Mycoplasma species and related organisms isolated from ruminants in Britain between 1990 and 2000.

Between 1990 and 2000, more than 1600 mycoplasmas and the related acholeplasmas were identified from ruminant animals by the Mycoplasma Group at the Veterinary Laboratories Agency--Weybridge. Mycoplasma bovis was the most commonly identified pathogen, mostly from pneumonic calves but occasionally from cattle with mastitis and arthritis. Mycoplasma canis was first isolated in Britain in 1995 from pneumonic calves and the number of isolates increased to 18 per cent of the total mycoplasmas isolated from cattle in 1999. The ELISA for antibodies to M. bovis detected 1971 positive samples (22 per cent) among 8959 serum samples, mainly from pneumonic cattle. Other mycoplasmas identified included Mycoplasma dispar from the lungs of cattle with respiratory disease, and Mycoplasma bovigenitalium from the reproductive tract of cows with vulvovaginitis and infertility. Mycoplasma bovirhinis and Acholeplasma species were found commonly but are thought to be more opportunistic than pathogenic. In sheep and goats, the majority of Mycoplasma species isolated were identified as Mycoplasma ovipneumoniae from pneumonic sheep, Mycoplasma conjunctivae from sheep with keratoconjunctivitis, and the ubiquitous Mycoplasma arginini.

Antiserum raised against gyrase A of Acholeplasma laidlawii reacts with phytoplasma proteins.

Part of the gyrase A gene (gyrA) of Acholeplasma laidlawii was cloned and incorporated directly downstream from a 6 x His tag segment of the pQE expression vector. The 23-kDa fusion protein was expressed as a 6 x His-tagged protein in Escherichia coli. The fusion protein was purified and used as an antigen for rabbit immunization. Western immunoblot analysis revealed that the antiserum raised against the gyrase A fragment had a specific affinity for a 108-kDa protein of A. laidlawii and cross-reacted with a 107.5-kDa protein of Acholeplasma axanthum, a 107-kDa protein of Acholeplasma granularum, and 95-97-kDa proteins of several phytoplasma-infected plants. The antiserum could also detect phytoplasmas in infected plant sap. These results demonstrate that the gyrase A protein (GyrA) of A. laidlawii shares antigenicity with the GyrA of other Acholeplasma species and also with those of phytoplasmas including some from a few groups with unrelated 16S rRNAs.

Use of antibodies against the P36 protein of Mycoplasma hyopneumoniae for the identification of M. hyopneumoniae strains.

Mycoplasma hyopneumoniae, the principal aetiological agent of porcine enzootic pneumonia, synthesizes a 36 kDa protein (P36) which is an early and strong immunogenic factor in experimentally and naturally infected swine. Polyclonal antibodies were made against the recombinant P36 protein in rabbits and used for the identification of M. hyopneumoniae by the immunoblot technique. The proteins from the M. hyopneumoniae reference strains and from 13 M. hyopneumoniae field strains isolated from naturally infected pigs in Switzerland, Hungary, France and Canada were analysed by the immunoblot technique using anti-P36 antibodies. All 13 field strains and the three reference J strains of M. hyopneumoniae, received from different collections and laboratories, exhibited a strong reaction with a protein of 36 kDa indicating that the P36 protein is a common M. hyopneumoniae antigen. None of the different porcine Mycoplasma species including M. flocculare, M. hyorhinis, M. hyosynoviae, A. axanthum, A. laidlawii and A. granularum showed any reaction on the immunoblot with the anti-P36 antibodies. In addition, we have found no reaction with anti-P36 antibodies using 47 different Mycoplasma or Acholeplasma species isolated from human, mice, rat, poultry, ruminant, dog and cat. In conclusion we have shown that P36 is a protein that is a common antigen of M. hyopneumoniae strains and is not found in other Mycoplasma or Acholeplasma species tested. Because of its high specificity, P36 protein, or antibodies made against this protein can be used for the identification of M. hyopneumoniae strains.

Rapid identification of mycoplasmas by indirect immunoperoxidase test using small square filter paper.

The indirect immunoperoxidase test using small, square filter paper was used for rapid identification of mycoplasmas. Colonies of type strains of 22 mycoplasma species, 3 acholeplasma species, and three Ureaplasma diversum serogroups were stained by this test with high sensitivity and specificity. All of 49 isolates from bovine materials and cell cultures were easily identified by this test, and the results agreed with those obtained by growth inhibition test. Use of filter paper made it possible to add different kinds of antisera or conjugates to the same agar plate simultaneously and also to save antiserum and conjugate. This test proved to be a simple and useful technique for rapid identification of many mycoplasma species grown on agar medium.

