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Cross-reactions between Mycoplasma hyopneumoniae and Mycoplasma flocculare--practical implications for the serodiagnosis of mycoplasmal pneumonia of swine.

Previous studies using hyperimmune antisera revealed significant serologic cross-reactions between Mycoplasma hyopneumoniae and M. flocculare. These findings, coupled with observations that M. flocculare infection may be widespread, indicated that antibodies to M. flocculare might be common in swine sera and thus cause nonspecific reactions in serodiagnostic tests for mycoplasmal pneumonia of swine (MPS). Information reported here indicates that this premise may be invalid. Twenty-one swine were infected experimentally with M. flocculare. Sera were collected every 2 weeks and tested against M. flocculare and M. hyopneumoniae antigens in an enzyme-linked immunosorbent assay (ELISA). Immunoassays against M. flocculare revealed that swine were slow to seroconvert, and that seroconversion was characterized by low titers. Significantly, none of the sera reacted with M. hyopneumoniae. M. hyopneumoniae reference antisera, and sera from swine naturally infected with M. hyopneumoniae and M. flocculare were also evaluated by an antibody inhibition procedure, i.e., sera were mixed with M. hyopneumoniae or M. flocculare antigen, incubated overnight and tested against M. hyopneumoniae antigen in the ELISA. Treatment with M. hyopneumoniae antigen markedly reduced or abolished seroreactivity, whereas treatment with M. flocculare antigen had little effect. These findings suggest that swine infected with M. flocculare usually do not develop sufficient levels of antibodies to detract from the specificity of the ELISA used to diagnose MPS.

Animals↗

[Studies of bovine mycoplasma mastitis. 1. Review of literature on the occurrence of bovine mastitis with Mycoplasma involvement].

The clinical pattern as well as the pathologico-anatomic or histological changes due to mycoplasma mastitis are neither specific nor pathognomic. Mastitis pathogens so far described included M. bovis, M. bovigenitalium, A. laidlawii, A. axanthum, M. alkalescens, M. canadense, M. dispar, M. bovirhinis, strains of Group 7 according to Leach, strain ST 6, and ureaplasma strains. In the GDR, enzootic mastitis has been confined to A. laidlawii and A. axanthum.

Animals↗

Chronological development of Mycoplasma hyorhinis and Mycoplasma hyosynoviae infections in cultures of a swine synovial cell strain.

The sequential development of Mycroplasma hyorhinis and Mycoplasma hyosynoviae was observed in cultures of a swine synovial fluid cell strain. An early transitory filamentous phase was observed with M. hyorhinis infection followed by the development of cell-associated, relatively large, round structures and some ring forms. Infection with M. hyorhinis was characterized by a generalized distribution of the organism and a severe cytopathic effect. Infection with M. hyosynoviae was represented by the development of circumscribed foci of small pleomorphic structures and a milder effect on the cells. At a high multiplicity of infection, this organism became associated with the cytoplasmic membranes of the cells.

Animals↗

Pathogenicity of Mycoplasma capricolum and Mycoplasma putrefaciens.

Mycoplasma capricolum causes high morbidity and mortality in goats. Young kids fed a one-time oral dose of greater than or equal to 1 X 10(5) colony-forming units of the GM13 isolate usually died during the septicemic phase. The cardinal lesions were a fibrinopurulent polyarthritis and an acute, diffuse interstitial pneumonia. Lactating goats exposed to low numbers of the organism via the teat canal experienced similar lesions and acute mastitis, agalactia, and hardened udders. The intramammary inoculation of M. putrefaciens caused only mastitis; infection could not be initiated by oral, intranasal, or i.m. exposure.

Animals↗

Experimental infection of ducklings with Mycoplasma gallisepticum and Mycoplasma anatis.

Two groups of day-old ducklings were infected with Mycoplasma gallisepticum and M anatis respectively by air sac inoculation. Clinical disease was not produced but air sacculitis was produced by both infections. M gallisepticum could be recovered readily from respiratory tissue 10 and 30 days after infections but RSA tests were negative. M anatis was not reisolated from these tissues or from the cloaca but a positive RSA test was observed on the serum of a few ducklings.

Air Sacs↗

Cloning and partial sequence analysis of a Mycoplasma synoviae DNA fragment encoding epitopes shared with the major adhesin P1 protein of Mycoplasma pneumoniae.

