Quantitative effects of in-line operations on Campylobacter and Escherichia coli through two Australian broiler processing plants

Campylobacter is an important food borne pathogen, mainly associated with poultry. A lack of through-chain quantitative Campylobacter data has been highlighted within quantitative risk assessments. The aim of this study was to quantitatively and qualitatively measure Campylobacter and Escherichia coli concentration on chicken carcasses through poultry slaughter. Chickens (n=240) were sampled from each of four flocks along the processing chain, before scald, after scald, before chill, after chill, after packaging and from individual caeca. The overall prevalence of Campylobacter after packaging was 83% with a median concentration of 0.8logCFU/mL. The processing points of scalding and chilling had significant mean reductions of both Campylobacter (1.8 and 2.9logCFU/carcase) and E. coli (1.3 and 2.5logCFU/carcase). The concentration of E. coli and Campylobacter was significantly correlated throughout processing indicating that E. coli may be a useful indicator organism for reductions in Campylobacter concentration. The carriage of species varied between flocks, with two flocks dominated by Campylobacter coli and two flocks dominated by Campylobacter jejuni. Current processing practices can lead to significant reductions in the concentration of Campylobacter on carcasses. Further understanding of the variable effect of processing on Campylobacter and the survival of specific genotypes may enable more targeted interventions to reduce the concentration of this poultry associated pathogen

Commonality among Fluoroquinolone-Resistant Sequence Type ST131 Extraintestinal Escherichia coli Isolates from Humans and Companion Animals in Australia ᰔ †

Escherichia coli sequence type 131 (ST131), an emergent multidrug-resistant extraintestinal pathogen, has spread epidemically among humans and was recently isolated from companion animals. To assess for humancompanion animal commonality among ST131 isolates, 214 fluoroquinolone-resistant extraintestinal E. coli isolates (205 from humans, 9 from companion animals) from diagnostic laboratories in Australia, provisionally identified as ST131 by PCR, selectively underwent PCR-based O typing and bla CTX-M-15 detection. A subset then underwent multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) analysis, extended virulence genotyping, antimicrobial susceptibility testing, and fluoroquinolone resistance genotyping. All isolates were O25b positive, except for two O16 isolates and one O157 isolate, which (along with six O25b-positive isolates) were confirmed by MLST to be ST131. Only 12% of isolates (25 human, 1 canine) exhibited bla CTX-M-15 . PFGE analysis of 20 randomly selected human and all 9 companion animal isolates showed multiple instances of >94% profile similarity across host species; 12 isolates (6 human, 6 companion animal) represented pulsotype 968, the most prevalent ST131 pulsotype in North America (representing 23% of a large ST131 reference collection). Virulence gene and antimicrobial resistance profiles differed minimally, without host species specificity. The analyzed ST131 isolates also exhibited a conserved, host species-independent pattern of chromosomal fluoroquinolone resistance mutations. However, eight (89%) companion animal isolates, versus two (10%) human isolates, possessed the plasmid-borne qnrB gene (P < 0.001). This extensive across-species strain commonality, plus the similarities between Australian and non-Australian ST131 isolates, suggest that ST131 isolates are exchanged between humans and companion animals both within Australia and intercontinentally

Long-term persistence of multi-drug-resistant Salmonella enterica serovar Newport in two dairy herds

Objective - To evaluate the association between maintaining joint hospital and maternity pens;and persistence of multi-drug-resistant (MDR) Salmonella enterica serovar Newport on 2 dairy farms. Design - Observational study. Sample Population - Feces and environmental samples from 2 dairy herds. Procedure - Herds were monitored for fecal shedding of S enterica Newport after outbreaks of clinical disease. Fecal and environmental samples were collected approximately monthly from pens housing sick cows and calving cows and from pens containing lactating cows. Cattle shedding the organism were tested serially on subsequent visits to determine carrier status. One farm was resampled after initiation of interventional procedures, including separation of hospital and maternity pens. Isolates were characterized via serotyping, determination of antimicrobial resistance phenotype, detection of the CMY-2 gene, and DNA fingerprinting. Results - The prevalence (32.4% and 33.3% on farms A and B, respectively) of isolating Salmonella from samples from joint hospital-maternity pens was significantly higher than the prevalence in samples from pens housing preparturient cows (0.8%, both farms) and postparturient cows on Farm B (8.8%). Multi-drug-resistant Salmonella Newport was isolated in high numbers from bedding material, feed refusals, lagoon slurry, and milk filters. One cow excreted the organism for 190 days. Interventional procedures yielded significant reductions in the prevalences of isolating the organism from fecal and environmental samples. Most isolates were of the C2 serogroup and were resistant to third-generation cephalosporins. Conclusions and Clinical Relevance - Management practices may be effective at reducing the persistence of MDR Salmonella spp in dairy herds, thus mitigating animal and public health risk

