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SummaryMycoplasma hyopneumoniae (M. hyopneumoniae) is the primary pathogen of enzootic pneumonia, a chronic respiratory disease in pigs. Infections occur worldwide and cause major economic losses to the pig industry. The present paper reviews the current knowledge on M. hyopneumoniae infections, with emphasis on identification and analysis of knowledge gaps for optimizing control of the disease. Close contact between infected and susceptible pigs is the main route of M. hyopneumoniae transmission. Management and housing conditions predisposing for infection or disease are known, but further research is needed to better understand M. hyopneumoniae transmission patterns in modern pig production systems, and to assess the impor-

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Sequences of the gene encoding the b-subunit of the RNA polymerase (rpoB) were used to delineate the phylogeny of the family Pasteurellaceae. A total of 72 strains, including the type strains of the major described species as well as selected field isolates, were included in the study. Selection of universal rpoB-derived primers for the family allowed straightforward amplification and sequencing of a 560 bp fragment of the rpoB gene. In parallel, 16S rDNA was sequenced from all strains. The phylogenetic tree obtained with the rpoB sequences reflected the major branches of the tree obtained with the 16S rDNA, especially at the genus level. Only a few discrepancies between the trees were observed. In certain cases the rpoB phylogeny was in better agreement with DNA-DNA hybridization studies than the phylogeny derived from 16S rDNA. The rpoB gene is strongly conserved within the various species of the family of Pasteurellaceae. Hence, rpoB gene sequence analysis in conjunction with 16S rDNA sequencing is a valuable tool for phylogenetic studies of the Pasteurellaceae and may also prove useful for reorganizing the current taxonomy of this bacterial family.The family Pasteurellaceae Pohl 1981 currently comprises 57 named bacterial species which have been isolated from man and various animals. The three genera Haemophilus, Actinobacillus and Pasteurella which originally formed the family have recently been joined by new genera, the most prominent being Mannheimia, which contains the species Mannheimia haemolytica (formerly Pasteurella haemolytica) (Angen et al., 1999). Three genera are currently formed by single species, these are Phocoenobacter uteri (Foster et al., 2000), Lonepinella koalarum (Osawa et al., 1995) and Histophilus somni (Angen et al., 2003). The latter genus has been proposed to include the three species incertae sedis 'Haemophilus somnus', 'Haemophilus agni' and 'Histophilus ovis'. Finally, Christensen et al. (2003a) have described the genus Gallibacterium, which currently consists of one species and one genomospecies.Members of the Pasteurellaceae are generally isolated from mucosal membranes and tissue of birds, turtles and mammals, including man. They show a strong host association and have probably co-evolved with their corresponding hosts (Bisgaard, 1993). While most of the species are commensals, there are a few that act as pathogens . These include the human pathogens Haemophilus influenzae, which causes neonatal meningitis and otitis media, and Actinobacillus actinomycetemcomitans, which causes juvenile periodontitis. Important animal diseases are caused, for example, by M. haemolytica (shipping fever of cattle), Pasteurella multocida (atrophic rhinitis in swine and fowl cholera) and Actinobacillus pleuropneumoniae (pleuropneumonia in pigs). In many of the pathogenic species of Pasteurellaceae, specific toxins belonging to the RTX toxin family are found . These toxins are often associated with specifically pathogenic isolates and seem to determine to some extent the host range of the pathoge...

A fourth type of RTX determinant was identified in Actinobacillus pleuropneumoniae and was designated apxlVA. When expressed in Escherichia coli, recombinant ApxlVA showed a weak haemolytic activity and cohaemolytic synergy with the sphingomyelinase (beta-toxin) of Staphylococcus aureus. These activities required the presence of an additional gene, ORF1, that is located immediately upstream of apxlVA. The apxlVA gene product could not be detected in A. pleuropneumoniae cultures grown under various conditions in vitro; however, pigs experimentally infected with A. pleuropneumoniae serotypes 1,5 and 7 started to produce antibodies that reacted with recombinant ApxlVA 14 d post-infection, indicating that apxlVA is expressed in vivo. In addition, sera from pigs naturally and experimentally infected with any of the serotypes all reacted with recombinant ApxlVA. The apxlVA gene from the serotype 1 A. pleuropneumoniae type strain Shope 4074T encodes a protein with a predicted molecular mass of 202 kDa which has typical features of RTX proteins including hydrophobic domains in the Nterminal half and 24 glycine-rich nonapeptides in the C-terminal half that bind Ca2+. The glycine-rich nonapeptides are arranged in a modular structure and there is some variability in the number of modules in the ApxIVA proteins of different serotypes of A. pleuropneumoniae. The deduced amino acid sequences of the ApxIVA proteins have significant similarity with the Neisseria meningitidis iron-regulated RTX proteins FrpA and FrpC, and to a much lesser extent with other RTX proteins. The apxlVA gene could be detected in all A. pleuropneumoniae serotypes and seems to be species-specif ic. Although the precise role of this new RTX determinant in pathogenesis of porcine pleuropneumonia needs to be determined, apxlVA is the first in vivo induced toxin gene that has been described in A. pleuropneumoniae.

A disposable microarray was developed for detection of up to 90 antibiotic resistance genes in gram-positive bacteria by hybridization. Each antibiotic resistance gene is represented by two specific oligonucleotides chosen from consensus sequences of gene families, except for nine genes for which only one specific oligonucleotide could be developed. A total of 137 oligonucleotides (26 to 33 nucleotides in length with similar physicochemical parameters) were spotted onto the microarray. The microarrays (ArrayTubes) were hybridized with 36 strains carrying specific antibiotic resistance genes that allowed testing of the sensitivity and specificity of 125 oligonucleotides. Among these were well-characterized multidrug-resistant strains of Enterococcus faecalis, Enterococcus faecium, and Lactococcus lactis and an avirulent strain of Bacillus anthracis harboring the broadhost-range resistance plasmid pRE25. Analysis of two multidrug-resistant field strains allowed the detection of 12 different antibiotic resistance genes in a Staphylococcus haemolyticus strain isolated from mastitis milk and 6 resistance genes in a Clostridium perfringens strain isolated from a calf. In both cases, the microarray genotyping corresponded to the phenotype of the strains. The ArrayTube platform presents the advantage of rapidly screening bacteria for the presence of antibiotic resistance genes known in gram-positive bacteria. This technology has a large potential for applications in basic research, food safety, and surveillance programs for antimicrobial resistance.

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