Adherence to various host cell lines of Mycoplasma bovis strains differing in pathogenic and cultural features.

Mycoplasma bovis is known to be responsible for pneumonia and arthritis in calves, as well as mastitis in dairy cows. Despite clear evidence of its pathogenic potential, little is known about mechanisms of cytadherence and the molecular factors involved. The purpose of this work was to compare adherence rates of M. bovis field strains to different host cell lines and study the effects of cloning and sub-culturing M. bovis strains on their adherence properties. Eighteen metabolically labeled M. bovis strains isolated from different pathological backgrounds were examined in adherence trials using four different host cell lines, i.e. embryonic bovine lung (EBL), embryonic bovine trachea (EBTr), Madin Darby bovine kidney (MDBK) and rabbit kidney (RK) cells. Although large interstrain variations in adherence rates (3.4-19.1%) were measured they could not be correlated to the pathological background (pneumonia, arthritis or mastitis). Adherence rates to the fibroblast cell line (EBTr) were significantly lower than those to the three epithelial cell lines (EBL, MDBK and RK). The only non-pathogenic strain (221/89) exhibited lower adherence rates than three isolates from clinical mastitis. Interestingly, adherence rates were significantly reduced after in vitro passaging. In contrast, no effect of single cloning of strains on adherence was observed. There was no general correlation between expression of variable surface proteins (Vsps) as monitored by immunoblotting and adherence rates, although alterations in Vsp expression profiles were seen as a consequence of passaging. As there is probably a large number of adhesins, variable and non-variable, on the surface of M. bovis cells the issue is very complex, and the most active components have yet to be identified.

Epitope mapping of immunogenic and adhesive structures in repetitive domains of Mycoplasma bovis variable surface lipoproteins.

The family of variable surface lipoproteins (Vsps) of the bovine pathogen Mycoplasma bovis includes some of the most immunogenic antigens of this microorganism. Vsps were shown to undergo high-frequency phase and size variations and to possess extensive reiterated coding sequences extending from the N-terminal end to the C-terminal end of the Vsp molecule. In the present study, mapping experiments were conducted to detect regions with immunogenicity and/or adhesion sites in repetitive domains of four Vsp antigens of M. bovis, VspA, VspB, VspE, and VspF. In enzyme-linked immunosorbent assay experiments, sera obtained from naturally infected cattle showed antibodies to different repeating peptide units of the Vsps, particularly to units R(A)1, R(A)2, R(A)4.1, R(B)2.1, R(E)1, and R(F)1, all of which were found to contain immunodominant epitopes of three to seven amino acids. Competitive adherence trials revealed that a number of oligopeptides derived from various repeating units of VspA, VspB, VspE, and VspF partially inhibited cytoadhesion of M. bovis PG45 to embryonic bovine lung cells. Consequently, putative adherence sites were identified in the same repeating units (R(A)1, R(A)2, R(A)4.1, R(B)2.1, R(E)1, and R(F)1) and in R(F)2. The positions and lengths of the antigenic determinants were mostly identical to those of adhesion-mediating sites in all short repeating units, whereas in the considerably longer R(F)1 unit (84 amino acid residues), there was only one case of identity among four immunogenic epitopes and six adherence sites. The identification of epitopes and adhesive structures in repetitive domains of Vsp molecules is consistent with the highly immunogenic nature observed for several members of the Vsp family and suggests a possible function for these Vsp molecules as complex adherence-mediating regions in pathogenesis.

Intraspecies polymorphism of vsp genes and expression profiles of variable surface protein antigens (Vsps) in field isolates of Mycoplasma bovis.

To assess the extent of interstrain variation, 50 isolates of Mycoplasma (M.) bovis including the type strain PG45 were examined for the presence of a family of variable membrane surface lipoproteins (Vsps) and their genes. Southern hybridization using a genomic fragment carrying three distinct vsp genes (vspAEF) revealed a striking heterogeneity, with only 2/50 strains having identical banding patterns. Cluster analysis of the data showed that most isolates from interrelated herds (groups 1, 2 and 3) were combined in a cluster of 50% homology, while isolates from distinct geographical regions (groups 4, 5 and 6) were linked only at 18% homology. Vsp antigen expression was monitored by Western immunoblotting using four specific monoclonal antibodies (MAbs). Resembling the findings at the DNA level, interstrain variation of Vsp expression among groups 1-3 was less pronounced than among non-interrelated isolates from groups 4-6. Ten out of 50 strains did not hybridize with the vspAEF gene probe at high-stringency conditions, 8/50 failed to react with any of the Vsp-related MAbs, and 6/50 proved negative in both assays. Interestingly, most of these isolates produced hybridization signals at low stringency suggesting major distinctions in their vsp gene structure. The extensive evidence obtained on interstrain vsp gene polymorphism and variation in Vsp expression could provide a basis for a future understanding of the pathogenic potential of individual M. bovis strains.

