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Campylobacter spp. is the leading cause of bacterial gastroenteritis worldwide and poultry are the primary reservoir. The aim of this study was to investigate the survival and/or growth of C. jejuni NCTC 11168 in broiler digestate prepared from commercial starter, grower and finisher feed formulations. Bolton broth and digestates were prepared, inoculated with C. jejuni NCTC 11168 (approximately 3 log10 CFU per ml) and incubated under microaerobic conditions at 42°C for 24 hours. Samples were taken at t = 0 (immediately after inoculation) and every 3 hours thereafter, serially diluted and plated onto mCCDA. C. jejuni grew as expected in Bolton broth (control) reaching the early stationary phase after approximately 15 hours. In contrast, although bacterial concentrations were maintained for at least 9 hours, none of the feed digestates supported the growth of C. jejuni, which were not detected after 15 hours. It is suggested that the nutrients available in the feed digestates are not enough to support C. jejuni growth and that additional factors may be at play in the avian gastrointestinal tract (GIT).

1. Campylobacteriosis is the leading cause of human bacterial gastroenteritis. Broilers are considered the most important source of human Campylobacter infection. In the 2008 European baseline surv...

The European Commission requested EFSA to assess if different thermal processes achieve a 5 log10 reduction in Enterococcus faecalis or Salmonella Senftenberg (775W) and (if relevant) a 3 log10 reduction in thermoresistant viruses (e.g. Parvovirus) as well as if different chemical processes achieve a 3 log10 reduction of eggs of Ascaris sp., in eight groups of Category 2 and 3 derived products and animal by-products (ABP). These included (1) ash derived from incineration, co-incineration and combustion; (2) glycerine derived from the production of biodiesel and renewable fuels; (3) other materials derived from the production of biodiesel and renewable fuels; (4) hides and skins; (5) wool and hair; (6) feathers and down; (7) pig bristles; and (8) horns, horn products, hooves and hoof products. Data on the presence of viral hazards and on thermal and chemical inactivation of the targeted indicator microorganisms and biological hazards under relevant processing conditions were extracted via extensive literature searches. The evidence was assessed via expert knowledge elicitation. The certainty that the required log10 reductions in the most resistant indicator microorganisms or biological hazards will be achieved for each of the eight groups of materials mentioned above by the thermal and/or chemical processes was (1) 99-100% for the two processes assessed; (2) 98-100% in Category 2 ABP, at least 90-99% in Category 3 ABP; (3) 90-99% in Category 2 ABP; at least 66-90% in Category 3 ABP; (4) 10-66% and 33-66%; (5) 1-33% and 10-50%; (6) 66-90%; (7) 33-66% and 50-95%; (8) 66-95%, respectively. Data generation on the occurrence and reduction of biological hazards by thermal and/or chemical methods in these materials and on the characterisation of the usage pathways of ABP as organic fertilisers/soil improvers is recommended. ispartof: EFSA JOURNAL vol:19 issue:12 ispartof: location:United States status: published

Background:Campylobacter is commonly transmitted to humans from chickens. Campylobacter jejuni is the species most frequently associated with human illness, and the most prevalent species recovered from poultry.Objective: The objective of this study was to analyse a sub-population of C. jejuni from two broiler flocks on the farm and at slaughter using whole-genome sequencing to gain insights into the changes in the Campylobacter population during broiler production, including changes in virulence and antimicrobial resistance profiles.Methods: In this study, ten composite faecal samples (n=10), obtained by pooling ten fresh faecal samples (n=10), were collected in the broiler house on two farms on days 14, 21, 28, and 34 (n=80) and ten composite (n=10) caecal samples were collected at the time of slaughter for each flock (n=20). These were tested for C. jejuni using the ISO 10272-2:2016 method. Seven isolates were randomly selected from each of the nine Campylobacter-positive sampling points (n=63) and were subjected to antimicrobial susceptibility tests. Their genomes were sequenced and the data obtained was used to characterise the population structure, virulence, antimicrobial resistance determinants and inter-strain variation.Results: The Farm 1 isolates had three MLST types (ST257-257, ST814-661 and ST48-48) while those on Farm 2 were ST6209-464 and ST9401. Interestingly, only the MLST types positive for most of the virulence genes tested in this study persisted throughout the production cycle, and the detection of antimicrobial resistance determinants (gyrA T86I and tetO) increased after thinning and at slaughter, with the detection of new strains.Conclusion: The persistence of the most virulent strains detected in this study throughout the production cycle has important implications for the risk to consumers and requires further investigation. The detection of new strains within the population corresponding with the time of thinning and transportation reflects previous reports and provides further evidence that these activities pose a risk of introducing new Campylobacter strains to broiler batches.

AimsCampylobacter fetus subsp fetus (CFF) can cause intestinal illness, particularly in immunocompromised humans, with the potential to cause severe systemic infections. CFF is a zoonotic pathogen with a broad host range among farm animals and humans, inducing abortion in sheep and cows. The current paper describes a strain of CFF isolated from a patient with prosthetic valve endocarditis in Mercy University Hospital, Cork, Ireland, during 2017. Only five cases of C. fetus as a cause of prosthetic valve endocarditis have been reported in the literature, with no reports of biofilm formation within the species.MethodsThe aetiological strain was speciated and subspeciated by the VITEK 2 NH card and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry. CFF biofilm formation was analysed using a crystal violet staining method. C. jejuni National Collection of Type Cultures (NCTC) 11168 was used as a positive control organism. Strains were incubated statically in Mueller-Hinton broth and Mueller-Hinton broth supplemented with 0.025% sodium deoxycholate for 3 and 7 days at 37°C, microaerobically.ResultsThe CFF strain formed stronger attached biofilms on polystyrene plates on day 3 (72 hours) than the C. jejuni NCTC 11168 control strain, but were weaker than the control strain on day 7 in Mueller-Hinton broth. Monoculture of this C. fetus isolate was found to exist in three defined forms of biofilms (attached, air–liquid interface and floccules).ConclusionsThis clinically significant C. fetus isolate showed considerable biofilm-forming capability, which we suggest conferred a survivalist advantage, contributing to the genesis of infective prosthetic valve endocarditis.

This study investigates the effect of stocking density and population dynamics on broiler growth rates and productivity, while further validating the ability of the biosecurity cubes (BC) to protect birds from Campylobacter. In our methodology, six BC were constructed in a commercial broiler house containing approximately 28,500 birds. During three trials, the BC were stocked at densities of 12, 14, 16, 18, 20 and 22 birds/m2, with the main flock (20 birds/m2) considered the control. Periodically, 10 birds per density were weighed and examined. The Campylobacter status of the birds was monitored via faecal samples using the ISO 10272: 2017. The stocking density for maximum calculated yield was 20 (trials 1 and 2) or 22 birds/m2 (trial 3), followed by 18, 16, 14 and 12. At the stocking rate of 20 birds/m2, the birds in the pen grew faster than those at the same density in the main flock achieving 2 Kg 3–6 days faster. Birds in the BC were observed to be generally healthier, and in some cases, remained Campylobacter negative, even after the main flock was infected. Our results conclude that dividing the flock into sub-flocks of approximately 20 birds/m2 using BC could increase productivity up to 20%, while preventing Campylobacter.

Farm-to-fork quantitative microbial risk assessments (QMRA) typically start with a preliminary estimate of initial concentration (Cinitial) of microorganism loading at farm level, consisting of an initial estimate of prevalence (P) and the resulting pathogen levels in animal faeces. An average estimation of the initial concentration of pathogens can be achieved by combining P estimates in animal populations and the levels of pathogens in colonised animals' faeces and resulting cumulative levels in herd farmyard manure and slurry (FYM&S). In the present study, 14 years of data were collated and assessed using a Bayesian inference loop to assess the likely P of pathogens. In this regard, historical and current survey data exists on P estimates for a number of pathogens, including Cryptosporidium parvum, Mycobacterium avium subspecies paratuberculosis (MAP), Salmonella spp., Clostridium spp., Campylobacter spp., pathogenic E. coli, and Listeria monocytogenes in several species (cattle, pigs, and sheep) in Ireland. The results revealed that Cryptosporidium spp. has potentially the highest mean P (Pmean) (25.93%), followed by MAP (15.68%) and Campylobacter spp. (8.80%) for cattle. The Pmean of E. coli is highest (7.42%) in pigs, while the Pmean of Clostridium spp. in sheep was estimated to be 7.94%. Cinitial for Cryptosporidium spp., MAP., Salmonella spp., Clostridium spp., and Campylobacter spp. in cattle faeces were derived with an average of 2.69, 4.38, 4.24, 3.46, and 3.84 log10 MPN g -1, respectively. Average Cinitial of Cryptosporidium spp., Salmonella spp., Clostridium spp., and E. coli in pig slurry was estimated as 1.27, 3.12, 3.02, and 4.48 log10 MPN g -1, respectively. It was only possible to calculate the average Cinitial of Listeria monocytogenes in sheep manure as 1.86 log10 MPN g -1. This study creates a basis for future farm-to-fork risk assessment models to base initial pathogen loading values for animal faeces and enhance risk assessment efforts. ispartof: SCIENCE OF THE TOTAL ENVIRONMENT vol:786 ispartof: location:Netherlands status: published

