Phenotypic and genotypic characterisation of Pasteurella multocida isolated from pigs at slaughter in New Zealand

AIMS: To examine pigs at slaughter in New Zealand for the presence of Pasteurella multocida, and to determine for isolates, their biochemical profi les, somatic and capsular types, and the presence or absence of the HSB and toxA genes, associated with haemorrhagic septicaemia (HS) and progressive atrophic rhinitis (PAR), respectively. METHODS: Swabs from 173 lungs, 158 palatine tonsils and 82 nasal passages of pigs at two abattoirs in New Zealand were cultured for P. multocida using conventional techniques, and isolated colonies were subjected to biochemical tests for identi- fi cation of biovars. Somatic serotyping was conducted using an agar gel immunodiffusion (AGID) test. Polymerase chain reaction (PCR) assays were used to confi rm phenotypic identifi cation of colonies using species-specifi c primers, capsule type using serogroup-specifi c primers and multiplex PCR, and to test for the presence of HSB and toxA genes. RESULTS: Pasteurella multocida was isolated from 11/173 (6.4%) lung, 32/158 (20.2%) palatine tonsil and 5/82 (6.1 %) nasal swab samples, a total of 48 isolates from 413 samples (11.6%). Isolation rates per farm ranged from 1–53% of tissue samples collected from pigs 5–6 months of age. On phenotypic characterisation, isolates were allocated to seven main biovars, viz 1, 2, 3, 5, 9, 12, and a dulcitol-negative variant of Biovar 8, the majority (30/48) being Biovar 3. Of the 42 isolates for which somatic serotyping was conducted, 10% were Serovar 1, 79% were Serovar 3, 2% were Serovar 6,1, 2% were Serovar 12, and 7% could not be typed. All 48 isolates were confi rmed as P. multocida using a species-specifi c PCR. In the capsular multiplex PCR, 92% of isolates were Capsular (Cap) type A, 2% were Cap D, and 6% could not be typed. None of the samples were positive for the HSB or toxA genes. CONCLUSION: Serovars or capsular types of P. multocida associated with HS or PAR in pigs were not detected. Establishment of species-specifi c, capsular and toxin PCR assays allowed the rapid screening of isolates of P. multocida, while serotyping provided an additional tool for epidemiological and tracing purposes.

Antibiotic resistance in Campylobacter jejuni and Campylobacter coli isolated from poultry in the South-East Queensland region

Objectives: The aim of this study was to determine the antimicrobial resistance patterns of 125 Campylobacter jejuni and 27 Campylobacter coli isolates from 39 Queensland broiler farms. Methods: Two methods, a disc diffusion assay and an agar-based MIC assay, were used. The disc diffusion was performed and interpreted as previously described (Huysmans MB, Turnidge JD. Disc susceptibility testing for thermophilic campylobacters. Pathology 1997; 29: 209–16), whereas the MIC assay was performed according to CLSI (formerly NCCLS) methods and interpreted using DANMAP criteria. Results: In both assays, no C. jejuni or C. coli isolates were resistant to ciprofloxacin or chloramphenicol, no C. coli were resistant to nalidixic acid, and no C. jejuni were resistant to erythromycin. In the MIC assay, no C. jejuni isolate was resistant to nalidixic acid, whereas three isolates (2.4%) were resistant in the disc assay. The highest levels of resistance of the C. jejuni isolates were recorded for tetracycline (19.2% by MIC and 18.4% by disc) and ampicillin (19.2% by MIC and 17.6% by disc). The C. coli isolates gave very similar results (tetracycline resistance 14.8% by both MIC and disc; ampicillin resistance 7.4% by MIC and 14.8% by disc). Conclusions: This work has shown that the majority of C. jejuni and C. coli isolates were susceptible to the six antibiotics tested by both disc diffusion and MIC methods. Disc diffusion represents a suitable alternative methodology to agar-based MIC methods for poultry Campylobacter isolates.

Leptospira weilii serovar Topaz, a new member of the Tarassovi serogroup isolated from a bovine source in Queensland, Australia

This paper reports on a Leptospira isolate of bovine origin and its identification as belonging to a previously unknown serovar, for which the name Topaz is proposed. The isolate (94-79970/3) was cultured from bovine urine from a north Queensland dairy farm in Australia. Strain 94-79970/3 grew at 30 °C in Ellinghausen McCullough Johnson Harris (EMJH) medium but failed to grow at 13 °C in EMJH medium or in the presence of 8-azaguanine. Serologically, strain 94-79970/3 produced titres against the Leptospira borgpetersenii serovar Tarassovi, the reference strain for the Tarassovi serogroup; however, no significant titres to any other serovars within the serogroup were obtained. Using 16S rRNA and DNA gyrase subunit B gene analysis, strain 94-79970/3 was identified as a member of the species Leptospira weilii. We propose that the serovar be named Topaz, after the location where the original isolate was obtained. The reference strain for this serovar is 94-79970/ 3 (=KIT 94-79970/35LT722).

