Genomic diversity, virulence and source of Campylobacter jejuni contamination in Irish poultry slaughterhouses by whole genome sequencing.

AIMS: The aim was to exploit whole genome sequencing (WGS) to assess genomic diversity, identify virulence genes and deduce the proportion of Campylobacter colonized broilers that directly contaminate their carcasses. METHODS AND RESULTS: Campylobacter jejuni isolates (107) from caeca and carcass neck skin samples (50 pairs from the same batch plus 7 individual caeca) sampled at three poultry slaughterhouses over a one-year period were selected for sequencing (MiSeq; Illumina). FastQ files were submitted to BioNumerics for analysis using the wgMLST scheme for allele calling. Campylobacter cgMLST and hierarchical clustering was performed by applying the single linkage algorithm. Sequence types (STs) were determined in silico from the WGS data and isolates were assigned into clonal complexes (CCs) using the Campylobacter PubMLST.org database. Virulence genes were determined by downloading core sequences from the virulence factor database (VFDB) and the National Center for Biotechnology Information (NCBI). A high degree of diversity was observed with 23 different STs identified. ST257 and CC-21 were the most common STs and CCs, respectively. cgMLST analysis suggested that 56% of carcass contamination was a direct result of contamination from caeca from the same batch. Virulence genes known to play a role in human C. jejuni infection were identified such as the wlaN gene and the genes associated with lipooligosaccharide synthesis, which were identified in 30% of isolates. CONCLUSIONS: Caecal colonization was the more plausible occurring source of C. jejuni contamination of broiler carcasses, compared with cross-contamination from another batch or the environment. The high rate of genetic diversity observed amongst caecal isolates is consistent with a wide variety of Campylobacter strains circulating in poultry flocks in Ireland. SIGNIFICANCE AND IMPACT OF STUDY: The results will further inform broiler processors and regulators about the influence and importance of on-farm colonization versus slaughterhouse cross-contamination and the relationship between C. jejuni in caeca and carcasses during processing.

The impact of key processing stages and flock variables on the prevalence and levels of Campylobacter on broiler carcasses.

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.

Towards the development of day one competences in veterinary behaviour medicine: survey of veterinary professionals experience in companion animal practice in Ireland.

BACKGROUND: Veterinary behaviour medicine should be a foundation subject of the veterinary curriculum because of its wide scope of applications to veterinary practice. Private practitioners are likely to be the primary source of information on animal behaviour for most pet owners, however studies indicate that behavioural issues are not frequently discussed during companion animal consultations and many practitioners lack confidence in dealing with behavioural problems, likely due to poor coverage of this subject in veterinary education.There is a need to identify learning outcomes to support day one competences in veterinary behaviour medicine and these should be informed by practice-based evidence. This study aimed to investigate the nature and frequency of behavioural queries experienced by veterinary professionals in Ireland, the provision of behavioural services at companion animal practices, behaviour referral practices and challenges associated with providing a behaviour service. METHODS: Two online surveys were developed, one for private veterinary practitioners (PVP) and one for veterinary nurses (VN). Invitations to participate were distributed using contact details from the Premises Accreditation Scheme database on the Veterinary Council of Ireland website. Thirty-eight PVPs and 69 VNs completed the survey. RESULTS: Results indicated that less than half of companion animal practices offer behavioural consults and under a third of practices provide training and socialization events. Over half of the practices surveyed have referred cases to a behavioural specialist.The majority of respondents encountered behavioural queries weekly. Ninety-eight percent reported receiving queries regarding dog behaviour. Toilet training and unruly behaviour were two issues encountered frequently. Behavioural issues in cats were also common. House soiling and destructive behaviour were the problems most frequently encountered by respondents.The two most commonly cited barriers to providing behavioural consultations were lack of in-house or personal expertise, and that clients were not willing to pay for these services.Furthermore over half of all veterinary professionals surveyed indicated that they had received inadequate undergraduate training in veterinary behaviour medicine. CONCLUSIONS: Behavioural problems in companion animals can affect the quality of life of pets and their owners. Our survey findings indicate that many veterinary professionals frequently encounter behavioural problems and identify an opportunity for improved provision in behaviour medicine in veterinary education.

Implementation of a national monitoring programme of Campylobacter in Irish broilers to measure progress of on-farm and primary processing control measures.