Although Salmonella spp., Escherichia coli O157, Listeria monocytogenes, Enterococcus faecalis and Clostridium spp. present a significant food safety and/or spoilage issue for the beef sector, there are limited data on their prevalence in Irish cattle. The objectives of this preliminary study were to investigate the distribution (percentage of farms positive) of Salmonella spp., E. coli O157, L. monocytogenes, E. faecalis and Clostridium spp. and the overall prevalence (%) of these bacteria in cattle on a small cohort of Irish beef farms. A total of 121 fresh bovine faecal samples were obtained on 10 randomly selected beef farms in the Northeast of Ireland and tested for the target pathogens using standard culture-based methods. Presumptive positives were confirmed using previously published polymerase chain reaction (PCR) methods. Salmonella were not detected in any of the samples. E. coli O157, L. monocytogenes, E. faecalis and Clostridium spp. were present on 50%, 40%, 100% and 100% of farms, respectively, with overall (all farms) prevalence rates in cattle of 9%, 8.2%, 61.9% and 87.6%, respectively. This study suggests that E. coli O157 may be more prevalent than previously thought and L. monocytogenes, E. faecalis and Clostridium spp. are widespread in Irish beef animals.

Phenotypic antimicrobial resistance testing of 160 Campylobacter jejuni isolates from broiler caeca and neck skin samples collected at key stages during processing from 32 batches was conducted to: (i) determine the prevalence of antimicrobial resistance in C. jejuni isolates (ii) investigate if C. jejuni strains with particular resistance profiles were more frequently recovered at the end of processing, (iii) to establish if multiple strains with different resistance profiles were present within individual batches of broilers and (iv) to determine the virulence profiles of isolates. C. jejuni isolates were tested for resistance to erythromycin (ERY), ciprofloxacin (CIP), tetracycline (TET), gentamicin (GEN), nalidixic acid (NAL) and streptomycin (STR) using a broth dilution phenotypic method. Resistance to TET was most prevalent (46%), followed by resistance to CIP and NAL (29%). All isolates were susceptible to ERY and GEN, while resistance to STR was low (2%). Strains with different resistance profiles were found within 8 individual batches of broilers. Both resistant and susceptible strains were present within 4 individual batches of broilers. Whole genome sequencing of 132 C. jejuni isolates was conducted to: (i) determine relationship between resistant phenotypes and genotypes, (ii) examine genomic diversity of strains present in caeca and in neck skin samples collected at different processing stages and (iii) gain insights into the genomic profiles, including the resistome and virulome of C. jejuni isolates. A high level of correlation between resistant phenotypes and genotypes was observed (99.2%). Resistance determinants identified in sequenced isolates using ResFinder included the tet(O) gene, tet(O/32/O) gene, gyrA p.T86I mutation and blaOXA-61 like genes. A high correlation between sequence type (ST) and antimicrobial resistance was observed. The 132 sequenced C. jejuni isolates were assigned to 10 sequence types (STs), which belonged to 7 clonal complexes, based on multi-locus sequence typing (MLST). ST-257 was prevalent with 58 isolates assigned to it, followed by ST-51 with 25 isolates, ST-10089 with 16 isolates, ST-48 with 13 isolates and ST-50 with 12 isolates. Four isolates were assigned to ST-354, while 1 isolate was assigned to STs 464, 42, 1489 and 6209. All sequenced isolates were assigned to 17 cgSTs. Virulence genes which might have a role in Campylobacter survival and environmental adaptation during key processing stages and a number of virulence genes associated with human infection and colonization of broilers were detected. This study is one of the first to provide data on genomic diversity of C. jejuni isolates from broilers collected during processing in Ireland.

Campylobacter spp. is a significant and prevalent public health hazard globally. Campylobacter jejuni is the most frequently recovered species from human cases and poultry are considered the most important reservoir for its transmission to humans. In this study, 30 Campylobacter jejuni isolates were selected from clinical (n = 15) and broiler (n = 15) sources from a larger cohort, based on source, virulence, and antimicrobial resistance profiles. The objective of this study was to further characterise the genomes of these isolates including MLST types, population structure, pan-genome, as well as virulence and antimicrobial resistance determinants. A total of 18 sequence types and 12 clonal complexes were identified. The most common clonal complex was ST-45, which was found in both clinical and broiler samples. We characterised the biological functions that were associated with the core and accessory genomes of the isolates in this study. No significant difference in the prevalence of virulence or antimicrobial resistance determinants was observed between clinical and broiler isolates, although genes associated with severe illness such as neuABC, wlaN and cstIII were only detected in clinical isolates. The ubiquity of virulence factors associated with motility, invasion and cytolethal distending toxin (CDT) synthesis in both clinical and broiler C. jejuni genomes and genetic similarities between groups of broiler and clinical C. jejuni reaffirm that C. jejuni from poultry remains a significant threat to public health.

If pathogens are present in feedstock materials and survive in anaerobic digestion (AD) formulations at 37 &deg, C, they may also survive the AD process to be disseminated in digestate spread on farmland as a fertilizer. The aim of this study was to investigate the prevalence of Salmonella spp., Escherichia coli O157, Listeria monocytogenes, Enterococcus faecalis and Clostridium spp. in AD feed and output materials and survival/growth in four formulations based on food waste, bovine slurry and/or grease-trap waste using International Organization for Standardization (ISO) or equivalent methods. The latter was undertaken in 100 mL Ramboldi tubes, incubated at 37 &deg, C for 10 d with surviving cells enumerated periodically and the T90 values (time to achieve a 1 log reduction) calculated. The prevalence rates for Salmonella spp., Escherichia coli O157, Listeria monocytogenes, Enterococcus faecalis and Clostridium spp. were 3, 0, 5, 11 and 10/13 in food waste, 0, 0, 2, 3 and 2/3 in bovine slurry, 1, 0, 8, 7 and 8/8 in the mixing tank, 5, 1, 17, 18 and 17 /19 in raw digestate and 0, 0, 0, 2 and 2/2 in dried digestate, respectively. Depending on the formulation, T90 values ranged from 1.5 to 2.8 d, 1.6 to 2.8 d, 3.1 to 23.5 d, 2.2 to 6.6 d and 2.4 to 9.1 d for Salmonella Newport, Escherichia coli O157, Listeria monocytogenes, Enterococcus faecalis and Clostridium sporogenes, respectively. It was concluded that AD feed materials may be contaminated with a range of bacterial pathogens and L. monocytogenes may survive for extended periods in the test formulations incubated at 37 &deg, C.

Campylobacter spp. are major causes of foodborne illness globally, and are mostly transmitted through the consumption and handling of poultry. Campylobacter infections have widely variable outcomes, ranging from mild enteritis to severe illness, which are attributed to host interactions and the virulence of the infecting strain. In this study, in order to investigate the effect of host stress on the growth and pathogenicity of C. jejuni, three strains associated with human infection and two strains from broilers were subject to growth, motility, adhesion and invasion assays, in response to exposure to catecholamines, epinephrine, norepinephrine and the glucocorticoid neuroendocrine hormones corticosterone, cortisol and cortisone which are associated with stress in humans and broilers. Catecholamines resulted in significantly increased growth, adhesion and invasion of Caco-2 cells. Corticosterone promoted growth in one of five strains, and cortisone resulted in a significant increase in motility in two out of five strains, while no significant differences were observed with the addition of cortisol. It was concluded that stress-associated hormones, especially catecholamines, may promote growth and virulence in Campylobacter.