The Epidemiology of Shiga Toxin-producing Escherichia coli in Australian Dairy Cattle

Shiga toxin-producing E. coli (STEC) have important public health and food safety implications. Cattle are the primary reservoir for STEC, which are transmitted to humans via contact with cattle or related food products. Dairy farms in particular have been incriminated as an important source of STEC. The broad aim of this study was to examine in depth the epidemiology of STEC on the dairy farm. The presence of STEC on three Australian dairy farms was surveyed. This aimed to provide data on the prevalence and nature of STEC on Australian dairy farms, as well as to examine in more detail the pre-harvest/slaughter ecology of STEC. STEC, E. coli O157:H7 and E. coli O26:H11 prevalences were similar to those from dairy farms in other countries. Replacement heifers were the most important source of STEC on the farms. Calves excreted STEC from an early age, with faecal prevalence peaking at weaning. Higher STEC prevalence was also associated with group housing of calves during weaning. Calf isolates were potential human pathogens based on serotype and virulence markers. Clonal relationships between isolates were analysed. Calf isolates were diverse and had a high clonal turnover. STEC isolated from within the same farm had a higher genetic similarity than those from different farms. Vertical and horizontal transmission were both identified among cattle. The farm environment was also identified as an important source of STEC. Reasons for increased levels of STEC excretion by calves were investigated. Two broad hypotheses for higher faecal shedding were proposed and examined individually. The first was that an animal is more likely to excrete STEC when its exposure to STEC is greater, thus promoting inoculation of the gastrointestinal tract. Calves were experimentally inoculated with a traceable STEC strain to examine the infection dynamics of STEC within cattle groups, and explore the effect of calf management procedures. Calves which were housed in groups and co-jointly fed and managed had a higher prevalence of the inoculation strain than animals housed individually. The test strain was readily isolated from the hides and saliva of inoculated calves, as well as their immediate environments. Calves become infected with STEC via the faecal-oral route, iv either by direct contact with other calves, or indirectly through contact with faecally contaminated materials. The second hypothesis was that individual animals are variably susceptible to intestinal colonisation by STEC, which leads to differing magnitudes and durations of STEC carriage. Factors influencing colonisation susceptibility to STEC and the mechanisms behind these factors were also examined. In order to compare enteric colonisation under a range of different conditions, a suitable experimental system was developed. In vitro organ culture of explanted ruminant colonic tissues provided a laboratory model that was representative of in vivo bacterial-mucosal attachment. The degree of STEC colonisation was enumerated using an immunofluorescent filtration technique. The quantitative colonisation assay was applied to determine the effects of host-dependant variables on STEC colonisation. Colonic tissues from weaning calves and adult cattle did not differ significantly in their susceptibility to colonisation; nor did tissues from cattle fed either high forage or high grain diets. Colonic explants from sheep, however, demonstrated significantly higher numbers of adherent STEC than bovine explants. It was therefore concluded that while species-specific differences in host tissues may mediate STEC carriage differences, this did not explain in vivo variability in age and diet related excretion. Factors that indirectly affect the susceptibility of host tissues to colonisation were examined. E. coli O157:H7 cultured in media designed to represent the enteric contents of a well-fed ruminant colonised the colonic mucosa in reduced numbers, indicating that age and diet may be correlated with differences in STEC carriage and excretion because of differing physiological augmentation of the intra-enteric environment. In conclusion, while group dynamics and management practices may increase STEC shedding prevalences for cattle via increased STEC exposure, factors that modulate an individual ruminant’s gastrointestinal carriage of STEC have a significant role in mediating STEC excretion. Either directly or indirectly, species, age and diet can affect the numbers of STEC that colonise the bowel wall, thereby influencing the magnitude and duration of STEC excretion. Both of these features of ruminant STEC ecology should be addressed in order to reduce the pre-slaughter/harvest presence of STEC

Characterisation and clonal relationships of Shiga-toxigenic Escherichia coli (STEC) isolated from Australian dairy cattle

