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This study investigated the prevalence of extraintestinal pathogenic E. coli (ExPEC)-associated sequence types (STs) from phylogenetic group B2 among 449 fluoroquinolone-susceptible dog clinical isolates from Australia. Isolates underwent PCR-based phylotyping and random amplified polymorphic DNA analysis to determine clonal relatedness. Of the 317 so-identified group B2 isolates, 77 underwent whole genome sequencing (WGS), whereas the remainder underwent PCR-based screening for ST complexes (STc) STc12, STc73, STc372, and ST131. The predominant ST was ST372 according to both WGS (31 % of 77) and ST-specific PCR (22 % of 240), followed by (per WGS) ST73 (17 %), ST12 (7 %), and ST80 (7 %). A WGS-based phylogenetic comparison of ST73 isolates from dogs, cats, and humans showed considerable overall phylogenetic diversity. Although most clusters were species-specific, some contained closely related human and animal (dog > cat) isolates. For dogs in Australia these findings both confirm ST372 as the predominant E. coli clonal lineage causing extraintestinal infections and clarify the importance of human-associated group B2 lineage ST73 as a cause of UTI, with some strains possibly being capable of bi-directional (i.e., dog-human and human-dog) transmission.

Mycoplasmas are cell wall-less bacteria that infect a variety of animals in a species-specific manner. In swine, Mycoplasma hyopneumoniae is the most virulent and presents the most disease and economic problems to the swine industry. Serological assays are commonly used to assess colonization and disease, but antigenic cross-reactivity between M. hyopneumoniae and other mycoplasma species, most notably Mycoplasma hyorhinis, Mycoplasma hyosynoviae and Mycoplasma flocculare, is a concern. The extent of cross-reactivity has not been thoroughly investigated. These studies were designed to identify M. hyopneumoniae proteins that are recognized by rabbit hyperimmune sera raised against the other swine mycoplasmas. Our results indicate extensive cross-reactivity between M. flocculare and M. hyopneumoniae , which explains previous reports seen with ELISA assays. Only three of the thirty-nine M. hyopneumoniae proteins tested showed no cross reactivity with the other three swine mycoplasmas, mhp182 (42 kDa C-terminal fragment), mhp638 and mhp684 (C-terminal fragment). Two proteins, mhp384 and mhp511, were cross-reactive with hyperimmune sera generated against three of the four species. None of the anti- M. hyorhinis hyperimmune sera reacted to any of the M. hyopneumoniae proteins. These results suggest that inapparent M. flocculare infections could produce positive responses in M. hyopneumoniae serological assays due to cross-reactivity, and that M. hyosynoviae infections are less likely to do so and M. hyorhinis infections unlikely to affect assay results.

Extraintestinal pathogenic Escherichia coli (ExPEC) can cause urinary tract and other types of infection in cats, but the relationship of cat ExPEC to human ExPEC remains equivocal. This study investigated the prevalence of ExPEC-associated sequence types (STs) from phylogenetic group B2 among fluoroquinolone-susceptible cat clinical isolates. For this, 323 fluoroquinolone-susceptible cat clinical E. coli isolates from Australia underwent PCR-based phylotyping and random amplified polymorphic DNA analysis to determine clonal relatedness. Of the 274 group B2 isolates, 53 underwent whole genome sequencing (WGS), whereas 221 underwent PCR-based screening for (group B2) sequence type complexes (STc) STc12, STc73, ST131, and STc372. Group B2 was the dominant phylogenetic group (274/323, 85 %), whereas within group B2 ST73 dominated, according to both WGS (43 % of 53; followed by ST127, ST12, and ST372 [4/53, 8 % each]) and ST-specific PCR (20 % of 221). In WGS-based comparisons of cat and reference human ST73 isolates, cat isolates had a relatively conserved virulence gene profile but were phylogenetically diverse. Although in the phylogram most cat and human ST73 isolates occupied host species-specific clusters within serotype-specific clades (O2:H1, O6:H1, O25:H1, O50/O2:H1), cat and human isolates were intermingled within two serotype-specific clades: O120:H31 (3 cat and 2 human isolates) and O22:H1 (3 cat and 5 human isolates). These findings confirm the importance of human-associated group B2 lineages as a cause of urinary tract infections in cats. The close genetic relationship of some cat and human ST73 strains suggests bi-directional transmission may be possible.