Campylobacteriosis is the leading cause of human bacterial gastroenteritis, very often associated with poultry consumption. Thermophilic Campylobacter (Campylobacter jejuni and Campylobacter coli) isolates (n = 158) recovered from broiler neck skin and caecal contents in Ireland over a one-year period, resistant to at least one of three clinically relevant antimicrobial classes, were screened for resistance determinants. All ciprofloxacin-resistant isolates (n = 99) harboured the C257T nucleotide mutation (conferring the Thr-86-Ile substitution) in conjunction with other synonymous and nonsynonymous mutations, which may have epidemiological value. The A2075G nucleotide mutation and amino acid substitutions in L4 and L22 were detected in all erythromycin-resistant isolates (n = 5). The tetO gene was detected in 100% (n = 119) of tetracycline-resistant isolates and three of which were found to harbour the mosaic tetracycline resistance gene tetO/32/O. Two streptomycin-resistant C. jejuni isolates (isolated from the same flock) harboured ant(6)-Ib, located in a multidrug resistance genomic island, containing aminoglycoside, streptothricin (satA) and tetracycline resistance genes (truncated tetO and mosaic tetO/32/O). The ant(6)-Ie gene was identified in two streptomycin-resistant C. coli isolates. This study highlights the widespread acquisition of antimicrobial resistance determinants among chicken-associated Campylobacter isolates, through horizontal gene transfer or clonal expansion of resistant lineages. The stability of such resistance determinants is compounded by the fluidity of mobile genetic element.

Campylobacter phage vB_CjeM_Los1 was recently isolated from a slaughterhouse in the Republic of Ireland using the host Campylobacter jejuni subsp. jejuni PT14, and full-genome sequencing and annotation were performed. The genome was found to be 134,073 bp in length and to contain 169 predicted open reading frames. Transmission electron microscopy images of vB_CjeM_Los1 revealed that it belongs to the family Myoviridae, with tail fibres observed in both extended and folded conformations, as seen in T4. The genome size and morphology of vB_CjeM_Los1 suggest that it belongs to the genus Cp8virus, and seven other Campylobacter phages with similar size characteristics have also been fully sequenced. In this work, comparative studies were performed in relation to genomic rearrangements and conservation within each of the eight genomes. None of the eight genomes were found to have undergone internal rearrangements, and their sequences retained more than 98% identity with one another despite the widespread geographical distribution of each phage. Whole-genome phylogenetics were also performed, and clades were shown to be representative of the differing number of tRNAs present in each phage. This may be an indication of lineages within the genus, despite their striking homology.

Farmyard manure and slurry (FYM&S) is a valuable feedstock for anaerobic digestion (AD) plants. However, FYM&S may contain high concentrations of pathogens, and complete inactivation through the AD process is unlikely. Thus, following land application of digestate, pathogens may contaminate a range of environmental media posing a potential threat to public health. The present study aimed to combine primary laboratory data with literature-based secondary data to develop an Excel-based exposure assessment model (ADRISK) using a gamma generalised linear model to predict the final microorganism count in the digestate. This research examines the behaviour of a suite of pathogens (Cryptosporidium parvum, norovirus, Mycobacterium spp., Salmonella spp., Listeria monocytogenes, Clostridium spp., and pathogenic Escherichia coli) and indicators (total coliforms, E. coli, and enterococci) during mesophilic anaerobic digestion (M-AD) at 37 °C, pre/post-AD pasteurisation, and after a period of storage (with/without lime) for different feedstock proportions (slurry:food waste: 0:1, 1:3, 2:1, and 3:1). ADRISK tool simulations of faecal indicator bacteria levels across all scenarios show that the digestate can meet the EU standard without pasteurisation if the AD runs at 37 °C or a higher temperature with a higher C:N ratio (recipe 3) and a hydraulic retention time ≥ 7 days. The storage of digestate also reduced levels of microorganisms in the digestate. The Irish pasteurisation process (60 °C for 4 days), although more energy-intensive, is more effective than the EU pasteurisation (70 °C for 1 h) specification. Pre-AD pasteurisation was more effective for C. parvum, norovirus, Mycobacterium thermoresistibile. However, post-AD literature-based pasteurisation is most likely to assure the safety of the digestate. The information generated from this model can inform policy-makers regarding the optimal M-AD process parameters necessary to maximise the inactivation of microorganisms, ensuring adverse environmental impact is minimised, and public health is protected. ispartof: SCIENCE OF THE TOTAL ENVIRONMENT vol:800 ispartof: location:Netherlands status: published

This study investigated bacteriophage (phage) mediated transfer of the vtx2 gene from a donor Escherichia coli (C600φ3538(Δvtx2::cat)) to enteropathogenic (EPEC), enterotoxigenic (ETEC), enteroaggregative (EAEC), enteroinvasive (EIEC) and diffusely adherent (DAEC) E. coli strains in LB broth, milk, ground beef and lettuce. Two bacterial concentrations for both the E. coli donor and recipient strains, 3 and 5 log10 CFU/ml (LB broth and milk)/g (beef) or/cm2 (lettuce), were used. When transductants were obtained, the location of insertion of the phage (insertion sites wrbA, yehA, sbcB, yecE and/or Z2577) in the E. coli chromosome was investigated by PCR. The vtx2 gene was readily transferred to EAEC O104:H4 (E99518) in all matrices and inserted into the chromosome at the sbcB locus. At higher cell concentrations, transductants were also obtained with ETEC E4683, ETEC E8057 (insertion site unknown) and DAEC O75:H- E66438 (insertion site unknown) in LB broth and milk. It was concluded that the vtx2 gene may be transferred by bacteriophage to different E. coli pathotypes in laboratory and food matrices, resulting in the spread of the vtx2 gene and the emergence of novel foodborne pathogens.

Previous studies demonstrated that commercial broiler flocks could be protected from Campylobacter colonisation using a bird pen, termed the “biosecurity cube”, constructed from four polycarbonate sheets (1m high x 2.5m long x 6 mm thick) supported at the corners by 4 × 1m high wooden columns. However, this design had issues with airflow and potential for upscaling. A biosecurity cube composed of four galvanised steel mesh panels (3.44m long x 1.25m high) was therefore developed onto which different barrier materials, preventing contact between the test birds and the main flock, were attached. The objective of this study was to test a range of barrier materials including cardboard, wire mesh, polyurethane film and later (at the suggestion of broiler industry personnel) flyscreen mesh. Initial studies suggested that while the cardboard and wire mesh were ineffective, the polyurethane film protected the birds. Further validation (over 2 separate trials, 7 cubes for each barrier material) demonstrated that polyurethane and flyscreen mesh were effective. It was concluded that a biosecurity pen infrastructure based on galvanised steel mesh panels surrounded by polyurethane film or flyscreen mesh was effective at protecting the birds from Campylobacter but upscaling studies will be undertaken before full implementation.

Aims Silage is grass, preserved by fermentation and used as winter feed for cattle. The impact of a range of current grass silage preparation practices on the survival of Escherichia coli C600φ3538(Δvtx2 ::cat) and on the induction, release and infectivity of free phage were investigated. Methods and results Wilted and fresh grass samples, from plots with and without slurry application, were ensiled with or without formic acid. Each treatment combination was inoculated with approximately 6 log10 CFU per g E. coli C600φ3538(Δvtx2 ::cat) (donor strain) and E. coli C600::kanamycinR (recipient strain) in test-tube model silos and incubated in the dark at 15°C. The physico-chemical (pH, ammonia, ethanol, lactic acid and volatile fatty acids) and microbiological (total viable counts, TVC, total Enterobacteriaceae counts, TEC, E. coli counts, ECC and lactic acid bacteria, LAB) properties of each fermentation were monitored throughout the experiment as were the concentrations of E. coli C600φ3538(Δvtx2 ::cat), E. coli C600::kanamycinR , free phage and transductants, using culture and PCR-based methods. Over the course of the experiment the pH of the grass samples typically decreased by 2 pH units. TVC, TEC and ECC decreased by up to 2·3, 6·4 and 6·2 log10 CFU per g, respectively, while the LAB counts remained relatively stable at 5·2-7·1 log10 CFU per g. Both donor and recipient strains decreased by approximately 5 log10 CFU per g. Free phages were detected in all treatments and transductants were detected and confirmed by PCR in the silo containing wilted grass, pretreated with slurry and ensiled without formic acid. Conclusions Verocytotoxigenic E. coli may survive the ensiling process and the conditions encountered are sufficient to induce vtx2 bacteriophage leading to low levels of phage-mediated vtx2 gene transfer. Significance and impact of the study These studies suggest that the ensiling of grass may create an environment which facilitates the emergence of new verocytotoxigenic E. coli.