BACKGROUND: Campylobacter is the most common food-borne pathogen in the European Union. In 2018, the crude incidence rate in Ireland was 63.6 per 100,000 population. Chicken is considered an important source of infection for humans. In 2015, the Campylobacter Stakeholders' Group (CSG) was established to reduce Campylobacter contamination levels in Irish broiler flocks. AIMS: This work aimed to describe the Campylobacter monitoring programme that was established by the CSG, to analyse the results of this testing between 2019 and 2022, and to assess progress. METHODS AND RESULTS: This paper describes the monitoring programme that was established by the CSG, which harmonized Campylobacter enumeration testing across all Irish broiler processors and allowed comparability of results for trend analysis. An analysis of the 2019-2022 data is presented here and compared to previous studies of Campylobacter levels in Irish broilers. An analysis of the 2019-2022 data showed a significant reduction in levels in both caeca and neck skin when the results from 2022 were compared to those from 2019 to 2020. Campylobacter spp. were detected in 37% of caecal samples from first depopulation (pre-thin) broilers and 30% of neck skin samples in 2022, with just 4% of carcases (in neck skin samples) with ≥1000 colony-forming units per gram detected in 2022. Campylobacter levels detected in Irish broilers, in the present monitoring programme were less than those reported in previous studies in both caecal and carcase samples, although not directly comparable for statistical significance because of differences in study methods. CONCLUSIONS: The cooperation between stakeholders and regulators of the Irish broiler chicken industry over the past decade has facilitated a coordinated approach to monitoring of Campylobacter levels in broilers, and implementation of control measures. This has enabled a steady reduction in the levels of Campylobacter in Irish chicken.

Evaluation of methods for detection of Campylobacter in raw milk: A multi-country study.

Raw milk is considered a high-risk source of Campylobacter due to faecal contamination from healthy cattle and farm environments, thus linking raw milk consumption to global outbreaks. Detection of Campylobacter in raw milk poses challenges due to low contamination levels and antibacterial properties of the milk. Culture-based protocols for Campylobacter detection in milk vary, mainly with regard to pH adjustment and the choice of enrichment broth. This European collaborative study was organised by the EU Reference Laboratory (EURL) for Campylobacter together with eight EU National Reference Laboratories (NRL) for Campylobacter with the purpose to evaluate methods for culture-based detection of Campylobacter in raw cow's milk. The study was divided into two parts, an interlaboratory part and an intralaboratory part, both organised around the same two protocols. The aim of protocol 1 was to evaluate the impact of pH adjustment and storage of the milk on the culturability of Campylobacter over time. Aliquots of the spiked milk were adjusted either to pH 7.0 or pH 7.6 or left unadjusted. The milk was stored up to 48 h at refrigerated temperature and Campylobacter was quantified according to ISO 10272-2 on day 0, 1 and 2. The aim of protocol 2 was to evaluate which enrichment broth, Bolton broth (BB) or Preston broth (PB), showed highest sensitivity in detection of Campylobacter. The spiked milk was enriched in BB and PB as described in ISO 10272-1:2017 or ISO 10272-1:2017/Amd1.2023. In the interlaboratory part, each milk batch was collected locally by each participating NRL/EURL and inoculated with the same Campylobacter strain. In the follow-up intralaboratory part, the EURL-Campylobacter repeated the tests in protocol 1 and 2 but used different Campylobacter strains and strains subjected to thermal stress prior to inoculation. The results show that pH adjustment of raw milk has a negligible impact on culture-based detection of Campylobacter, regardless of strain and level of environmental stress. The composition of milk and properties of the inoculated strain influence culture-based detection of Campylobacter over storage time, and strains subjected to additional stress prior to inoculation in milk are reduced in culturability much faster than the same strains prepared under normal conditions. Finally, the study showed that PB without Campylobacter growth supplement is less effective than BB in detecting Campylobacter in raw milk.

A coprological survey of parasites of wild carnivores in Ireland.

The increasing movement of people to wilderness areas, shrinking of wildlife habitats and the resulting urbanisation of wildlife has led to growing concerns about the transfer of parasitic diseases, particularly from contaminated faeces. Faecal samples from wild carnivores in Ireland were examined for the presence of protozoan and nematode parasites. Red fox (Vulpes vulpes) samples (n = 91) were positive for Uncinaria stenocephala (38%), Eucoleus aerophilus (26%), Toxocara canis (20%), Trichuris vulpis (4%) and Isospora-like oocysts (9%). Badger (Meles meles) samples (n = 50) were positive for Uncinaria criniformis (40%), E. aerophilus (6%) and Isospora-like oocysts (16%). No parasites were observed in pine marten (n = 48; Martes martes) faeces. Approximately 5% of American mink (Mustela vison) samples were positive for Cryptosporidium by polymerase chain reaction (identified as Cryptosporidium andersoni (n = 3) and 'mink' genotype (n = 1)). The results suggest that wild carnivores in Ireland have a range of parasites, although it is unclear from the present study to what extent these infections are associated with morbidity. While it can be expected that, via their faeces, wild carnivores contribute to the spread of these parasites, they are unlikely the primary source of environmental contamination. Therefore, they should not always be the principal target of control measures.