Polyclonal antibodies specific for the adhesin P1 protein of Mycoplasma pneumoniae were used to screen an expression library of M. synoviae genomic DNA constructed in the expression vector lambda gt11. Following several cycles of immunoscreening using the anti-P1 antiserum, a lambda gt11 recombinant clone containing 3.9 kilobase pairs (kbp) of M. synoviae DNA was identified and isolated from the expression library. Expression of the recombinant clone (designated MS-1) in Escherichia coli Y1089 followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the crude E. coli lysates revealed the presence of two novel proteins. Two antibodies that recognize the adhesin polypeptide--chicken anti-M. synoviae antibodies and anti-P1 antiserum--reacted with both proteins on immunoblots. Partial sequence analysis of the M. synoviae DNA in clone MS-1 and computer comparison of the predicted amino-acid sequences with existing protein sequence files revealed homology with the adhesin P1 protein of M. pneumoniae.

Adhesins, Bacterial↗

A comparison of the virulence of three strains of Mycoplasma gallisepticum and one strain of Mycoplasma gallinarum in chicks, turkey poults, tracheal organ cultures and embryonated fowl eggs.

The virulence of three strains of Mycoplasma gallisepticum (S6 of low broth passage, S6 of high broth passage and A514) and of one strain of M gallinarum was investigated in specific pathogen free chicks, turkey poults, chick embryo tracheal organ cultures and embryonated fowl eggs. One strain of M gallisepticum, S6, of low passage, caused high mortality in newly hatched chicks, and turkey poults, high embryo mortality, and rapid suppression of ciliary activity in tracheal organ cultures. The other strains of M gallisepticum and M gallinarum had relatively less effect. In view of its greater sensitivity, chick embryo mortality is preferable to tracheal organ culture for assessing the virulence of experimental strains of M gallisepticum.

Animals↗

Induction of pneumonia in gnotobiotic calves following inoculation of Mycoplasma dispar and ureaplasmas (T-mycoplasmas).

Cloned cultures of Mycoplasma dispar and of Ureaplasma sp were inoculated endobronchially into gnotobiotic calves. The resulting pneumonia was estimated to involve 2 to 10 per cent of the lung although no clinical signs were observed. The histopathological lesions consisted of infiltration with mononuclear cells, cuffing (peribronchiolar lympho-reticular hyperplasia), catarrhal bronchiolitis and atelectasis. The results indicate that both M dispar and Ureaplasma sp are capable of causing sub-clinical cuffing pneumonia in calves.

Animals↗

Chronic arthritis of rabbits induced by mycoplasmas. III. Induction with nonviable Mycoplasma arthritidis antigens.

Preexposure of rabbits to Mycoplasma arthritidis predisposed them to subsequent development of chronic inflammation in knee joints challenged with nonviable M arthritidis antigens. Rabbits sensitized by hyperimmunization developed a more severe arthritis than those sensitized by infection. Unsensitized rabbits responded minimally to intraarticular injection of nonviable organisms. These results suggested that an immune response to a persisting mycoplasmal antigen might be capable of sustaining chronic inflammation in rabbits with arthritis induced by viable M arthritidis.

Animals↗

Electron microscopic studies of three gliding Mycoplasmas, Mycoplasma mobile, M. pneumoniae, and M. gallisepticum, by using the freeze-substitution technique.

Freeze-substitution technique was applied to thin-sectioning electron microscopy of Mycoplasma mobile, M. pneumoniae, and M. gallisepticum, all of which can glide in the direction of the tapered cell end. M. mobile presented a flask-like cell morphology. An additional layer was found around the tapered end. The cell images of M. pneumoniae showed a protruding membrane extension, the attachment organelle, composed of a low density space inside the cells and featuring a filamentous dense core anchored to the terminal end. The detailed structures were more obvious than those observed by the conventional chemical fixation. The cells of M. gallisepticum presented irregular dense granules, in contrast to regular particles, which can be observed in the images of chemically fixed thin sections, in the rear portion of the cells.

Freezing↗

Characterization of the mycoplasma membrane proteins. VI. Composition and disposition of proteins in membranes from aging Mycoplasma hominis cultures.

Membranes of Mycoplasma hominis cells from cultures progressing from the mid to the end of the logarithmic phase of growth became richer in protein, poorer in phospholipids and cholesterol, heavier in density, and more viscous as determined by EPR. The membrane-bound ATPase activity declined steeply. Electrophoretic analysis failed to show marked changes in membrane protein composition on aging, apart from an increase in the staining intensity of one protein band (Mr approximately 130 000) concomitant with a decrease in the staining intensity of several minor protein bands of high molecular weight. To test for possible changes in the disposition of the various membrane proteins on aging of cultures, a comparison was made of the susceptibility of membrane proteins of intact cells and isolated membranes to trypsinization and lactoperoxidase-mediated iodination. The iodination values and the percent of membrane protein released by trypsinization of intact cells were similar in cells from cultures of different ages, indicating no significant changes in the organization of the proteins on the outer surface. On the other hand, trypsinization and iodination of isolated membranes were found to be most markedly affected by the culture age, indicating significant changes in the organization of the proteins on the inner membrane surface. Thus, the iodination values of isolated membranes decreased by almost two fold, while the percentage of protein released from the membrane by trypsin increased from 28% to 50% during the experimental period. It is suggested that aging in M. hominis cultures is accompanied by a continuous increase in the packing density of the protein molecules on the inner surface of the cell membrane.