Chemical structures of acholeplasmal lipoglycans and their relation to biological interactions.

The partial characterization of the structure of the lipoglycan (LG) from Acholeplasma axanthum is added to the previous complete structural analysis of the lipoglycan from A. granularum. The terminal sequence of A. axanthum LG is Glcp(beta 1----2)-Glcp(beta 1----2)-Glcp(beta 1----6)-; of A. granularum Glcp(beta 1----2)-Glcp(alpha 1----4)-Glcp(beta 1----4)-. These specific residues define the major antigenic determinants of the LG as determined by blockage of hemagglutination of LG coated erythrocytes by specific oligosaccharides and binding of radiolabeled LG to specific immunoglobulins. The binding of LG to mammalian cells occurs by an interaction between specific eucaryotic cell receptors and the internal sequence of the oligosaccharide chain of LG. Size and sugar chains of LG rather than fatty acid residues appears to define the binding site on the LG.

Identification of the major antigenic determinants on lipoglycans from Acholeplasma granularum and Acholeplasma axanthum.

The major antigenic determinants of the lipoglycans from Acholeplasma granularum and Acholeplasma axanthum were found to be the oligosaccharide sequences Glcp(beta 1 leads to 2)-Glcp(alpha 1 leads to 4)-Glcp and Glcp(beta 1 leads to 2)-Glcp(beta 1 leads to 2)-Glcp, respectively. The disaccharides sophorose and maltose inhibited hemagglutination by specific antiserum of sheep erythrocytes coated with lipoglycan from A. granularum. Only sophorose possessed this capacity with the lipoglycan from A. axanthum. Periodate oxidation destroyed the ability of both lipoglycans to interact with specific antibody, a result compatible with structural data on the lipoglycans.

Serological investigation of horse sera for antibodies against mycoplasmas and acholeplasmas.

Sera from horses with respiratory disease (RD) have been investigated using the complement fixation test, indirect hemagglutination test, enzyme immune assay, and the metabolic inhibition test, and sera from mares after abortion, using the complement fixation test, indirect hemagglutination test and enzyme immune assay, for antibodies against Mycoplasma equirhinis, M subdolum, M. equigenitalium, M. pulmonis, M. felis, Acholeplasma laidlawii, A. hippikon and A. equifetale. Antibodies were found against all mycoplasma and acholeplasma species tested, more often against acholeplasmas. The antibody pattern was quite similar for horses with RD and for mares after abortion. The results of the four serological tests performed showed only a limited correlation and the percentage of sera with antibodies detected by the four tests used differed widely.

Variation in the susceptibility of bovine mycoplasmas to killing by the alternative complement pathway in bovine serum.

All but one of nine Acholeplasma strains were susceptible to killing by gnotobiotic-calf serum while all but one of fifteen Mycoplasma strains were resistant. The mechanism of killing was examined using inhibitors of complement, viz. EDTA. MgEGTA. epsilon-amino caproic acid, and also by desalting serum and adding MgCl2 and CaCl2. The results indicated that, when strains were killed by serum from gnotobiotic calves it was by the alternative complement pathway. However, experiments with mycoplasmas and Escherichia coli indicated that the effect of MgEGTA was not identical for bovine and human sera. Sensitivity to killing by the alternative complement pathway in bovine serum appeared to be a property of avirulent strains and resistance to killing may be regarded as a virulence determinant of mycoplasmas.

Endotoxin-like activities of mycoplasmal lipopolysaccharides (lipoglycans).