A novel species of mastrevirus (family Geminiviridae) isolated from Digitaria didactyla grass from Australia.

Mastreviruses (family Geminiviridae) that infect monocotyledonous plants occur throughout the temperate and tropical regions of Asia, Africa, Europe and Australia. Despite the identification of a very diverse array of mastrevirus species whose members infect African monocots, few such species have been discovered in other parts of the world. For example, the sequence of only a single monocot-infecting mastrevirus, Chloris striate mosaic virus (CSMV), has been reported so far from Australia, even though earlier biological and serological studies suggested that other distinct mastreviruses were present. Here, we have obtained the complete nucleotide sequence of a virus from the grass Digitaria didactyla originating from Australia. Analysis of the sequence shows the virus to be a typical mastrevirus, with four open reading frames, two in each orientation, separated by two non-coding intergenic regions. Although it showed the highest levels of sequence identity to CSMV (68.7%), their sequences are sufficiently diverse for the virus to be considered a member of a new species in the genus Mastrevirus, based on the present species demarcation criteria. We propose that the name first used during the 1980s be used for this species, Digitaria didactyla striate mosaic virus (DDSMV).

Evidence of bat origin for Menangle virus, a zoonotic paramyxovirus first isolated from diseased pigs

Menangle virus (MenPV) is a zoonotic paramyxovirus capable of causing disease in pigs and humans. It was first isolated in 1997 from stillborn piglets at a commercial piggery in New South Wales, Australia, where an outbreak of reproductive disease occurred. Neutralizing antibodies to MenPV were detected in various pteropid bat species in Australia and fruit bats were suspected to be the source of the virus responsible for the outbreak in pigs. However, previous attempts to isolate MenPV from various fruit bat species proved fruitless. Here, we report the isolation of MenPV from urine samples of the black flying fox, Pteropus alecto, using a combination of improved procedures and newly established bat cell lines. The nucleotide sequence of the bat isolate is 94% identical to the pig isolate. This finding provides strong evidence supporting the hypothesis that the MenPV outbreak in pigs originated from viruses in bats roosting near the piggery. © 2012 Printed in Great Britain.

First Report of a Toxic Nodularia spumigena (Nostocales/Cyanobacteria) Bloom in Sub-Tropical Australia. II. Bioaccumulation of Nodularin in Isolated Populations of Mullet (Mugilidae)

Fish collected after a mass mortality at an artificial lake in south-east Queensland, Australia, were examined for the presence of nodularin as the lake had earlier been affected by a Nodularia bloom. Methanol extracts of muscle, liver, peritoneal and stomach contents were analysed by HPLC and tandem mass spectrometry; histological examination was conducted on livers from captured mullet. Livers of sea mullet (Mugil cephalus) involved in the fish kill contained high concentrations of nodularin (median 43.6 mg/kg, range 40.8-47.8 mg/kg dry weight; n = 3) and the toxin was also present in muscle tissue (median 44.0 mu g/kg, range 32.3-56.8 mu g/kg dry weight). Livers of fish occupying higher trophic levels accumulated much lower concentrations. Mullet captured from the lake 10 months later were also found to have high hepatic nodularin levels. DNA sequencing of mullet specimens revealed two species inhabiting the study lake: M. cephalus and an unidentified mugilid. The two mullet species appear to differ in their exposure and/or uptake of nodularin, with M. cephalus demonstrating higher tissue concentrations. The feeding ecology of mullet would appear to explain the unusual capacity of these fish to concentrate nodularin in their livers; these findings may have public health implications for mullet fisheries and aquaculture production where toxic cyanobacteria blooms affect source waters. This report incorporates a systematic review of the literature on nodularin measured in edible fish, shellfish and crustaceans.

The persistence of biofilm-associated antibiotic resistance of Staphylococcus aureus isolated from clinical bovine mastitis cases in Australia

The aim of this investigation was to determine the persistence of biofilm-associated antibiotic resistance developed by methicillin-sensitive Staphylococcus aureus (MSSA), of different capsular types, during biofilm formation. Because of superiority of the tissue culture plate (TCP) over the Congo Red Agar (CRA) method for measuring biofilm formation, it was used to determine the persistence of the antibiotic resistance developed by the isolates in biofilms. The antibiotic resistance was found to persist for 3-4 wk post-propagation as planktonic subcultures. Interestingly, some strains even developed resistance to vancomycin and/or teicoplanin. However, no association of either biofilm formation or persistent antibiotic resistance with the major capsular phenotype was observed. These observations highlight the potential significance of (a) determining the antibiograms of S. aureus subcultured from biofilms developed in vitro using the TCP method as well as from planktonic cultures for formulation of an optimal therapeutic strategy, and (b) continuing to identify predominant non-capsular antigens contributing to biofilm formation, regardless of the capsular phenotype for the development of an effective potentially broad-spectrum vaccine for prevention of bovine mastitis caused by S. aureus.