The apxIVA gene, a recently discovered RTX determinant of Actinobacillus pleuropneumoniae, was shown to be species-specific. DNA hybridization experiments using probes for various regions of apxIVA revealed that the 3'-terminus of this gene was present in all 14 serotypes of A. pleuropneumoniae but absent from phylogenetically related species. A primer pair spanning this region specifically amplified a 422bp fragment in PCR experiments with DNA from the reference strains of the 14 serotypes and 194 field strains isolated from various geographic locations worldwide. DNA sequence analysis of PCR products derived from all serotypes were identical except in serotypes 3, 8, and 10, which showed minor differences. The PCR did not amplify any product when DNA from 17 different bacterial species closely related to A. pleuropneumoniae was used as template. In addition, the PCR was negative with DNA of several Actinobacillus sp. which were initially characterized as A. pleuropneumoniae using routine phenotypic and serological analyses but which were subsequently shown by 16S rRNA sequence analysis to belong to yet undefined Actinobacillus species. The sensitivity of the PCR was determined to be 10pg of A. pleuropneumoniae DNA. A set of nested primers amplified a 377bp fragment specifically with A. pleuropneumoniae DNA. DNA titration experiments using the flanking and nested primer pairs showed an improved level of sensitivity to approximately 10fg of genomic DNA. The nested PCR was used to monitor the spread of A. pleuropneumoniae in pigs experimentally infected with a virulent serotype 1 strain and housed in a controlled environment facility. A. pleuropneumoniae DNA could be detected by nested PCR in nasal swab samples of infected pigs receiving either a high dose (5x10(5)) or a low dose (1x10(4)) challenge and in unchallenged cohorts that were contact-infected by the inoculated animals. Furthermore, PCR confirmed the presence of A. pleuropneumoniae in 16/17 homogenates from necrotic lung lesions, while the bacterium was successfully recovered from 13 of these lesions by culture.

In Mycoplasma hyopneumoniae (Mhp) infection of swine, the host immune response is considered a major driver of lung pathology; however the underlying inflammatory mechanisms are not well understood. The serine protease plasmin is being increasingly recognised as a significant player in inflammatory processes. Here we compare plasmin activity in tracheobronchial lavage fluid (TBLF) from pigs experimentally challenged with Mhp that were either unvaccinated (n=10), or vaccinated with the commercial vaccine Suvaxyn® M.hyo (n=10). TBLF collected immediately prior to challenge and at 21d and 35d post-challenge was also assayed for levels of proinflammatory cytokines (TNF-α, IL-1β and IL-6), and for bacterial load (by qPCR). Clinical signs, pathology, cytokine analyses and qPCR all indicated that vaccinated pigs had significantly reduced disease relative to unvaccinated animals. Plasmin activity increased significantly in TBLF collected at 21d post-challenge compared to pre-challenge TBLF in unvaccinated (P0.05). A significant correlation was observed between bacterial load and plasmin activity in the 21d (r=0.66; P

An ELISA for the detection of serum antibodies to Mycoplasma hyopneumoniae in pigs and based on a 43 kDa purified protein derived from the cytoplasmic membrane of M. hyopneumoniae strain J is described. This ELISA (MHPP ELISA) was compared with another recently described (Auspharm ELISA, Sheldrake and Romalis 1992) that is based on column-purified sonicated proteins of strain J. Using sample to negative ELISA ratios of 3 and 4 as cutoffs for inconclusive and positive reactors respectively (compared to 2 and 3 for the Auspharm ELISA), the two tests had high specificity (MHPP 99.6%; Auspharm 100%) in 280 SPF pigs. In 176 pigs from commercial herds with endemic M. hyopneumoniae, the MHPP ELISA showed a higher sensitivity than the Auspharm ELISA in both high lung score (LS ≥ 5) (85.5% vs. 69.9%) and low lung score (0 < LS < 5) (57.9% vs. 49%) pigs when the positive cutoff for each test was selected. The sensitivity when the inconclusive cutoff was selected was similar in both tests (85%; 85.7%) when low and high lung score pigs were pooled. Although the MHPP also gave more positive reactors in 36 pigs from M. hyopneumoniae-infected herds with no lung pathology at slaughter than the Auspharm ELISA (11 vs. 4), the total number of inconclusive and positive reactors in these pigs was similar for both tests (18 vs. 14). The MHPP ELISA gave significantly higher ELISA ratios in infected pigs (up to 17.9) than the Auspharm ELISA (up to 9), and earlier seroconversion in naturally-infected (6–8 weeks vs. 9–10 weeks) and experimentally-infected pigs (2–4 weeks vs. 4–6 weeks post infection).

Swine erysipelas vaccines are routinely used to protect pigs against peracute and acute/urticarial forms of Erysipelothrix. Between 1995 and 1998, 34 swine herds across four Australian states experienced vaccine failure. Forty-four isolates of Erysipelothrix rhusiopathiae of serovars 2, 1a, 1b and 1b × 21 were recovered from 15 of these 34 vaccine breakdown herds. These isolates were characterised by restriction fragment length polymorphism (RFLP) analyses using Rsa I and Alu I on whole cell DNA and for the presence of plasmid DNA. Results were compared with those of 20 isolates from 16 herds unaffected by vaccine breakdown and 13 isolates representing 10 reference strains. The majority of breakdown herds possessed isolates of serovar 2 (9/15 herds), followed by serovar 1a (5 herds). No geographic predominance of a single serovar was evident. The identification of 10 Rsa I profiles from whole cell DNA among the 44 isolates from 15 breakdown herds indicated that a single, new clonal lineage of E. rhusiopathiae was not responsible for vaccine failure. Rsa I RFLP analyses detected a further 14 distinct profiles among 20 field strains unassociated with vaccine breakdowns, and none matched profiles of the 10 serovar reference strains for serovars 1a, 1b, 2 or 21. This technique is recommended for epidemiological studies of E. rhusiopathiae strains.