Lipoglycans (previously designated lipopolysaccharides) from several species of Acholeplasma and from Thermoplasma acidophilum were examined for endotoxin-like activities as measured by the standard rabbit fever test and the Limulus amoebocyte lysate assay. The lipoglycans from Acholeplasma granularum, Achloplasma laidlawii, Acholeplasma modicum, and Acholeplasma oculi caused a febrile response at concentrations of 1 ng/ml per kg or greater, whereas with control Escherichia coli EC-2 lipopolysaccharides, 6.25 ng/ml per kg was required. Similar results were obtained in the Limulus amoebocyte lysate test. The minimum concentrations in nanograms per milliliter required to stimulate formation of a solid clot were: Acholeplasma axanthum, 0.22; A. granularum, 0.85; A. modicum, 0.51; A. laidlawii, 1.05; A. oculi, 0.74. Standard E. coli 1B lipopolysaccharide required a concentration of 0.125 ng/ml. Thermoplasma lipoglycan was least active, requiring 4.25 ng/ml. Clotting of the Limulus lysate proceeds by the activation by lipopolysaccharide plus Ca(2+) of a proenzyme which cleaves an arginine-lysine peptide bond of the coagulogen. The clotting and amidase activities are inactivated by deoxycholate and can be reactivated by addition of lipopolysaccharide and Ca(2+). As with E. coli 1B lipopolysaccharide, acholeplasmal lipoglycans were shown to restore both clotting and amidase activities of the deoxycholate-inactivated Limulus clotting enzyme. The degree of restoration of amidase activity by mycoplasmal lipoglycans relative to E. coli lipopolysaccharide (1.00) were: A. axanthum, 1.71; A. modicum, 1.22; A. granularum, 0.61; and Thermoplasma, 0.37. The coagulating enzyme, restored with either E. coli lipopolysaccharide or mycoplasmal lipoglycans, was able to react with the synthetic peptide benzoyl-Ile-Glu-(gamma-OCH(3))-Gly-p-nitroaniline (an analog of the coagulogen) or with the purified coagulogen itself to form the clot. The mycoplasmal lipoglycans alone were incapable of promoting these reactions when incubated with the synthetic peptide or with the purified coagulogen, thereby ruling out the contamination of these lipoglycans with proteases capable of cleaving the same Arg-Lys peptide bond of the coagulogen. These results show that acholeplasmal lipoglycans possess endotoxin-like activities. Their passive or active role in disease remains to be established.

Humoral immune response of rabbits to acholeplasmal lipoglycans.

Lipoglycans extracted from Acholeplasma species with hot aqueous phenol were immunogenic for rabbits when introduced by an appropriate method. All lipoglycans examined elicited antibody associated with a heavy, 2-mercaptoethanol-sensitive immunoglobulin fraction when inoculated intravenously adsorbed to autologous rabbit erythrocytes. This antibody was specific for the Acholeplasma species from which the lipoglycan was extracted. Extensive immunization of these animals with acholeplasmal lipoglycans produced significant increases in sheep erythrocyte hemolysin. Some, but not all, Acholeplasma species yielded lipoglycans that were immunogenic when emulsified with Freund complete adjuvant and introduced via the footpad into rabbits. Such animals produced antibodies corresponding to the M and G immunoglobulin classes that reacted with both homologous and heterologous acholeplasmal lipoglycans by precipitation in immunodiffusion as well as passive hemagglutination. None of the animals inoculated demonstrated a significant anamnestic response after booster injections either intravenously or via the footpads.

[Investigation of mare sera for antibodies against acholeplasmas and mycoplasmas with ther enzyme linked immunosorbent assay (ELISA) (author's transl)]. / Untersuchung von Stutenseren auf Antikörper gegen Acholeplasmen und Mykoplasmen mit dem Enzyme Linked Immunosorbent Assay (ELISA).

After abortion sera were taken from 58 thoroughbred and other mares of the northwestern part of Germany and investigated by ELISA (enzyme linked immuno-sorbent assay) for antibodies against Mycoplasma equirhinis, M. subdolum, M. equigenitalium, M. pulmonis, M. felis, Acholeplasma laidlawii, A. hippikon, and A. equifetale. Reactions at serum dilutions of 1:32 and higher were considered as positive. At serum dilution 1:32 no antibodies were found in 11 sera. The remaining sera showed antibodies against one or more of the mycoplasma antigens investigated. The number of multiple reactions decreased with an increasing dilution of the sera. Titers were found between 1:32 and 1:256. In one case a titer of 1:2048 against M. equigenitalium antigen was found. Most often antibodies against A. laidlawii were observed i.e. in 37 sera. These antibodies also showed the highest titers. Only 3 sera contained antibodies against A. hippikon. Antibodies against M. felis and A. equifetale were found in 26 sera. Between 10 and 15 sera showed antibodies against the remaining mycoplasma species.

[Bidimensional immunoelectrophoretic study of the antigenic composition of the membrane in various mycoplasma strains]. / Etude par immunoélectrophorèse bidimensionnelle de la composition antigénique de la membrane de quelques souches de mycoplasms

Membrane antigenic composition of Acheloplasma laidlawii PG9, A. granularum BTS-39, and Mycoplasma fermentans PG 18(G) was determined by means of bidimensional immunoelectrophoresis in the presence of sodium desoxycholate 0.5%. Depending upon the mycoplasma species from which membranes were obtained, 7 to 15 antigens were evidenced. Using sodium desoxycholate presents the advantage over non-ionic detergents to dissolve better the mycoplasmic membrane antigenic complexes. A comparative study of five strains belonging to the above-noted species confirms the serological heterogeneity of the Mycoplasmateles order and shows variability at the membrane antigenic composition level of Acheloplasma laidlawii.