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Study of Salmonella, Toxoplasma, Hepatitis E virus, Yersinia, Porcine Reproductive and Respiratory Syndrome virus, antimicrobial resistance in Campylobacter and extended spectrum beta lactamase E. coli in UK pigs at slaughter Section 8: Project Report to Defra The objectives of project OZ0150, a four month prevalence study of Salmonella and of Toxoplasma, Hepatitis E virus (HEV), Yersinia, Porcine Reproductive and Respiratory Syndrome virus (PRRSv), Antimicrobial Resistance (AMR) in Campylobacter coli and E. coli, and Extended Spectrum Beta Lactamase (ESBL) E. coli in UK pigs at slaughter, have been met. Details of the objectives, as set out in the contract, are given in Appendix I. A peer-review publication on the prevalence findings from project OZ0150 will be taken forward by Animal Health and Veterinary Laboratories Agency (AHVLA), a separate short communication on ESBL E. coli and a paper on the HEV results will be prepared by AHVLA and Public Health England (PHE) respectively, otherwise there are no outstanding actions (IP, Knowledge Exchange) resulting from this research. Isolates and samples have been stored for further work. The following report is prepared, albeit in more detail, in the format of a draft publication: Introduction This paper describes a prevalence study of seven important organisms and antimicrobial resistance (AMR) in certain bacteria in UK pigs, several of which are of public health significance. This work extended a prevalence study for Salmonella in pigs, to take the opportunity to estimate the prevalence of Toxoplasma, HEV, Yersinia, PRRSv and ESBL E. coli and to investigate the extent and pattern of AMR in Campylobacter coli. This is the first UK-wide prevalence estimate for Toxoplasma, HEV, PRRSv and ESBL E. coli in slaughter pigs. Salmonella A series of prevalence surveys of poultry and pigs have been conducted within the European Union (EU) over the last decade with the aim of obtaining baseline and comparable data for all Member States concerning foodborne zoonoses of interest; two of these surveys, conducted in 2006/07 and 2008, respectively, focused on Salmonella in finisher pigs and breeding herds (Commission Decision2005/636/EC and 2006/668/EC, respectively). The results from finishing pigs showed that UK levels of Salmonella were above the EU average with a prevalence from lymph nodes of 21.8% and carcase contamination of 15.1% (versus 10.3% and 8.3%, respectively, across the EU) (EFSA Journal, 2008; Marier et al., 2008). Salmonella is the second most commonly reported cause of food poisoning, behind Campylobacter, in the UK. In 2009, 108,614 human cases of salmonellosis were reported in the EU (EFSA, 2011a). Under-ascertainment of infectious intestinal disease is well recognised, hence the true population burden of infections is likely to be far greater (Tam et al., 2011; Wheeler et al., 1999). There has been a reduction in the number of reported human cases of Salmonella over the past five years, which is in part due to the successful implementation of Salmonella national control plans in the poultry sector. However, given the reduction in risk from poultry meat and eggs, the role of pork and pork products and the

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relative number of human cases of salmonellosis attributed to such products may rise, even though the actual numbers may change little. Pork and pork products have been identified as a significant source of Salmonella, with an estimated 22% of cases in the Netherlands and 14% of cases in Denmark potentially attributed to this source (EFSA, 2006). More recently, the European Food Safety Authority (EFSA) estimated that over 50% of Salmonella infections across the EU may be attributable to pork (EFSA, 2012) and for the UK the contribution has been estimated at 11.7% (Pires et al., 2012). Planning of the study reported here began with the decision to test for Salmonella, in preparation for an anticipated future EU-wide national control programme (NCP) for Salmonella in pigs at slaughter (Paiba et al., 2011). The aim of an NCP together with the application of the microbiological criteria in foodstuffs regulations is to protect public health by reducing the risk of foodborne disease due to Salmonella in consumers from pig meat and associated products. However, testing in the study reported herein was expanded, by cross agency collaboration, to include other potentially zoonotic pathogens of humans: Toxoplasma, HEV, Yersinia, ESBL E. coli and AMR in Campylobacter. The addition of PRRSv, which is not a zoonosis, to the testing portfolio was approved in March 2013. Toxoplasma The intra-cellular protozoan, Toxoplasma gondii, is widely prevalent in humans, warm-blooded animals and birds throughout the world (VanWormer et al., 2013). An estimated 350,000 people become infected with Toxoplasma each year in the UK, of which 10-20% are symptomatic (ACMSF, 2011). Although the clinical signs are usually mild, infection can be associated with serious sequelae including eye disease and disability. People who are immunocompromised and pregnant women newly infected with Toxoplasma are particularly vulnerable; in the latter, miscarriage, stillbirth and deformities