This study investigated the fate of Salmonella Typhimurium and Escherichia coli O157 on hot boned versus conventionally chilled beef. Beef samples were individually inoculated with S. Typhimurium ATCC 14028, S. Typhimurium 844, E. coli O157 EDL 933 or E. coli T13. Half the samples were subject to the same time-temperature chilling profile used for conventionally chilling beef carcasses while the other half was subject to hot boned conditions. The surface pH (5.5) and aw (0.95 to 0.97) were stable. S. Typhimurium and E. coli O157 counts, which decreased by up to 1.0 and 1.5log10cfucm-2, respectively, were statistically similar (P>0.05), regardless of the chilling regime applied, with the exception of E. coli O157 EDL 933, where the counts on hot boned beef were significantly (P

The true prevalence of tet(A), which codes for a tetracycline efflux pump, in thermophilic Camplyobacter spp. requires clarification after reports emerged in Iran (2014) and Kenya (2016) of the novel detection of tet(A) in Campylobacter. During our investigation of antibiotic resistance mechanisms in a sample of Irish thermophilic Campylobacter broiler isolates, it was determined that 100% of tetracycline-resistant isolates (n = 119) harboured tet(O). Accessory tetracycline-resistance mechanisms were considered as tetracycline minimum inhibitory concentrations ranged from 4 to ≥ 64 mg/L. Primers previously reported for the detection of tet(A) in Campylobacter failed to produce an amplicon using a positive control strain (Escherichia coli K12 SK1592 containing the pBR322 plasmid) and a selection of Campylobacter isolates. Accordingly, we designed new tet(A)-targeting primers on SnapGene2.3.2 that successfully generated a 407 bp product from the positive control strain only. Further in silico analysis using BLASTn and SnapGene2.3.2 revealed that previously reported Campylobacter tet(A) sequences deposited on GenBank shared 100% homology with Campylobacter tet(O). We postulate that this gave rise to the erroneous report of a high tet(A) prevalence among a pool of Kenyan broiler Campylobacter isolates that were tested using primers designed based on these apparent tet(A) sequences. In conclusion, further work would be required to determine whether the homology between tet(A) potentially present in Campylobacter and known tet(A) genes would be sufficient to allow amplification using the primers designed in our study. Finally, the existence of tet(A) in thermophilic Campylobacter spp. remains to be demonstrated.

Campylobacter spp. is a leading cause of foodborne illness globally. The pathogen colonises the gastrointestinal tract of the host, where small concentrations of neuroendocrine hormones are also secreted. Epinephrine and norepinephrine are neuroendocrine hormones involved in the stress response that have been shown to promote the expression of virulence factors in pathogens including E. coli, Salmonella spp., and Campylobacter spp. In our study Campylobacter jejuni strains from human infection and broiler farms that were supplemented with epinephrine and norepinephrine showed increased growth characterised by shorter lag phases and higher maximum OD595, and enhanced pathogenicity characterised by increased motility, attachment to and invasion of Caco-2 cells. The data obtained suggests that host stress may promote C. jejuni proliferation and pathogenicity.

This study examined the impact of key processing stages and flock variables on the prevalence of Campylobacter on broiler carcasses. Overall, the prevalence of Campylobacter was 62% in caeca, and 68%, 65% and 62% in neck skin samples collected after evisceration, final wash and carcass chilling, respectively. Campylobacter were found in 32% of caeca, and 52%, 40% and 32% of neck skin samples collected after evisceration, final wash and carcass chilling, respectively from first thin broiler batches. Final thin broiler batches were more frequently contaminated with prevalences of 83% found in caeca, 80% in neck skin samples collected after evisceration and 83% found in neck skin samples collected after both final wash and carcass chilling stages (p < 0.05). Thinning status had a significant effect on Campylobacter counts with significantly higher counts observed in samples from final thin batches (p < 0.05). Highest Campylobacter concentrations in neck skin samples were observed at the evisceration stage in both first and final thin samples, with counts ranging from 2.0 to 3.8 log10 CFU/g and 2.3 to 4.8 log10 CFU/g in first and final thin batches, respectively. All first thin samples had counts below the European Union (EU) Process Hygiene Criterion threshold level of 3 log10 CFU/g after chilling while 52% of final thin batches had counts above this limit.

Application of novel decontamination strategies such as Ultraviolet (UV) irradiation are required to mitigate the risks associated with Campylobacter jejuni in food. This study evaluated the use of a light-emitting diode (LED) based technology to inactivate C. jejuni NCTC 11168 in Maximum Recovery Diluent (MRD) at wavelengths of 280, 300 and 365 nm and combinations. To assess the survival curves, two linear (Log linear (LL) and Linear and Shoulder) and two non-linear models (Weibull and Double Weibull) were fitted. UV exposures showed different antimicrobial effects where a combination of 280/300 nm was the most effective treatment with a 4Dt value of 5 s observed in a bacterial suspension of 5 log CFU/mL. Moreover, the LL model was the most robust model to describe the inactivation kinetics of Campylobacter when exposed to UV and therefore, modelling tools could be applied to predict the efficiency of UV light in a model solution. Industrial relevance: Light-based technologies like UV light are identified in the literature as potential alternatives to assure the decontamination of surfaces, liquids and solid food. However, some of these techniques require further investigation. The present study evaluated the use of a LED system and effect of combined wavelengths in the inactivation of Campylobacter through predictive modelling. This technique was observed to predict and explain kinetics of inactivation of Campylobacter and could be key in the scaling-up process of UV light at industrial level.

peer-reviewed This study compared the impact of three indirect heating systems to direct gas flame heaters on a selection of flock performance and environmental indicators in commercial broiler units. No statistically significant differences (P≥0.05) were found in flock mortality rates, bird weight, water consumption, stress response, carbon dioxide, ammonia, temperature, relative humidity, litter quality, within-flock Campylobacter levels or mean Campylobacter counts when flock data from any of the three indirect heating systems were compared to flocks reared in houses with direct heating systems. Differences in litter quality were observed between upper and lower litter layers in all houses, regardless of heating type, which may have implications for bird health and welfare. Carbon dioxide concentrations in houses with direct heating systems were significantly higher than those in houses with indirect heating systems during the first 10 days of bird life (P≤0.05). This was due to the increased use of heating systems during this period of the flock cycle. Differences in CO2 concentrations had no effect on flock performance, possibly due to the fact that concentrations did not exceed known safe levels. A statistically significant increase in stress response was observed in birds as a result of partial depopulation (thinning) within houses, irrespective of heating system type used (P≤0.05). Stress associated with thinning may have consequences for bird welfare and food safety. In conclusion, the results of our study suggest that indirect heating systems do not appear to negatively impact on flock performance, stress response, within-flock Campylobacter levels or mean Campylobacter counts and do not appear to significantly alter environmental conditions within broiler houses when compared to houses equipped with direct heating systems. Indirect systems are a viable alternative for heating broiler houses in terms of flock performance, bird welfare and food safety.

The objective of this study was to test the hypothesis that farm staff are the primary vector of Campylobacter transmission into broiler flocks. On 3 different farms and 5 different flocks (3 flocks on farm 1 and 1 flock on each of farms 2 and 3) a small section of the broiler house (3 × 2 m (farm 1) and 1 m × 1 m (farms 2 and 3)) was sectioned off using Perspex or plastic sheeting. This ‘biosecure cube’ (BC) was populated with 25–125 chicks (test birds), a small subset of the general population of up to 30,000 (control) birds in the broiler house. The BC area incorporated the water and feed-lines thus the test and control birds had access to the same feed, water and air. However, unlike in the general broiler house, the farm staff had no direct access to this sub-population. Dead birds were aseptically removed by the researchers. The birds were tested for Campylobacter (faecal and/or caecal samples), on the day of chick arrival and every 7 days thereafter. In farm 1-flock 1 the general broiler population was Campylobacter-positive after 21 days but the test birds remained negative until day 35. The general broiler population in the other 4 flocks were Campylobacter positive as early as day 14, but in all cases the test birds remained negative. Moreover BC broilers were significantly (P

Summary Verocytotoxigenic (vtx) Escherichia coli (VTEC) are zoonotic foodborne pathogens with the vtx operon encoded by lambdoid bacteriophage (phage). Despite much research on the host bacteria, similar data on the persistence of verocytotoxin converting phage and the ecological niches where transduction occurs are lacking and novel VTEC of important public health significance, have and continue to emerge. This study investigated the survival of a temperate vtx bacteriophage (24B::kanamycinR) in water (raw farm, pasteurized farm, laboratory tap and autoclaved purified water) and soil (sandy loam and loam soil). It also examined the persistence of an anti-VTEC lytic phage (e11/2) in the same matrices as this may be one option for controlling the emergence of novel VTEC, especially in farm ecological niches where other control options, such as chemical, heat or high pressure treatments, are not feasible. Samples inoculated with 24B::kanamycinR and e11/2 bacteriophage (8 log10 pfu/ml or pfu/g) separately were incubated at 4°C and 14°C, representative Irish Winter and Summer temperatures, respectively, and tested every 2 days for 40 days. The transduction of 24B::kanamycinR was also continuously assessed. Both phages survived with reductions observed, regardless of matrix or storage temperature. Moreover, 24B::kanamycinR was able to transduce its host E. coli strain. It was therefore concluded that aquatic and soil environments on farms may serve as a vtx phage reservoir and transduction point but anti-VTEC phage is a possible biocontrol option.