Bacterial Proteins↗

The growth response of Mycoplasma hyopneumoniae and Mycoplasma flocculare based upon ATP-dependent luminometry.

Cultures of Mycoplasma hyopneumoniae and M. flocculare in Friis' broth grew faster and to higher titers in air than in 8% CO2; cultures in air grew better when shaken than when stationary. Under the optimal conditions, both species have generation times of about 10 h and achieve maximum titers of at least 10(9) organisms per ml. Maximum growth was reached near pH 7.0, before the phenol red indicator had noticeably changed colour. Changes in growth were readily detected by an ATP assay based upon the luciferin-luciferase reaction. Concentrations of ATP fell rapidly after peak growth. Although the addition of 27 mM glucose to the medium did not change the pattern of growth and gas chromatography gave no evidence of the production of volatile or non-volatile end-products, washed harvested cells of both species metabolized [14C]-glucose. The addition of 29 mM arginine to the medium inhibited growth.

Adenosine Triphosphate↗

Variation and genetic control of surface antigen expression in mycoplasmas: the Vlp system of Mycoplasma hyorhinis.

Surface antigenic diversity in the swine pathogen Mycoplasma hyorhinis is generated by random combinatorial expression and high-frequency phase variation of multiple, size-variant membrane surface lipoproteins (Vlps) which represent the major coat proteins of this wall-less procaryote. The distinctive structural basis for Vlp variation was revealed in a family of several related but divergent vlp genes. These occur in one cluster as single chromosomal copies, each encoding a conserved domain for membrane insertion and lipoprotein processing, and a divergent external domain that changes size by deletion or insertion of repetitive intragenic coding sequences while retaining a distinctive charge motif. Lack of detectable changes in restriction fragment patterns or DNA sequence of vlp structural genes during phase transitions between ON and OFF expression states ruled out long range genomic rearrangements and frameshift mutations as a means of controlling Vlp phase variation. However, highly homologous vlp promoter regions contain a homopolymeric tract of contiguous adenine residues [poly(A)] upstream of the transcriptional start site which is subject to frequent mutations altering its length. These mutations are the only sequence changes detected during phase transitions, and are highly correlated with the expression state of each vlp gene. This suggests a mechanism of transcriptional control regulating Vlp phase variation by critical changes within the poly(A) region affecting the spacing between the -10 and -35 hexamers or a putative regulator binding site. The multiple levels of structural and antigenic diversity embodied in the vlp gene family may provide essential adaptive capabilities for this wall-less microbial pathogen.

Animals↗

Characterization of membrane components of the flask-shaped mycoplasma Mycoplasma mobile.

The cell membrane of Mycoplasma mobile was isolated by either ultrasonic or French press treatment of intact cells. The membrane fraction contained all of the cellular lipids, but only one-third of cellular proteins and had a density of 1.14 g ml-1. The soluble fraction contained the NADH dehydrogenase activity of the cells, as well as a protein with an apparent molecular mass of 55 kDa that was phosphorylated in the presence of ATP. Lipid analyses of M. mobile membranes revealed that membrane lipid could be labelled by radioactive glycerol, oleate and to a much higher extent by palmitate but not by acetic acid. The membrane lipid fraction was composed of 54% neutral and 46% polar lipid. The major constituents of the neutral lipid fraction were free fatty acid, free cholesterol and cholesterol esters (45, 25 and 20%, respectively, of total neutral lipid fraction). The free cholesterol count was 13% (w/w) of total membrane lipids with a cholesterol:phospholipid molar ratio of about 0.9. Among the polar lipids, both phospho- and glycolipids were detected. The phospholipid fraction consisted of a major de novo-synthesized phosphatidylglycerol (approximately 63% of total phospholipids), plus exogenous phosphatidylcholine and sphingomyelin incorporated in an unchanged form from the growth medium. The glycolipid fraction was dominated by a single glycolipid (approximately 90% of total glycolipids) that was preferentially labelled by palmitic acid and showed a very high saturated:unsaturated fatty acids ratio.

Bacterial Proteins↗

Defining the mycoplasma 'cytoskeleton': the protein composition of the Triton X-100 insoluble fraction of the bacterium Mycoplasma pneumoniae determined by 2-D gel electrophoresis and mass spectrometry.