of the child can occur. Tissue cysts are highly infectious for humans and other animals and, in addition to direct transmission from cat faeces or material from aborting sheep, undercooked meat has been identified as an important source of human infection. In the recent ACMSF report on Toxoplasma, it was noted that there is little data on Toxoplasma meat contamination in the UK and “further data on seroprevalence in farm animals would be useful in monitoring the effectiveness of control measures in animal husbandry and testing of a larger range of meat samples would be useful in identifying the main sources of risk. Further studies are therefore recommended to establish seroprevalence in UK livestock species”. A recent serological survey has been carried out in sheep (Hutchinson et al., 2011) but there have not been any UK-wide surveys of Toxoplasma in pigs. Hepatitis E virus The hepatitis E virus (HEV) is one of the five viruses in humans which principally cause hepatitis (an inflammation of the liver). The clinical illness in humans is called hepatitis E. Within the family HEV there are four genotypes, identified as G1-4, based upon sequence phylogeny. Each genotype has different hosts, causes different patterns of disease of varying severity and has different global distribution (Aggarwal and Naik, 2009). Within each genotype viruses differ and it is possible to separate them into recognisable groups,

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sometimes referred to as sub genotypes though the significance of subgenotyping is now called into question (Smith et al., 2013). Genotype 1 is found through Asia and Africa and infects humans only Genotype 2 is found in Mexico and parts of Africa and infects humans only Genotype 3 is found throughout the world and infects both humans and animals Genotype 4 is found through South-East Asia and infects both humans and animals Until recently hepatitis E has been considered in the UK as an exotic infection only acquired by travel to those areas of the world where HEV is endemic. It is now recognised that the majority of cases of hepatitis E occur in people who have not travelled outside the UK, these are by definition indigenous to the UK. Public Health England has had an enhanced surveillance programme running in England and Wales since 2003 to investigate cases of hepatitis E (Ijaz et al., in press). In developed countries, including the UK, it is now widely accepted that the virus in humans represents a zoonosis acquired through the consumption of undercooked or raw meat and also perhaps consumption of shellfish (Cacopardo et al., 1997; Said et al., 2009). For reasons which are unclear secondary spread of HEV from person to person is very rare. Since 2010, there has been in England and Wales a year on year increase in the number of patients with hepatitis E. In 2012 there were 579 cases, with the majority (71%) occurring in people who have not travelled outside England and Wales. All of these cases were infected with genotype 3 (G3) HEV (Ijaz S et al., in press). Further data in support of a widespread infection burden in the UK come from the modelling of antibody prevalence data from a study in the general population (Ijaz et al., 2009) and from on-going analysis of HEV incidence data in blood donors (Beale et al., 2011). Both data sets indicate that there are likely to be at least 60 000 new human HEV infections each year in England but that very few of these cause disease in people. More importantly since 2010 there has also been a change in the genetic profile of G3 viruses in cases of hepatitis E to the extent that there are now two groups within G3 viruses, an original group 1 present from the time sequencing was undertaken in the mid 2000s and the emergent increasingly common group 2 which presently accounts for two thirds of the clinical G3 cases (Ijaz et al., in press). Genotypes 3 and 4 are principally animal viruses and are most commonly found infecting pigs, deer and wild boar. Consumption of inadequately cooked meat from these three species has been recognised as sources of infection in humans (Colson et al., 2010; Tei et al., 2003; Masuda et al., 2005). Routine sequence analysis of infections in humans identified by diagnostic testing and their phylogenetic analysis can give information on potential sources of infections, especially when viruses are closely related genetically (Meng et al., 1997). As such, phylogenetic analysis forms an essential part of the current enhanced surveillance of hepatitis E undertaken by PHE. In a previous study of one UK pig herd, HEV seroprevalence (anti-HEV antibody) was roughly estimated to be 85% for the 40 pigs tested (Banks et al., 2004). In another UK study there was evidence of HEV RNA in sausages (6 out of 63 sausages tested) on sale in the UK (Berto et al., 2012). Five of the six positive sausages were in one of the 11 batches of sausages tested. A more recent publication in autumn 2013 of HEV cases in England and Wales showed that consumption of processed pork products is significantly associated with an increased risk of acquiring hepatitis E (Said et al., 2013). These observations were important drivers to the study of HEV in British swine, including the enumeration undertaken in this study.