Four antimicrobials, namely; Articoat DLP 02 (AR), Artemix Consa 152/NL (AX), Auranta FV (AFV) and sodium octanoate (SO) were examined for their effectiveness, both before and after heat treatments, against bacterial strains Bacillus cereus, Pseudomonas fluorescens, Escherichia coli, Staphylococcus aureus and the microflora obtained from commercial beef steaks. Minimum inhibitory concentrations (MIC) using AR, AX, AFV and SO against these microbes were then obtained using the 96-well plate method. SO was the most effective against all bacterial strains, demonstrating the lowest MIC compared to the other antimicrobials. These antimicrobials were then successively incorporated into beef-derived gelatine films and these films were subsequently tested for structural, mechanical and barrier properties. Significantly (p < 0.05) enhanced water vapour barrier properties were determined only for antimicrobial films containing AX or SO when compared to control films. On the basis of FTIR spectra, significant changes in the structure of SO-containing films were determined when compared with control gelatin films. It was shown that active antimicrobial agents could potentially serve as commercial antimicrobial coatings for application onto conventional plastic-based food packaging.

Studies were undertaken to investigate the effect of oxidative stress conditions (exposure to hydrogen peroxide, H2O2) on [1] the expression of 14 Campylobacter jejuni virulence-associated genes associated with motility and/or invasion (flaA, flaB, flhA, flhB, ciaB, iamA), adhesion (cadF), cytotoxin production (cdtA, cdtB, cdtC) as well as some of the regulators of these genes (rpoN, fliA, luxS, cj1000), in 10 C. jejuni strains (5 poultry and 5 human) and [2] the ability of these cells to adhere to and invade Caco-2 cells. Using 16S rRNA as the reference gene (preliminary research demonstrated that this gene was stably expressed), the expression of the 14 virulence associated genes was investigated under normal and oxidative stress conditions using reverse transcription PCR. A Caco-2 cell tissue culture assay was used to examine adhesion and invasion. The response to oxidative stress was strain-dependent. Two strains showed significant (p

Listeria monocytogenes is an important bacterial pathogen in seafood products, but limited information is currently available on the thermal resistance of relevant isolates in seafood. Thermal inactivation studies were undertaken (i) to provide much needed thermal inactivation data for L. monocytogenes in crab meat and (ii) to investigate whether tryptone soya broth (TSB) is representative of crab meat in thermal inactivation studies involving L. monocytogenes. D-values were obtained for a cocktail of two crab isolates (serotypes 1/2a and 4b) at 50, 55, and 60°C. In crab meat, D-values were 174.4, 28.2, and 1.6 min, respectively. Similar D-values of 176.4, 28.8, and 1.4 min were obtained in TSB. The corresponding z-values were 4.9°C (crab meat) and 4.8°C (TSB), respectively. The conclusions were that (i) current pasteurization conditions (e.g., 70°C for 2 min) would achieve complete destruction of any L. monocytogenes present in crab meat and (ii) TSB could be used as a model matrix for assessing the thermal inactivation of L. monocytogenes in crab meat.

Anaerobic digestion (AD) has been identified as one of the cleanest producers of green energy. AD typically uses organic materials as feedstock and, through a series of biological processes, produces methane. Farmyard manure and slurry (FYM&S) are important AD feedstock and are typically mixed with agricultural waste, grass and/or food wastes. The feedstock may contain many different pathogens which can survive the AD process and hence also possibly be present in the final digestate. In this study, a semi-quantitative screening tool was developed to rank pathogens of potential health concern emerging from AD digestate. A scoring system was used to categorise likely inactivation during AD, hazard pathways and finally, severity as determined from reported human mortality rates, number of global human-deaths and infections per 100,000 populations. Five different conditions including mesophilic and thermophilic AD and three different pasteurisation conditions were assessed in terms of specific pathogen inactivation. In addition, a number of scenarios were assessed to consider foodborne incidence data from Ireland and Europe and to investigate the impact of raw FYM&S application (without AD and pasteurisation). A sensitivity analysis revealed that the score for the mortality rate (S3) was the most sensitive parameter (rank coefficient 0.49) to influence the final score S; followed by thermal inactivation score (S1, 0.25) and potential contamination pathways (S2, 0.16). Across all the scenarios considered, the screening tool prioritised Cryptosporidium parvum, Salmonella spp., norovirus, Streptococcus pyogenes, enteropathogenic E. coli (EPEC), Mycobacterium spp., Salmonella typhi (followed by S. paratyphi), Clostridium spp., Listeria monocytogenes and Campylobacter coli as the highest-ranking pathogens of human health concern resulting from AD digestate in Ireland. This tool prioritises potentially harmful pathogens which can emerge from AD digestate and highlights where regulation and intervention may be required., Graphical abstract Unlabelled Image, Highlights • Health risks from spreading animal waste and/or anaerobic digestate. • Semi-quantitative screening tool developed to rank pathogens. • Scoring pathogens on thermal survivability, exposure pathways, severity or human mortality rate in untreated patients.

Food safety criteria for Listeria monocytogenes in ready‐to‐eat (RTE) foods have been applied from 2006 onwards (Commission Regulation (EC) 2073/2005). Still, human invasive listeriosis was reported to increase over the period 2009–2013 in the European Union and European Economic Area (EU/EEA). Time series analysis for the 2008–2015 period in the EU/EEA indicated an increasing trend of the monthly notified incidence rate of confirmed human invasive listeriosis of the over 75 age groups and female age group between 25 and 44 years old (probably related to pregnancies). A conceptual model was used to identify factors in the food chain as potential drivers for L. monocytogenes contamination of RTE foods and listeriosis. Factors were related to the host (i. population size of the elderly and/or susceptible people; ii. underlying condition rate), the food (iii. L. monocytogenes prevalence in RTE food at retail; iv. L. monocytogenes concentration in RTE food at retail; v. storage conditions after retail; vi. consumption), the national surveillance systems (vii. improved surveillance), and/or the bacterium (viii. virulence). Factors considered likely to be responsible for the increasing trend in cases are the increased population size of the elderly and susceptible population except for the 25–44 female age group. For the increased incidence rates and cases, the likely factor is the increased proportion of susceptible persons in the age groups over 45 years old for both genders. Quantitative modelling suggests that more than 90% of invasive listeriosis is caused by ingestion of RTE food containing > 2,000 colony forming units (CFU)/g, and that one‐third of cases are due to growth in the consumer phase. Awareness should be increased among stakeholders, especially in relation to susceptible risk groups. Innovative methodologies including whole genome sequencing (WGS) for strain identification and monitoring of trends are recommended., This publication is linked to the following EFSA Supporting Publications article: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2018.EN-1352/full

Despite over 30 years of research, campylobacteriosis is the most prevalent foodborne bacterial infection in many countries including in the European Union and the United States of America. However, relatively little is known about the virulence factors in Campylobacter or how an apparently fragile organism can survive in the food chain, often with enhanced pathogenicity. This review collates information on the virulence and survival determinants including motility, chemotaxis, adhesion, invasion, multidrug resistance, bile resistance and stress response factors. It discusses their function in transition through the food processing environment and human infection. In doing so it provides a fundamental understanding of Campylobacter, critical for improved diagnosis, surveillance and control.

