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Summary A total of 227 field samples from naturally exposed foals aged between 3 weeks and 6 months were used in an evaluation of a peptide-based enzyme-linked immunosorbent assay (ELISA) for diagnosis of Rhodococcus equi infection. A biotinylated peptide derived from the virulence-associated protein A (VapA) of R. equi, a horse pathogen, was synthesized and designated as PN11-14. The peptide corresponds to the N-terminal B-cell epitope TSLNLQKDEPNGRASDTAGQ of the VapA protein. Based upon a serum immunoglobulin (Ig)G titre of 512 as a positive cut-off value for the R. equi infection, the ELISA provided the overall sensitivity of 47.62%, specificity of 69.67% and an accuracy of 59.47% with a positive predictive value of 57.47% for true R. equi pneumonia. The assay was improved by detecting VapA-specific IgGb antibodies against N-terminal B-cell epitope of the VapA protein rather than IgG antibodies. The VapA-IgGb ELISA showed the overall sensitivity of 70.47%, specificity of 72.13% and accuracy of 71.36% with a positive predictive value of 68.52%. Diagnosis of R. equi disease in 6-week-old foals showed that the VapA-IgGb ELISA provided an increasing trend (P = 0.0572) in sensitivity of 82.4% in comparison with the VapA-IgG ELISA which showed the sensitivity of 58.8%. However, differences in specificity of both tests were statistically insignificant (P = 0.357) as analysed by the McNemar test. These results indicated that detection of VapA-specific IgGb antibodies may be a better predictor of R. equi disease in foals.

1. SUMMARY Dogs and cats have become an integral part of modern society in the developed world and as such attention is given to their care and welfare. At some stage of their lives many cats and dogs suffer from skin and other superficial staphylococcal infections such as pyoderma and otitis externa and are therefore treated with antibiotics. This practice has led to the emergence of resistant staphylococci in these animals. Antimicrobial susceptibility studies in companion animals have revealed that staphylococci isolated from cats and dogs exhibit similar resistance patterns to human staphylococci and that resistance development is influenced by the frequency of use of certain antibiotics. This review examines the antibiotic resistance patterns of the most frequently isolated staphylococci from cats and dogs and the mechanisms underlying resistance. The possible transfer of methicillin-resistant staphylococci from cats and dogs to humans has been identified as a potential public health issue. Typing tools used for epidemiological differentiation of pathogenic and nonpathogenic staphylococci and for species identification studies may help to elucidate the importance or otherwise of this problem.

The aim of this study was to evaluate the usefulness of the previously identified B-cell epitope TSLNLQKDEPNGRASDTAGQ of the VapA protein of Rhodococcus equi and its association with R. equi pneumonia. A modified peptide designated PN11-14 corresponding to the epitope was recognized by all sera from experimentally infected foals with virulent R. equi ATCC103+ containing the virulence plasmid but not by its plasmid-cured derivative ATCC103- strain. Marked levels of VapA-specific immunoglobulin (Ig)G were detected in all sera from the ATCC103+ infected foals at 2 weeks after the infection. One control animal had high titres as determined by the peptide enzyme-linked immunosorbent assay (ELISA), indicating the ELISA may not absolutely differentiate between foals with R. equi pneumonia and healthy exposed foals in farms where the prevalence of disease is high. However, numbers of animals used were small. Further evaluation of the peptide ELISA with field samples is necessary to determine whether the assay is diagnostically useful. This study showed that levels of passive transfer of maternal IgG antibodies to the epitope in newborn foals could be measured. Interestingly, the maternally derived antibodies were found to significantly (P

A total of 129 Pseudomonas spp. and 90 Aeromonas spp. were isolated from nine rainbow trout (Oncorhynchus mykiss) farms in Australia. All the isolates were tested for sensitivity to 15 antibiotics and the multiresistant strains were tested for sensitivity to seven heavy metals. Minimal inhibitory concentrations (MICs) were determined by the agar dilution method. In Pseudomonas spp., resistance to amoxicillin, cefalothin, ceftiofur, ticarcillin, chloramphenicol, florfenicol, streptomycin, nitrofurantoin and trimethoprim was widespread, whereas resistance to cefotaxime and oxolinic acid was less common and only single isolates were resistant to tetracycline and sulfamethoxazole; all isolates were sensitive to ciprofloxacin and gentamicin. In Aeromonas spp., resistance to amoxicillin and cefalothin was widespread, resistance to ticarcillin, tetracycline and streptomycin was common, whilst resistance to ceftiofur, florfenicol and sulfamethoxazole was less common. Single isolates were resistant to chloramphenicol, nitrofurantoin and trimethoprim, and all isolates were sensitive to cefotaxime, oxolinic acid, ciprofloxacin and gentamicin. Multiple resistance was also observed. Most isolates were tolerant to different concentrations of various heavy metals, as evidenced by their MICs ranging from 6.25 microg/mL to >3200 microg/mL. These results confirm our previous findings that bacteria resistant to antibiotics are present in fish and sediments from aquaculture in Australia. In addition, we have found resistance to heavy metals in fish and sediment isolates. Much of the antibiotic resistance detected is likely to be intrinsic, although resistance to oxytetracycline, streptomycin and sulfonamides suggests either contamination from run-off from farms or perhaps off-label use of antibiotics in a situation where no antibiotics are licensed for use in aquaculture.

