five) used in the study, only MDCK cell culture was found to support the efficient growth of all the tested mycoplasmas (Mycoplasma arginini, M. bovis, M. fermentans, M. gallinaceum, M. gallisepticum, M. synoviae, M. hominis, M. hyorhinis, M. orale, M. salivarium, and Acholeplasma laidlawii) known to be most frequently associated with contamination of cell substrates and cell lines in research laboratories or manufacturing facilities. The infection of MDCK cells with serial dilutions of each mycoplasma species demonstrated that these common cell line contaminants can be detected reliably after 7-day enrichment in MDCK cell culture at contamination levels of 0.05 to 0.25 CFU/ml. The High Five insect cell line was also found to be able to support the efficient growth of most mycoplasma species tested, except for M. hyorhinis strain DBS1050. However, mycoplasma growth in insect cell culture was demonstrated to be temperature dependent, and the most efficient growth was observed when the incubation temperature was increased from 28°C to between 35 and 37°C. We believe that this type of mycoplasma enrichment is one of the most promising approaches for improving the purity and safety testing of cell substrates and other cell-derived biologics and pharmaceuticals.     

General N-and O-Linked Glycosylation of Lipoproteins in Mycoplasmas and Role of Exogenous Oligosaccharide

The lack of a cell wall, flagella, fimbria, and other extracellular appendages and the possession of only a single membrane render the mycoplasmas structurally simplistic and ideal model organisms for the study of glycoconjugates. Most species have genomes of about 800 kb and code for few proteins predicted to have a role in glycobiology. The murine pathogens Mycoplasma arthritidis and Mycoplasma pulmonis have only a single gene annotated as coding for a glycosyltransferase but synthesize glycolipid, polysaccharide and glycoproteins. Previously, it was shown that M. arthritidis glycosylated surface lipoproteins through O-linkage. In the current study, O-linked glycoproteins were similarly found in M. pulmonis and both species of mycoplasma were found to also possess N-linked glycans at residues of asparagine and glutamine. Protein glycosylation occurred at numerous sites on surface-exposed lipoproteins with no apparent amino acid sequence specificity. The lipoproteins of Mycoplasma pneumoniae also are glycosylated. Glycosylation was dependent on the glycosidic linkages from host oligosaccharides. As far as we are aware, N-linked glycoproteins have not been previously described in Gram-positive bacteria, the organisms to which the mycoplasmas are phylogenetically related. The findings indicate that the mycoplasma cell surface is heavily glycosylated with implications for the modulation of mycoplasma-host interactions.     

Interaction of cationic antimicrobial peptides with Mycoplasma pulmonis

We investigated the mode of action underlying the anti-mycoplasma activity of cationic antimicrobial peptides (AMPs) using four known AMPs and Mycoplasma pulmonis as a model mycoplasma. Scanning electron microscopy revealed that the integrity of the M. pulmonis membrane was significantly damaged within 30 min of AMPs exposure, which was confirmed by measuring the uptake of propidium iodine into the mycoplasma cells. The anti-mycoplasma activity of AMPs was found to depend on the binding affinity for phosphatidylcholine, which was incorporated into the mycoplasma membrane from the growth medium and preferentially distributed in the outer leaflet of the lipid bilayer.     

Acholeplasma laidlawii PG8 ultramicroforms amplificate selectivelyrrnB nucleotide sequences

Mycoplasmas are frequent contaminants ofin vitro animal cell cultures. Despite a broad spectrum of modern methods, detection of mycoplasmas remains a serious problem. The situation is complicated by the fact that mycoplasmas may be presented in cell cultures or biological samples by viable but unculturable forms (ultramicroforms). We found that the DNA ofAcholeplasma laidlawii PG8 ultramicroforms showed selective amplification of therrnB nucleotide sequences while vegetative cells of the mycoplasma showed amplification both forrrnA andrrnB sequences. The role of enzyme deproteinization in PCR results was also shown. The results presented in this report indicate that the optimisation of primer sequences as well as PCR regime may be crucial steps in detection and differentiation of vegetative forms and ultramicroforms ofA. laidlawii.