This study tested 24 Campylobacter isolates for the presence of 35 virulence genes using the polymerase chain reaction. The target genes included those involved in motility (flaA, flaB, flhA, flhB, flgB, flgE2, fliM, fliY), chemotaxis (cheA, cheB, cheR, cheW, cheY, cheZ), cell adhesion (cadF, dnaJ, jlpA, pldA, racR, virB11), invasion (iamA, ciaB, ceuE), cytotoxin production (cdtA, cdtB, cdtC, wlaN), capsule (kpsM), multidrug and bile resistance (cmeA, cmeB, cmeC), stress response/survival (katA, sodB), and the iron uptake system (cfrA, fur). The motility genes (with the exception of flaB), the CmeABC efflux system, cdtABC genes, and the sodB gene were commonly distributed among Campylobacter strains while the virB11 and wlaN genes were rarely detected. Interestingly, the findings suggest that flaB is not essential for full motility and C. coli lacking the flhA gene may be highly invasive. This study provides additional information on the distribution of Campylobacter virulence factors and the effect of their presence/absence on adhesion and invasion. It will inform future studies designed to elucidate the exact mechanisms of pathogenesis in Campylobacter.

Research focussing on the use of chemicals to decontaminate poultry carcasses to reduce pathogenic and spoilage organisms has increased in recent years. The objective of this study was to evaluate the efficacy of 12% (w/v) trisodium phosphate (TSP), 2% (w/v) citric acid (CA) and 5% (w/v) capric acid sodium salt (CP) in reducing Campylobacter, total viable counts and total Enterobacteriaceae counts on poultry. These chemicals were also used in various combinations (TSP þ CA, TSP þ CP and CA þ CP) to determine if sequential treatments would enhance microbial reductions. TSP (1.9e2.3 log10 cfu cm 2 ) and CP (2.2 e2.4 log10 cfu cm 2 ) gave the largest Campylobacter jejuni reductions while TSP was the most effective at reducing TVC (0.9 log10 cfu cm 2 ) and TEC (0.9 log10 cfu cm 2 ). TSP þ CP was the most effective combination treatment (2.9 log10 cfu cm 2 ) for reducing C. jejuni counts and was significantly (P < 0.05) greater than any of the single chemical treatments, with the exception of CP treatment against strain 1146 (2.4 log10 cfu cm 2 ). The TVC and TEC populations proved more resistant to combination treatments as only TSP þ CP showed a significantly (p < 0.05) enhanced reductive efficacy in comparison to single CP treatment. This study provides further data on the efficacy of a number of potential chemicals used alone and in combination for the decontamination of raw poultry.

Studies were undertaken to investigate the effect of different modified atmospheric packaging (MAP) gaseous combinations on Campylobacter and the natural microflora on poultry fillets. Skinless chicken fillets were stored in gaseous mixtures of 10%, 30%, 50%, 70% and 90% CO2 balanced with N2, 80:20% O2:N2 and 40:30:30% CO2:O2:N2 and control conditions (air) at 2 °C. Samples were analysed periodically for (previously inoculated) Campylobacter, total viable counts (TVC) (mesophiles), TVC (psychrophiles), Enterobacteriaceae, Pseudomonas and lactic acid bacteria (LAB) over 17 days of storage. The carbon dioxide solubility was determined by monitoring the changes in the headspace volume over time using a buoyancy technique and performing calculations based on volumetric measurements and the Henry's constant. Henry's constant was also used to estimate the oxygen solubility in the chicken fillets. The presence of O2 in the MAP gaseous mixtures increased the rate of Campylobacter decline on poultry fillets but in general the counts obtained in aerobic versus anaerobic packs were not significantly (P > 0.05) different. CO2 inhibited the growth of TVC, TEC, LAB and Pseudomonas but only at MAP gaseous combinations containing 50–90% CO2 where concentrations of up to 2000 ppm CO2 were recorded in the fillets after 5 days. Under these conditions a shelf-life in excess of 17 days at 2 °C was obtained. Although, dissolved O2, at levels of 33 ppm in 80:20% O2:N2 packs after 3 days, reduced Campylobacter, it also favoured the growth of the other microbes on the chicken. The optimum gaseous mixture for achieving the combined objectives of reducing Campylobacter and extending shelf was therefore 40:30:30 CO2:O2:N2, which achieved a shelf-life in excess of 14 days.

Cost-effective and acceptable decontamination technologies that can be readily applied to the broiler-processing industry have been proposed as a strategy to reduce pathogenic organisms on carcasses and improve food safety. The objective of this study was to determine the potential of combining chemical and ultrasonication processes to reduce Campylobacter, total viable counts (TVCs) and total Enterobacteriaceae counts (TECs) on broiler carcasses. Drumsticks were inoculated with Campylobacter jejuni and immersed in 12 % (w/v) trisodium phosphate (TSP), 2 % (w/v) citric acid (CA) or 5 % (w/v) capric acid sodium salt (CP) solutions for 1 min, while ultrasonication was performed at 40, 60 or 80 kHz. In addition, chemicals were administered in sequential combination (TSP + CA, TSP + CP or CA + CP) while ultrasonication was performed at the frequencies mentioned. The sequential treatment of TSP and CP with ultrasonication at 80 kHz achieved the greatest reductions for C. jejuni (4.5–4.6 log10 colony forming units (CFU)/cm2) and TVC (1.9 log10 CFU/cm2). Enterobacteriaceae were susceptible to the sequential application of CA and CP and ultrasonication at 80 kHz (2 log10 CFU/cm2). This study demonstrated that combining chemical decontaminants with ultrasonication can significantly (p

Aims The objectives of this study were the following: (i) to investigate if Campylobacter negative flocks at first thinning remain negative at second thinning; (ii) to determine if the caecal counts in birds infected during first thinning remain lower than in birds that were positive at first thinning; and (iii) to determine if reducing the time between first and second thinning to a maximum of 4 days would reduce both the incidence and prevalence of broiler caecal contamination. Methods and Results Twenty-two flocks were tested at first and second thinning using ISO methodologies. Of the 14 that had a 4-day duration between first and second thinning, nine flocks were Campylobacter negative at first thinning. By second thinning, all 14 flocks were positive and Campylobacter counts ranged from 5·5 to 6·6 log10 CFU g−1 regardless of the status at first thinning. The other eight flocks were all Campylobacter positive at first thinning with counts ranging from 0·8 to 6·1 log10 CFU g−1 which increased to 5·1 to 6·9 log10 CFU g−1 by second thinning (3–10 days). PCR speciation and MLST genotyping suggested the majority of isolates were Camp. jejuni belonging to STs 257, 814, 6763 and 6764. Conclusions It was concluded that; thinning introduces Campylobacter into broiler flocks; caecal counts in birds at second thinning are similar, regardless of flock status at first thinning and reducing the time between first and second thinning to a maximum of 4 days is not an effective control strategy. Significance and Impact of the Study This study informs Campylobacter control strategy at primary production, suggesting all post-first thinning broilers should be treated as ‘high risk’ regardless of Campylobacter status at first thinning or duration between first and second thinning.

Research was undertaken to investigate cross-contamination of the domestic kitchen environment during poultry fillet preparation using a streptomycin-resistant strain of Pseudomonas fluorescens as a model organism. The potential role of a cook-in-the-bag technology to control this cross-contamination was also investigated. Poultry fillets were inoculated with P. fluorescens (6.06 log10 CFU/cm2). Six people were challenged to unpack, defrost, cut and cook without contaminating the preparation environment. After preparation, the chopping board, knife blade, dishcloth, refrigerator handle, oven handle, oven buttons, draining board, tap, microwave handle, microwave buttons, plate, tinfoil and press handle were tested for the presence of the P. fluorescens strain, before and after washing. The experiment was then repeated with a precut cook-in-the-bag product. In a separate experiment, the effect of freezing and frozen storage (−20C) on Campylobacter and the sensory attributes of chicken fillets were investigated. The cook-in-the-bag approach considerably reduced the incidence and levels of cross-contamination in the domestic kitchen. Freezing significantly (P

Campylobacteriosis is the most common foodborne bacterial illness in Europe and most cases are associated with the consumption of poultry. Interventions are urgently required to reduce Campylobacter counts on poultry carcasses. While trisodium phosphate (TSP), citric acid (CA) and lactic acid (LA) are effective poultry carcass decontamination treatments, their direct application on carcasses is not permitted. This study examined their effectiveness in killing Campylobacter in cloacal contents before testing their efficacy as cloacal wash treatments immediately before defeathering. In laboratory experiments, fresh broiler cloacal contents inoculated with a 5 strain cocktail of Campylobacter jejuni (3) and Campylobacter coli (2) was treated with TSP (5, 10 & 20% w/v), CA (1, 5 & 10% w/v) and LA (1, 5 & 10% v/v)and surviving cells enumerated after 0, 4 and 10 min on mCCDA. The same chemical treatments were applied as a cloacal wash in a commercial broiler plant using naturally contaminated broiler carcasses.Carcass Campylobacter, TVC (psychrophile and mesophile) and TEC were determined immediately after defeathering and evisceration. TSP (20%, w/v), CA (5 & 10%, w/v) and LA (5 & 10%, w/v) reduced Campylobacter counts in broiler cloacal contents by approximately 2.0e2.5 log10 cfu g1 after 4 min.However, only an LA (5%, v/v) cloacal wash achieved a significant (P < 0.05) reduction in carcass Campylobacter counts (0.66 log10 cfu cm-2) and this was obtained post evisceration. In general none of the treatments affected psychrophilic or mesophilic TVC with the exception of CA (5 & 10%, w/v), where post-evisceration counts were significantly (P < 0.05) reduced by 0.88 log10 cfu cm-2 and 0.56 log10 cfu cm-2, respectively. None of the treatments significantly reduced TEC. This study provides further data supporting the application of cloacal washing but only as part of an overall package of measures designed to reduce Campylobacter contamination on broiler carcasses during processing.