A total of 136 Shiga toxin-producing Escherichia coli (STEC) isolated during a longitudinal survey of three Australian dairy farms were examined to determine their virulence factors, serotype and genomic relationships. This study aimed to assess the potential of these STEC to cause disease in humans and to analyse the on-farm ecology of STEC. Virulence factors (sis, eae, ehxA) were used as determinants of potential to be enterohaemorrhagic E. coli (EHEC) and were examined using polymerase chain reaction (PCR). Among the cattle groups tested, calves, both before and during weaning, shed the most putative EHEC and were the main source of serotypes commonly associated with human disease. E, coli O157:H7 and E, coli O26:H11 represented 9.4 and 7.8% of cattle STEC isolates respectively, with other putative EHEC serotypes reported for the first time from cattle. Based on serotype and virulence factors, 20% of STEC were putative EHEC. Pulsed-field gel electrophoresis (PFGE) was used to compare the genomic profiles of STEC from dairy farms. Isolates common to cattle and the farm environment were identified. Multiple strains of STEC with high clonal turnover were detected in the faeces of cattle, and isolates appeared to be specific to individual farms. To fully assess the pre-slaughter EHEC risk factors on-farm, examination of STEC virulence is as important as determination of STEC prevalence. (C) 2001 Elsevier Science B.V. All rights reserved

Intestinal Shiga Toxin-Producing Escherichia coli Bacteria Mitigate Bovine Leukemia Virus Infection in Experimentally Infected Sheep

Ruminants often carry gastrointestinal Shiga toxin (Stx)-producing Escherichia coli (STEC). Stxs belong to a large family of ribosome-inactivating proteins (RIPs), found in many plants and some bacteria. Plant RIPs, secreted into extracellular spaces, limit the spread of viruses through plant tissues by penetrating and killing virally infected cells. Previously, we showed Stx activity against bovine leukemia virus (BLV)-infected cells in vitro and hypothesized that STEC bacteria have antiviral activity in ruminant hosts. Here, we investigated the impact of STEC on the initial phases of BLV infection in sheep. Sheep were treated with biweekly oral doses of E. coli O157:H7 (an STEC) or an isogenic stx mutant strain. A different group of sheep were similarly treated with five naturally occurring ovine STEC isolates or stx -negative E. coli . Intestinal STEC bacteria were enumerated and identified by standard fecal culture and DNA hybridization. Oral STEC treatment did not always result in carriage of STEC, although many animals consistently presented with >10 4 CFU/g feces. BLV viremia was assessed by spontaneous lymphocyte proliferation (SLP) in cultures of blood mononuclear cells and by syncytium formation in cocultures of the same with F-81 indicator cells. SLP was lower ( P < 0.05) and syncytia were fewer ( P  < 0.05) in STEC-treated sheep than in untreated sheep. Both lower SLP and fewer syncytia positively correlated with fecal STEC numbers. Average weight gain post-BLV challenge was higher in STEC-treated sheep than in untreated sheep ( P < 0.05). These results support the hypothesis that in ruminants, intestinal STEC bacteria have antiviral activity and mitigate BLV-induced disease

Low numbers of intestinal Shiga toxin-producing E. coli correlate with a poor prognosis in sheep infected with bovine leukemia virus

Healthy ruminants carry intestinal Shiga toxin (Stx)-producing Escherichia coli (STEC). Stx has antiviral activities in vitro and STEC numbers correlate with reduced early viremia in sheep experimentally infected with bovine leukemia virus (BLV). This study assessed the impact of intestinal STEC on BLV-induced disease for one year post-BLV-challenge. High STEC scores (CFU/g feces × frequency of STEC-positive samples) correlated with good health, whereas poor weight gain, distress, and tumor development occurred only among animals with low STEC scores. STEC carriage was associated with increased percentages of B cells in peripheral blood

Carriage of Shiga-toxigenic Escherichia coli by native marsupials in Australia

Shiga-toxigenic strains of Escherichia coli (STEC) are zoonotic pathogens with human health, meat processing and trade impacts. Cattle are the principal reservoirs of STEC, although other animals can be carriers. The STEC status of Australian native marsupials has not been formatively described to date. The aim of the current study was to investigate carriage of STEC by native Australian marsupials in Southeast Queensland. Faeces from a variety of marsupial species, stratified by gastrointestinal morphology and dietary type, were screened for stx, stx and other STEC virulence genes by PCR. Positive samples were cultured to isolate STEC for characterisation. A number of macropods from both captive and wild habitats had evidence of STEC in their faeces. Rates of stx carriage by macropods (8.6%) were comparable, though generally low, compared to cattle. Eastern grey kangaroos had the highest rate of stx presence in faeces (10.3%). Hindgut-fermenting and monogastric marsupials had no evidence of STEC shedding. Based on virulence marker possession and serotype, the human pathogenic potential of isolates was low. This is the first report of Australian marsupials carrying STEC. Australian native macropods may act as reservoirs for STEC strains, but the potential significance to public health and/or livestock epidemiology remains questionable