Fluoroquinolone-resistant extraintestinal pathogenic Escherichia coli, including O25b-ST131, isolated from faeces of hospitalized dogs in an Australian veterinary referral centre

To determine rates of carriage of fluoroquinolone-resistant Escherichia coli and extraintestinal pathogenic E. coli (ExPEC) among dogs in a specialist referral hospital and to examine the population structure of the isolates. Fluoroquinolone-resistant faecal E. coli isolates (n232, from 23 of 123 dogs) recovered from hospitalized dogs in a veterinary referral centre in Sydney, Australia, over 140 days in 2009 were characterized by phylogenetic grouping, virulence genotyping and random amplified polymorphic DNA (RAPD) analysis. The RAPD dendrogram for representative isolates showed one group B2-associated cluster and three group D-associated clusters; each contained isolates with closely related ExPEC-associated virulence profiles. All group B2 faecal isolates represented the O25b-ST131 clonal group and were closely related to recent canine extraintestinal ST131 clinical isolates from the east coast of Australia by RAPD analysis. Hospitalized dogs may carry fluoroquinolone-resistant ExPEC in their faeces, including those representing O25b-ST131.

Antimicrobial susceptibility of Histophilus somni isolated from clinically affected cattle in Australia

This study investigated antimicrobial resistance traits, clonal relationships and epidemiology of Histophilus somni isolated from clinically affected cattle in Queensland and New South Wales, Australia. Isolates (n = 53) were subjected to antimicrobial susceptibility testing against six antimicrobial agents (ceftiofur, enrofloxacin, florfenicol, tetracycline, tilmicosin and tulathromycin) using disc diffusion and minimum inhibitory concentration (MIC) assays. Clonal relationships were assessed using repetitive sequence PCR and descriptive epidemiological analysis was performed. The H. somni isolates appeared to be geographically clonal, with 27/53 (47%) isolates grouping in one cluster from one Australian state. On the basis of disc diffusion, 34/53 (64%) isolates were susceptible to all antimicrobial agents tested; there was intermediate susceptibility to tulathromycin in 12 isolates, tilmicosin in seven isolates and resistance to tilmicosin in one isolate. Using MIC, all but one isolate was susceptible to all antimicrobial agents tested; the non-susceptible isolate was resistant to tetracycline, but this MIC result could not be compared to disc diffusion, since there are no interpretative guidelines for disc diffusion for H. somni against tetracycline. In this study, there was little evidence of antimicrobial resistance in H. somni isolates from Australian cattle. Disc diffusion susceptibility testing results were comparable to MIC results for most antimicrobial agents tested; however, results for isolates with intermediate susceptibility or resistance to tilmicosin and tulathromycin on disc diffusion should be interpreted with caution in the absence of MIC results.

Human-associated fluoroquinolone-resistant Escherichia coli clonal lineages, including ST354, isolated from canine feces and extraintestinal infections in Australia

Phylogenetic group D extraintestinal pathogenic Escherichia coli (ExPEC), including O15:K52:H1 and clonal group A, have spread globally and become fluoroquinolone-resistant. Here we investigated the role of canine feces as a reservoir of these (and other) human-associated ExPEC and their potential as canine pathogens. We characterized and compared fluoroquinolone-resistant E. coli isolates originally identified as phylogenetic group D from either the feces of hospitalized dogs (n = 67; 14 dogs) or extraintestinal infections (n = 53; 33 dogs). Isolates underwent phylogenetic grouping, random amplified polymorphic DNA (RAPD) analysis, virulence genotyping, resistance genotyping, human-associated ExPEC O-typing, and multi-locus sequence typing. Five of seven human-associated sequence types (STs) exhibited ExPEC-associated O-types, and appeared in separate RAPD clusters. The largest subgroup (16 fecal, 26 clinical isolates) were ST354 (phylogroup F) isolates. ST420 (phylogroup B2); O1-ST38, O15:K52:H1-ST393, and O15:K1-ST130 (phylogroup D); and O7-ST457, and O1-ST648 (phylogroup F) were also identified. Three ST-specific RAPD sub-clusters (ST354, ST393, and ST457) contained closely related isolates from both fecal or clinical sources. Genes encoding CTX-M and AmpC β-lactamases were identified in isolates from five STs. Major human-associated fluoroquinolone-resistant ± extended-spectrum cephalosporin-resistant ExPEC of public health importance may be carried in dog feces and cause extraintestinal infections in some dogs.