The aim of this study was to investigate the molecular basis for observed high-level quinolone and macrolide resistance in poultry Campylobacter isolates. Seventeen Campylobacter isolates displaying high-level resistance to nalidixic acid, ciprofloxacin and/or erythromycin were investigated. Minimum inhibitory concentrations were initially determined using both the broth microdilution and E-test methods. The contribution of target gene mutations and active efflux to the observed resistances were then investigated using PCR and sequencing methods. High-level resistance to nalidixic acid was attributed to amino acid substitutions Thr-86-Ile and Asn-203-Ser in GyrA in some but not all isolates. Contrary to previous reports, the Thr-86-Ile substitution did not confer universal resistance to all quinolones. Strains displaying a high level of resistance to erythromycin carried the 23S rRNA transition mutation A2075G and/or carried mutations in the L4 and/or L22 ribosomal-encoding proteins. Interestingly and in contrast to previous studies, not all of the isolates carrying substitutions within the bhairpin region of the L22 ribosomal protein displayed erythromycin resistance. With the exception of a single isolate, efflux did not contribute to either quinolone or macrolide resistance. This study further expands our understanding of the molecular basis of quinolone and macrolide resistance in Campylobacter spp. and suggests that other factors, remaining to be elucidated, may also contribute to the resistant phenotypes observed.

Thirty-nine Shiga toxin-producing Escherichia coli (STEC) O113 Irish farm, abattoir, and clinical isolates were analyzed in conjunction with eight Australian, New Zealand, and Norwegian strains for H (flagellar) antigens, virulence gene profile (eaeA, hlyA, tir, espA, espB katP, espP, etpD, saa, sab, toxB, iha, lpfA(O157/OI-141,) lpfA(O113,) and lpfA(O157/OI-154)), Shiga toxin gene variants (stx(1c), stx(1d), stx(2), stx(2c), stx(2dact), stx(2e), stx(2f,) and stx(2g)) and were genotyped using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). All of the Irish strains were O113:H4, regardless of source, while all non-Irish isolates carried the H21 flagellar antigen. The stx(1) gene was present in 30 O113:H4 strains only, whereas the stx(2d) gene was common to all isolates regardless of source. In contrast, eaeA was absent, while hlyA was found in the Australian, New Zealand, Norwegian, and two of the Irish human clinical isolates. saa was present in the O113:H21 but not in the O113:H4 serotype. To the best of the author's knowledge, this is the first report of clinically significant STEC lacking both the eaeA and saa genes. PFGE analysis was inconclusive; however, MLST grouped the strains into three sequence types (ST): ST10, ST56, and ST223. Based on our findings, it was concluded that the stx(2d) gene is common in STEC O113, which are generally eaeA negative. Furthermore, STEC O113:H4 is a new, emerging bovine serotype of human clinical significance.

The objectives of this study were to evaluate current cleaning practices in broiler houses by testing a range of sites after cleaning and disinfection and to test the efficacy of the most commonly used methods in a commercial broiler house after flock harvesting. Cleaning procedures on 20 broiler houses (10 separate farms) were examined by testing a range of sampling points (feeders, drinkers, walls, etc.) for total viable count (TVC), total Enterobacteriaceae count (TEC) and Campylobacter spp. after cleaning and disinfection, using culture based methods. In a second experiment, the six most commonly used commercially available disinfectants and/or detergent products were evaluated. The results of the first study demonstrated that critical areas in 12 of the 20 broiler houses were not effectively cleaned and disinfected between flocks as the tarmac apron, ante-room, house door, feeders, drinkers, walls, columns, barriers and/or bird weighs were Campylobacter positive. Thermal fogging with the combination of potassium peroxymonosulfate, sulfamic acid and sodium chloride (5%, v/v) or the glutaraldehyde and quaternary ammonium complex (0.3%, v/v) were the most effective treatments while other disinfectant treatments were considerably less effective. It was therefore concluded that farmers should review their broiler house cleaning and disinfection procedures if Campylobacter cross-contamination between successive flocks is to be prevented.

Introduction : Campylobacter jejuni is the leading bacterial food-borne pathogen within the European Union, and poultry meat is an important vehicle for its transmission to humans. However, there is limited knowledge about how this organism persists in broiler litter and faeces. The aim of this study was to assess the impact of a number of environmental parameters, such as temperature, humidity, and oxygen, on Campylobacter survival in both broiler litter and faeces. Materials and methods : Used litter was collected from a Campylobacter- negative broiler house after final depopulation and fresh faeces were collected from transport crates. Samples were confirmed as Campylobacter negative according to modified ISO methods for veterinary samples. Both sample matrices were inoculated with 9 log 10 CFU/ml C. jejuni and incubated under high (≥85%) and low (≤70%) relative humidity conditions at three different temperatures (20°C, 25°C, and 30°C) under both aerobic and microaerophilic atmospheres. Inoculated litter samples were then tested for Campylobacter concentrations at time zero and every 2 hours for 12 hours, while faecal samples were examined at time zero and every 24 hours for 120 hours. A two-tailed t-test assuming unequal variance was used to compare mean Campylobacter concentrations in samples under the various temperature, humidity, and atmospheric conditions. Results and discussion : C. jejuni survived significantly longer ( P ≤0.01) in faeces, with a minimum survival time of 48 hours, compared with 4 hours in used broiler litter. C. jejuni survival was significantly enhanced at 20°C in all environmental conditions in both sample matrices tested compared with survival at 25°C and 30°C. In general, survival was greater in microaerophilic compared with aerobic conditions in both sample matrices. Humidity, at the levels examined, did not appear to significantly impact C. jejuni survival in any sample matrix. The persistence of Campylobacter in broiler litter and faeces under various environmental conditions has implications for farm litter management, hygiene, and disinfection practices. Keywords: humidity; temperature; aerobic; microaerophilic; broilers; atmosphere; Campylobacter jejuni (Published: 28 june 2016) Citation: Infection Ecology and Epidemiology 2016, 6: 31685 - http://dx.doi.org/10.3402/iee.v6.31685

Background : Campylobacter jejuni is the leading bacterial food-borne pathogen within the European Union (EU), and poultry meat is the primary route for transmission to humans . Material and methods : This study examined the impact of partial depopulation (thinning), season, and farm performance (economic, hygiene, and biosecurity) on Campylobacter prevalence in Irish broilers over a 13-month period. Ten caecal samples were taken per flock, for a total of 211 flocks from 23 farms during the duration of the study. Campylobacter was isolated and enumerated according to modified published ISO methods for veterinary samples. Biosecurity was evaluated through a questionnaire based on risk factors for Campylobacter identified in previous studies. Hygiene compliance was assessed from audit records taken over the course of 1 year. All information relating to biosecurity and hygiene was obtained directly from the processing company. This was done to ensure farmers were unaware they were being monitored for Campylobacter prevalence and prevent changes to their behaviour. Results and discussion : Farms with high performance were found to have significantly lower Campylobacter prevalence at first depopulation compared with low-performance farms across all seasons ( P ≤0.01). Peak Campylobacter levels were observed during the summer season at first thin in both the high- and low-performance groups. Campylobacter prevalence was found to increase to ≥85% in both high- and low-performance farms across all seasons at final depopulation, suggesting that Campylobacter was introduced during the first depopulation. On low-performance farms, four biosecurity interventions were found to significantly reduce the odds of a flock being Campylobacter positive (physical step-over barrier OR=0.17, house-specific footwear OR=0.13, absence of water body within 0.5 km OR=0.13, two or more broiler houses on a farm OR=0.16), compared with farms without these interventions. For high-performance farms, no single biosecurity intervention was identified as significant as this group had full compliance with multiple factors. High-performance farms had significantly better feed conversion ratios compared with low-performance farms (1.61 v 1.67 ( P ≤0.01)). No differences in flock mortality rates were observed ( P ≥0.05). This highlights the impact of season, biosecurity, partial depopulation, and farm performance on Campylobacter prevalence in Irish broilers. Keywords: Ireland; poultry; food safety; season ; Campylobacter; FCR; thinning (Published: 10 May 2016) Citation: Infection Ecology and Epidemiology 2016, 6: 31454 - http://dx.doi.org/10.3402/iee.v6.31454