Preliminary Data on the Occurrence of Anisakis spp. in European Hake (Merluccius merluccius) Caught Off the Portuguese Coast and on Reports of Human Anisakiosis in Portugal.

Parasitic nematodes of the genus Anisakis are among the most important biological hazards associated with seafood. A survey of Anisakis spp. in European hake (Merluccius merluccius) was undertaken as this species is a staple of the Portuguese diet. Moreover, a literature review of cases of anisakiosis reported from Portugal, a country with one of the highest levels of fish consumption in the world, was also carried out. Seventy-five European hake caught in the Atlantic Ocean off the northern coast of Portugal were analyzed to determine the infection levels and site distribution of Anisakis spp. Isolated nematode larvae were identified to species level by molecular analysis. Two sets of samples were collected. Firstly, a total of 46 Anisakis spp. L3 larvae were collected with a prevalence of 76.7% (95% CI 61.5-91.8%) and intensity (mean ± SD, range) of 2.0 ± 1.2 (1-5). Most larvae were found on the liver (45.7%) and on the gonads (32.6%), but none in the muscle. The molecular analysis showed the presence of both A. simplex s.s. (70%) and A. pegreffii (30%). For the second sample, analyzed using the UV-Press method, a total of 473 Anisakis spp. were found, with a prevalence of 95.6% (95% CI 89.5-100.0%), intensity (mean ± SD, range) of 11.3 ± 9.7 (1-41), density of 0.05 ± 0.04 (0-0.16) worms/muscle weight in g, and density of 0.54 ± 0.50 (0-2.53) worms/viscera weight in g. Surprisingly, only three very recent cases of human anisakiosis in Portugal have been reported in the literature. Data from this study contribute towards an updating of the existing epidemiological picture in an area characterized by very high seafood consumption and changing eating habits.

Prevalence and levels of Campylobacter in broiler chicken batches and carcasses in Ireland in 2017-2018.

In 2008, an EU wide baseline survey of broilers revealed a high Campylobacter prevalence. To assist with industry-wide controls, updated data were required. The primary objective of this study was to establish up-to-date data on Campylobacter carriage and carcass contamination in Irish broilers. Monthly samples were collected from the three largest broiler processing plants in Ireland over a twelve-month period. Samples were taken from both first and final thin birds (partial and full depopulation) from 358 batches of broilers. From each batch, a composite sample of 10 caecal contents (n = 358) and 5 neck skins (n = 1790) were collected and numbers of Campylobacter in each sample were determined. Of the 1790 neck skin samples tested, 53% were Campylobacter positive. Campylobacter was detected in the caecal contents of 66% of all batches tested. Depopulation and/or age had a significant effect on Campylobacter prevalence with 67% of final thin broilers yielding Campylobacter-positive neck skin samples in contrast to 38% of first thin broilers that yielded positive neck skin samples (P ≤ 0.002). A significant seasonal variation was observed in the rate of Campylobacter-positive caecal samples with higher prevalence seen in July (85%) than the colder months of November (61%), December (50%), January (61%) March (57%) and April (59%). Neck skin samples were 7 times more likely to be Campylobacter positive if the caecal contents from the same batch were positive (odds ratio = 7.1; P ≤ 0.0001). The decrease in Campylobacter prevalence observed in neck skin and caecal contents demonstrates the improvements and progress made in reducing prevalences of this important enteropathogen in the Irish poultry industry since the 2008 EU baseline survey. It also provides further supporting data on the impact of thinning, the processing environment and season on Campylobacter prevalence.

Experimental Fasciola hepatica infection alters responses to tests used for diagnosis of bovine tuberculosis.

Fasciola hepatica is a prevalent helminth parasite of livestock. Infection results in polarization of the host's immune response and generation of type 2 helper (Th2) immune responses, which are known to be inhibitory to Th1 responses. Bovine tuberculosis (BTB) is a bacterial disease of economic and zoonotic importance. Control polices for this disease rely on extensive annual testing and a test-and-slaughter policy. The correct diagnosis of BTB relies on cell-mediated immune responses. We established a model of coinfection of F. hepatica and Mycobacterium bovis BCG to examine the impact of helminth infection on correct diagnosis. We found the predictive capacity of tests to be compromised in coinfected animals and that F. hepatica infection altered macrophage function. Interleukin-4 and gamma interferon expression in whole-blood lymphocytes restimulated in vitro with M. bovis antigen was also altered in coinfected animals. These results raise the question of whether F. hepatica infection can affect the predictive capacity of tests for the diagnosis of BTB and possibly also influence susceptibility to BTB and other bacterial diseases. Further studies on the interplay between helminth infection and BTB are warranted.