Molecular characterisation of the Mycoplasma cynos haemagglutinin HapA.

Mycoplasma (M.) cynos is a proven pathogen of dogs causing respiratory infections including pneumonia. We examined 19 M. cynos strains isolated from different organs of dogs in Austria, Denmark and Israel. All strains agglutinated mammalian and chicken erythrocytes. Using erythrocytes of chickens or dogs as specific ligands we isolated an approximately 65 kDa protein from cell-free supernatants of 3 M. cynos strains, which showed an apparent capacity for haemagglutination. The N-terminal sequence of a 25 kDa fragment of this protein was identified as NNEMTPKVTVEAKSMELLLSVEK. The identical amino acid sequence is encoded by the gene MCYN_0308 in the genome of M. cynos C142. This gene belongs to a family of some 20 genes which encode putative lipoproteins with proline-rich regions (PRR) in the first third of their molecules. We termed the 65 kDa haemagglutinin HapA and sequenced hapA gene homologues of 16 M. cynos strains. Analyses of hapA gene homologues revealed similar but not identical sequences, some having insertions and/or deletions in the PRR. We produced a recombinant HapA protein (rHapA) and also mouse monoclonal antibodies (mAbs) recognizing HapA. However, enzyme immunoassays using native M. cynos colonies and mAbs 5G2 or 3B7 showed variable expression of HapA in all M. cynos strains. This was further confirmed by Western blot analyses which showed different HapA quantities and also size-variation of HapA among strains. Analyses of cDNA of the expressed hapA genes showed that besides the hapA gene cultures of M. cynos (strains 105, 2002, 2297) can also express other forms of hap genes. In addition, in cloned cultures of strain 2297 altered HapA epitopes for mAbs 5G2 and 3B7 with distinct hapA gene mutations that resulted in altered HapA amino acid sequence were found. Most of the dogs examined had serum antibodies to rHapA. In conclusion, we characterized the M. cynos haemagglutinin HapA protein and encoding gene hapA, a factor involved in cytadherence to host cells and therefore important for M. cynos infection, and showed that expression of HapA is varied in M. cynos by two distinct mechanisms; differential gene expression and nucleic acid substitution within hapA homologues.

Ornithobacterium rhinotracheale has neuraminidase activity causing desialylation of chicken and turkey serum and tracheal mucus glycoproteins.

Neuraminidases (sialidases) are virulence factors of several poultry pathogens. Ornithobacterium rhinotracheale is a well known poultry pathogen causing respiratory disease in chickens and turkeys all over the world. We investigated whether O. rhinotracheale has neuraminidase enzymatic activity (NEAC). We tested NEAC in 47 O. rhinotracheale strains isolated from turkeys and chickens in eight countries. All strains showed relatively strong NEAC and considerable levels of NEAC were detected also in "cell-free supernatants" of their pelleted cells. Zymography using neuraminidase-specific chromogenic substrate indicated that a protein with molecular mass of ~40kDa and isoelectric point (pI) of ~8.0 is a putative neuraminidase of O. rhinotracheale. Notably, the genome of the type strain of O. rhinotracheale, DSM 15997 contains a gene (Ornrh_1957) encoding a putative neuraminidase with such Mw (39.5 kDa) and pI (8.5). We sequenced a corresponding genomic region of 20 O. rhinotracheale strains and found five distinct types of the neuraminidase gene (termed nanO) sequences. Most diversified nanO sequence was found in two strains isolated from chickens in Hungary in 1995. Their nanO sequences differ from that of the type strain (LMG 9086(T)) in 27 nucleotides. O. rhinotracheale neuraminidase showed capacity to cleave sialic acid from chicken and turkey glycoproteins. It cleaved sialic acid from SAα(2-6)gal moiety of their serum proteins, including immunoglobulin G (IgG) and transferrin. O. rhinotracheale also desialylated chicken and turkey tracheal mucus glycoprotens with SAα(2-3)gal moieties. This study provides the first evidence that O. rhinotracheale has neuraminidase which can desialylate glycoproteins of its natural hosts.