Staphylococci are part of the normal microflora of humans and animals and some are potential pathogens that have become resistant to almost all known antibiotics. Despite the widespread reports of penicillin resistance in cat and dog staphylococci, the mechanism underlying penicillin resistance has not been examined. This study was aimed at investigating the molecular basis of resistance to penicillin in cat and dog staphylococcal isolates that showed phenotypic resistance to beta-lactam antibiotics. An 861 bp fragment of the structural blaZ gene which codes for beta-lactamase production in staphylococci was amplified by polymerase chain reaction (PCR) and the products were sequenced. Sequenced fragments were analysed by protein signature typing and sequences were compared to published blaZ sequences of human and bovine staphylococcal strains held in a public database. Four known protein signature types (1, 3, 5 and 6) and one new type (12) were identified in this study. When sequences were compared with published blaZ sequences, gene phylogenetic analysis revealed three major groups. The four variants of beta-lactamases types (A, B, C and D) belonged to each major group except for types A and D which were both in group II. These findings confirm that the blaZ gene is responsible for beta-lactamase production leading to subsequent resistance to beta-lactam antibiotics in feline and canine staphylococci and that the gene shows similar diversity and relatedness as found with blaZ sequences obtained from human and bovine staphylococci.

Aims: To carry out a preliminary assessment of the occurrence of resistance to antimicrobials in bacteria that has been isolated from a variety of aquaculture species and environments in Australia. Method and Results: A total of 100 Gram-negative (Vibrio spp. and Aeromonas spp. predominantly) and four Gram-positive bacteria isolated from farmed fish, crustaceans and water from crab larval rearing tanks were obtained from diagnostic laboratories from different parts of Australia. All the isolates were tested for sensitivity to 19 antibiotics and Minimal Inhibitory Concentrations were determined by the agar dilution method. Plasmid DNA was isolated by the alkali lysis method. Resistance to ampicillin, amoxycillin, cephalexin and erythromycin was widespread; resistance to oxytetracycline, tetracycline, nalidixic acid and sulfonamides was common but resistance to chloramphenicol, florfenicol, ceftiofur, cephalothin, cefoperazone, oxolinic acid, gentamicin, kanamycin and trimethoprim was less common. All strains were susceptible to ciprofloxacin. Multiple resistance was also observed and 74·4% of resistant isolates had between one and ten plasmids with sizes ranging 2–51 kbp. Conclusions: No antibiotics are registered for use in aquaculture in Australia but these results suggest that there has been significant off-label use. Significance and impact of study: Transfer of antibiotic resistant bacteria to humans via the food chain is a significant health concern. In comparison with studies on terrestrial food producing animals, there are fewer studies on antibiotic resistance in bacteria from aquaculture enterprises and this study provides further support to the view that there is the risk of transfer of resistant bacteria to humans from consumption of aquaculture products. From the Australian perspective, although there are no products registered for use in aquaculture, antimicrobial resistance is present in isolates from aquaculture and aquaculture environments.

Rhodococcus equi is a significant pathogen in foals predominantly causing a pyogranulomatous bronchopneumonia. Many vaccine candidates have been tested for the prevention of R. equi disease in foals. However, none of these have been developed for widespread commercial use. Previous studies have shown that a Th1 immune response is imperative for the protection of foals against R. equi disease. In this study a DNA and a protein vaccine based upon the well-characterised R. equi virulence-associated protein VapA were developed. The vaccines were tested in the BALB/c murine model and the results showed that both vaccine candidates elicited a Th1-type response in the host. Upon coadministration of an IL-12 expression plasmid with the DNA vaccine, an increase in the Th1 response was observed. However, when mice were challenged with 1.5 x 10(7) virulent R. equi ATCC 33701 none of the vaccinated mice showed protection apart from the mice immunised with live R. equi. These results indicate that despite their immunogenicity the VapA-based DNA and recombinant protein vaccines developed in this study were unable to prevent bacterial replication following a high-dose systemic challenge with virulent R. equi in the BALB/c model.