Non-O157 Shiga toxin-producing Escherichia coli (STEC) strains are clinically significant food-borne pathogens. However, there is a dearth of information on serotype prevalence and virulence gene distribution, data essential for the development of public health protection monitoring and control activities for the meat and dairy industries. Thus, the objective of this study was to examine the prevalence of non-O157 STEC on beef and dairy farms and to characterize the isolates in terms of serotype and virulence markers. Bovine fecal samples ( n = 1,200) and farm soil samples ( n = 600) were collected from 20 farms throughout Ireland over a 12-month period. Shiga toxin-positive samples were cultured and colonies examined for the presence of stx 1 and/or stx 2 genes by PCR. Positive isolates were serotyped and examined for a range of virulence factors, including eaeA , hlyA , tir , espA , espB , katP , espP , etpD , saa , sab , toxB , iha , lpfA O157/OI-141 , lpfA O113 , and lpfA O157/OI-154 . Shiga toxin and intimin genes were further examined for known variants. Significant numbers of fecal (40%) and soil (27%) samples were stx positive, with a surge observed in late summer-early autumn. One hundred seven STEC isolates were recovered, representing 17 serotypes. O26:H11 and O145:H28 were the most clinically significant, with O113:H4 being the most frequently isolated. However, O2:H27, O13/O15:H2, and ONT:H27 also carried stx 1 and/or stx 2 and eaeA and may be emerging pathogens.

The aims of this study were to investigate the incidence of Salmonella, verocytotoxigenic Escherichia coli (VTEC)/Escherichia coli O157 and Campylobacter on four mixed farms and to characterize the isolates in terms of a range of virulence factors. Eighty-nine composite (five different samples from the same animal species combined) faecal [cattle (24), pigs (14), sheep (4), poultry (4), horses (7), deer (4), dogs (9), rodents (2) and wild birds (20)] samples, 16 composite soil samples plus 35 individual water samples were screened using culture-based, immunomagnetic separation and molecular methods. Salmonella was detected in bovine faeces, cattle and poultry house water. Salmonella serotypes/phage types included Dublin, Kiel and Typhimurium DT193, and most isolates were spvC, invA and rck positive. The pefA and rck genes were found exclusively in the non-Typhimurium strains, while Salmonella Dublin and Salmonella Kiel strains carried Salmonella genomic island I marker(s). VTEC/E. coli O157 were found in deer and dog faeces only. The E. coli O157 isolate was an enteroinvasive E. coli, while the VTEC isolate was untypable but carried the vt1, eaeA, hlyA, tir and eptD genes. This article reports the first confirmed carriage of E. coli O157 in Irish deer. Campylobacter species were not detected over the course of this study. It was concluded that [1] Salmonella, VTEC and Campylobacter have low (

A two-step real-time SYBR Green I multiplex polymerase chain reaction (PCR) assay with melting curve analysis was developed for rapid detection of 19 Salmonella serotypes frequently encountered in humans, animals, and animal-associated meat products within the European Union. The first-step single-tube reaction (Multiplex PCR I), consisting of five primer pairs, classified an initial test panel of eight Salmonella serotypes into five groups on the basis of characteristic amplicon melting temperatures produced by each strain. Following designation into groups, two subsequent triplex reactions (Multiplex PCR II-G1 and II-G3) allowed for further identification of five Salmonella serotypes by their melting peak temperatures. Primers for serotype differentiation were designed to target the genes encoding either phase 1 and 2 flagellar antigens fliC and fljB or unique serotype-specific loci. In addition, the assay simultaneously screened for the presence of the ampicilin-amoxicillin, chloramphenicol-florfenicol, streptomycin-spectinomycin, sulfanomides, and tetracycline (ACSSuT)-type multidrug resistance pattern, indicated by the floR gene, and for the Salmonella virulence plasmid encoded by the svp operon in Salmonella serotype Typhimurium. The established multiplex assays were successfully tested on 97 isolates, comprising 37 distinct Salmonella serotypes and 12 non-Salmonella strains. The two-step assay correctly detected 19 of 37 Salmonella serotypes and all non-Salmonella strains produced negative results. Of the 19 serotypes detected in the assays, 7 serotypes, including Salmonella serotypes Ohio, Goldcoast, Livingstone, Kedougou, Enteritidis, Kentucky, ACSSuT-type Salmonella serotype Typhimurium DT104 and DT104b, as well as non-ACSSuT-type Salmonella serotype Typhimurium strains, were definitively identified. The developed multiplex real-time SYBR Green I PCR assay represents a more rapid and reliable method for identification of large numbers of Salmonella serotypes prevalent throughout the European Union than assays using phenotypic serotyping methods.

Irish breeder and intensive broiler flocks together with the corresponding poultry farm environment were sampled for the presence of Campylobacter with the aim of identifying potential sources and transmission routes of poultry flock contamination. The genetic diversity of a subset of Campylobacter isolates was examined by analysis of the flaA-short variable region (SVR). Additional discrimination for a further subset of these isolates was achieved using multilocus sequence typing (MLST) analysis. Twenty-four flaA-SVR alleles and 15 FlaA peptides were detected among 92 Campylobacter jejuni and Campylobacter coli isolates. MLST data have been determined for 30 of 92 (32.6%) flaA-SVR-typed isolates with 13 sequence types (STs) present, which were assigned to seven clonal complexes. ST45 was the most common ST identified. Vertical transmission was not found to have played a role in transmission of the pathogen to the poultry flocks. Subtyping by flaA-SVR and MLST identified the practice of partial thinning of flocks as a potential source and route of flock contamination on one broiler farm and implicated a probable source of flock contamination on another. Although there have been several studies reported in the scientific literature, the findings from this study confirmed previous studies and suggested some new transmission pathways including via transport crates. Cross-contamination from adjacent cattle is a new development, and molecular evidence of the role of transport crates in introducing Campylobacter spp. into the broiler house is a recent finding. Further, this study reports the discovery of five new flaA-SVR allele types and eight new STs. These were widespread and persistent in the poultry environment. This new knowledge may explain why despite the on-farm Campylobacter data published to date, there are still no completely effective on-farm control measures to prevent Campylobacter contamination of broiler flocks.

Aims: To investigate the effect of diet on the survival of Salmonella in the bovine abomasum. Methods and Results: Five fistulated cows were randomly assigned to one of five diets denoted as: (i) 100% grass, (ii) grass + 5·3 kg DM concentrate, (iii) 100% grass silage, (iv) 100% hay and (v) maize/grass silage plus concentrates. Rumen fluid was harvested from each dietary treatment and inoculated with nonacid (NA) and acid-adapted (AA) 5-strain Salmonella cocktails. After 24-h incubation period, Salmonella were acid challenged to synthetic abomasum fluid (SAF, pH 2·5) for 5 h to determine their resistance to low pH. The study found that the volatile fatty acids composition and the pH profile of bovine rumen fluid were significantly altered (P

Aim: The aim of this study was to investigate the prevalence of Campylobacter species in a subset of intensive poultry flocks by examining samples collected in geographically disparate areas on the island of Ireland. Methods and Results: Faecal, water and environmental samples were collected from the interior of poultry houses on nine farms. Three cultural methods were used for Campylobacter isolation: direct plating, enrichment culture and a recovery method for emerging Campylobacter spp. Presumptive Campylobacter isolates were confirmed using biochemical tests and further identified to species level by multiplex PCR. All flocks sampled in this study were found to be contaminated with Campylobacter at the time of sampling. Structural and air samples taken from the interior of broiler houses were also found to be Campylobacter positive. All water samples were found to be Campylobacter negative. The Campycheck method was used for the isolation of emerging Campylobacter spp. Conclusions: Campylobacter spp. were recovered (as contaminants) from the poultry house interior, air and environmental samples in all intensive poultry flocks surveyed. Significance and Impact of the Study: This study highlights the need for improved biosecurity on selected poultry farms.