Mechanisms and factors involved in Mycoplasma bovis adhesion to host cells.

Mycoplasma (M.) bovis cytadhesion was studied using permanent embryonic bovine lung (EBL) cells as host system. Adherence rates were found to be strongly dependent on temperature and the mycoplasma-to-EBL ratio near the point of saturation of the attachment isotherm was determined to be 225 : 1. Mild trypsinization of viable M. bovis cells caused a measurable decrease of adherence indicating that surface proteins, among them the P26 antigen, played a major part as adhesion factors. Neuraminidase treatment of mycoplasmas led to a drastic reduction of adherence rates, which emphasizes the importance of sialyl moieties in adhesive interactions. The ability of the P26 antigen, a hydrophilic 32-kDa protein, to function as an adhesin was confirmed using a competitive adherence assay, in which the HPLC-purified protein was shown to reduce mycoplasma adhesion. These data complement previous findings obtained with the corresponding monoclonal antibody (MAb) 4F6. In further inhibition experiments, it could be demonstrated that MAb 1E5, which is directed against a common epitope of at least three members of the Vsp (variable surface protein) family of M. bovis, was also capable of decreasing mycoplasma attachment to EBL cells. This is the first evidence of possible involvement of Vsps in cytadhesion. In an effort to identify more putative adhesion proteins of this organism, the reverse adherence screening assay was used, a procedure based on the specific binding of labelled mammalian tissue culture cells to Western-blotted mycoplasmal proteins.

Comparison of Mycoplasma bovis strains based on SDS-PAGE and immunoblot protein patterns.

Whole-cell protein patterns generated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were compared for 34 isolates of Mycoplasma bovis. A high degree of similarity between most of the strains was established with strain-to-strain differences mainly confined to quantitative variations of certain protein bands, particularly in the molecular weight regions of 64-68, 55 and 26 kD. Two of the isolates provided more deviating patterns. Hydrophobic membrane protein fractions of the strains as prepared by Triton X-114 phase partitioning were compared by SDS-PAGE, which confirmed some of the characteristic strain features found with whole-cell proteins. The immunoblot analysis revealed that up to 20 of the 50-55 discrete protein bands detected in SDS-PAGE patterns were recognized to be antigenic by rabbit and bovine hyperimmune sera. It is concluded that the same set of major antigens is present in all strains investigated, but amounts of individual constituents may be differing.

A group of indole-negative bovine strains with high deoxyribonucleic acid homology to Pasteurella multocida.

A group of nine bovine Pasteurella strains not producing indole were investigated for their taxonomic relationships with Pasteurella multocida, Pasteurella haemolytica and Pasteurella canis. For all strains, DNA-DNA hybridization has revealed a high genetic relatedness at the species level to P. multocida and significantly lower homologies of only 18-41% towards P. haemolytica and 11-15% towards P. canis. Guanine plus cytosine values of 38.0 to 42.1 mol% and several phenotypic characters have been found to be different from the established pattern for P. multocida subspecies. It is suggested that the strains represent a new taxon, possibly another P. multocida subspecies.

[Demonstration of iron transport activity in Pasteurella multocida cultures]. / Nachweis von Eisen-Transport-Aktivität in Pasteurella multocida-Kulturen.

It has been established that Pasteurella multocida cultures possess pronounced iron transport activities to accumulate the iron necessary for growth. Experiments with Fe-59 confirmed that the bacterial cells are able to acquire iron without direct contact from high molecular iron substrates, such as iron dextrane, ferritine or transferrine. Microbial siderophores of the hydroxamate and phenolate types, such as desferrioxamin B and enterobactine as well as other iron chelators (phenanthroline, citrate and nitrilotriacetate) decrease the bacterial cell growth or iron incorporation and are not relevant for iron transport in P. multocida. The direct analytical identification of siderophores using the reactions by Csaky (hydroxamate type) and Arnow (phenolate type) has proved unsuccessful. The importance of the mannan cell wall polysaccharide is discussed with respect to the iron transport. Thus in terms of iron accumulation, P. multocida is similar to Yersinia, which also possess an efficient transport system for iron not involving siderophores.