After treating Mycoplasma pneumoniae cells with the nonionic detergent Triton X-100, an undefined, structured protein complex remains that is called the 'Triton X-100 insoluble fraction' or 'Triton shell'. By analogy with eukaryotic cells and supported by ultrastructural analyses it is supposed that this fraction contains the components of a bacterial cytoskeleton-like structure. In this study, the composition of the Triton X-100 insoluble fraction was defined by electron microscopic screening for possible structural elements, and by two-dimensional (2-D) gel electrophoresis and MS to identify the proteins present. Silver staining of 2-D gels revealed about 100 protein spots. By staining with colloidal Coomassie blue, about 50 protein spots were visualized, of which 41 were identified by determining the mass and partial sequence of tryptic peptides of individual proteins. The identified proteins belonged to several functional categories, mainly energy metabolism, translation and heat-shock response. In addition, lipoproteins were found and most of the proteins involved in cytadherence that were previously shown to be components of the Triton X-100 insoluble fraction. There were also 11 functionally unassigned proteins. Based on sequence-derived predictions, some of these might be potential candidates for structural components. Quantitatively, the most prevalent proteins were the heat-shock protein DnaK, elongation factor Tu and subunits alpha and beta of the pyruvate dehydrogenase complex (PdhA, PdhB), but definite conclusions regarding the composition of the observed structures can only be drawn after specific proteins are assigned to them, for example by immunocytochemistry.

Bacterial Proteins↗

Application of an arbitrarily-primed polymerase chain reaction to mycoplasma identification and typing within the Mycoplasma mycoides cluster.

An arbitrarily-primed polymerase chain reaction (AP-PCR) was developed using a primer pair, Mlip1 and Mlip4, for members of the Mycoplasma mycoides cluster, a group containing important pathogens of small and large ruminants. Parameters that influence the reproducibility of this assay were optimized: magnesium, primer and template concentrations, and pH. AP-PCR fingerprinting, carried out on a number of strains of each of the six species or subspecies belonging to the mycoides cluster, allowed the typing of strains within each group. The AP-PCR assay showed that the cluster can be divided into two groups: (i) high and (ii) no genomic polymorphism variation. In addition, specific polymorphic bands for members of species or subspecies included in this cluster were amplified by this AP-PCR method, thus allowing their identification.

Bacterial Typing Techniques↗

In vitro susceptibility of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis to fifty-one antimicrobial agents.

By a tube dilution assay technique, 51 antimicrobial agents were singly tested against 9 strains of Mycoplasma hyopneumoniae and 1 strain of M. hyorhinis for the purpose of obtaining information useful for selecting an agent for testing in vivo against porcine mycoplasmal pneumonia. Based on determining minimal inhibitory concentrations and chemically grouping the agents into nine classes, all M. hyopneumoniae strains were found resistant to penicillins and peptides and susceptible to sulfonamides and tetracyclines, and, in other classes, were either susceptible or resistant depending on the particular agent being tested. Strains were susceptible to the same 33 of the 51 agents. Minimal inhibitory concentrations ranged from 0.06 to 9.20 mug/ml. M. hyorhinis was susceptible to 19 of the 33 agents that M. hyopneumoniae was susceptible to. Minimal inhibitory concentrations ranged from 0.03 to 8.10 mug/ml. All strains of M. hyopneumoniae differed from M. hyorhinis in that they were susceptible to cephaloglycin and nitrofurazone.

Anti-Bacterial Agents↗

Mycoplasma-dependent activation of normal lymphocytes: role of arginine and nonviable mycoplasma antigen in the induction of lymphocyte-mediated cytotoxicity for syngeneic mouse target cells.

The ability of arginine supplementation to abrogate or potentiate Mycoplasma arthritidis-mediated cytotoxicity induction by normal CBA mouse lymphocytes was investigated. Also, nonviable antigens of M. arthritidis were assayed for their ability to induce a cytotoxic response. The presence of additional arginine in the culture medium had no significant effect on the amount of cytotoxicity produced. In some cases, an increase in cytotoxicity was seen in the presence of additional arginine. Amino acid analysis of the culture supernatants showed that the arginine in the nonsupplemented medium was exhausted during the assay, whereas ample amounts of arginine were present in the supplemented medium. However in both cases the amount of cytotoxicity was comparable. Nonviable antigens of M. arthritidis were unable to induce a significant cytotoxic response in the presence of normal lymphocytes. The same antigens exhibited a suppressive response when tested for blastogenesis in the presence of normal lymphocytes. A discussion of lymphocyte involvement is presented.

Animals↗