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Yersinia spp. Yersiniosis caused by the bacterium Yersinia enterocolitica is a zoonotic gastrointestinal disease. Infection with Y. enterocolitica can cause fever, abdominal pain and diarrhoea. Chronic illness and reactive arthritis have also been reported as sequelae of Y. enterocolitica infection (Porter et al., 2013; Saebø and Lassen, 1992). The number of confirmed human cases of Y. enterocolitica and other Yersinia spp. in the UK has declined in recent years with 55 confirmed cases in 2012 (Health Protection Scotland data, PHE data, UK Zoonoses Report 2012). The number of cases in the UK are low compared to other European countries due to the low consumption of raw pork in the UK (Rosner et al., 2010). Pigs are considered to be the primary reservoir of human pathogenic Y. enterocolitica strains, mainly because of the high prevalence of such strains in pigs and the high genetic similarity between human and porcine isolates (Fredriksson-Ahomaa et al., 2006). Estimates of Y. enterocolitica carriage in pigs have been reported previously as 1.8% (O’Sullivan et al., 2011), 10.2% (Milnes et al., 2008), 10.8% (Bonardi et al., 2013), 19.5% (Liang et al., 2012) and 26.1% (McNally et al., 2004). Yersinia was identified in the recent EFSA opinion on meat inspection in pigs as one of the four major public health hazards (EFSA, 2011b) and Member States are recommended to gather prevalence data for pigs at slaughter at regular intervals (EFSA, 2009a), hence the data collected by this study will help to address this data gap. Porcine reproductive and respiratory syndrome virus Porcine reproductive and respiratory syndrome virus (PRRSv) was first confirmed in the UK in 1991 and is now considered endemic (Edwards et al., 1992). In 2003/2004, a cross sectional study of 103 British pigs found that 35 herds (34.0%) were seronegative, 41 (39.8%) were seropositive and 27 (26.2%) were vaccinated (Evans et al., 2008). A more recent study by Velasova et al. (2012) estimated that there was active PRRSv circulation on 35.1% of farms. Porcine reproductive and respiratory syndrome virus is not zoonotic. However, the economic impact of this disease to pig farming is significant with direct and indirect costs associated with production losses, increased mortality, treatment, and disruption to breeding programmes. The cost of PRRSv to a 500-sow herd in the first year of infection has been estimated at £52,000 and £94,000 in the growing herd and breeding herd respectively (Richardson, 2011). Better understanding of the current prevalence and the epidemiology of PRRSv in the UK is needed in order to target control measures appropriately and assess the effectiveness of interventions. Antimicrobial Resistance in Campylobacter coli Campylobacter is a key area for public health targeting. The number of confirmed cases of human campylobacteriosis in the EU has shown a statistically significant increasing trend between 2008 and 2011 (EFSA and ECDC, 2013). There has also been a rise in the number of confirmed cases reported in England and Wales, from 44,544 in 2004 to 64,608 in 2011, which represents an increase of 44% (PHE data). Campylobacter jejuni and Campylobacter coli are the most common species to cause human infections, with approximately 7% of human infections caused by the latter in England and Wales (Gillespie et al., 2002). To date, studies indicate that the majority of infections are due to poultry, poultry handling or poultry products (Sheppard et al., 2009). Although a previous slaughter pig survey reported a high prevalence of thermophilic Campylobacter in caecal contents (Milnes et al., 2008),

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multi Locus Sequence Typing (MLST) of C. coli isolates has shown that pig sequence types are less commonly found in humans, with only 6% of human C. coli cases estimated to be attributable to pigs (Roux et al., 2013). Directive 2003/99/EC, requires EU Member States to provide comparable monitoring data on the occurrence of antimicrobial resistance in zoonotic bacteria. Detailed specifications for carrying out such harmonised monitoring on AMR have been prepared by EFSA (EFSA, 2007). The specifications include a list of antimicrobials and epidemiological cut-off values (discriminating between wild type bacteria and those showing any acquired resistance) to be used, as well as a specified concentration range to determine the susceptibility of Campylobacter to the antimicrobials tested. The target number of Campylobacter isolates to be included in the antimicrobial resistance monitoring from each Member State is 170 per year for each study population. The results of the antimicrobial resistance monitoring are assessed and reported in the annual EU Summary Report on Antimicrobial Resistance (available on the EFSA website). This study provided an opportunity to obtain C. coli isolates for AMR testing and reporting to the EU so as to comply with the harmonised requirements. The method used was not the same as performed in previous studies. Extended Spectrum Beta Lactamase (ESBL) E. coli The monitoring and reporting of AMR data from Escherichia coli within the EU is encouraged but voluntary. The emergence of E. coli with resistance to extended spectrum β-lactam antibiotics (ESBLs) has been reported in farm animals both in the UK (Horton et al., 2011; Randall et al., 2011; Snow et al., 2011; Teale et al., 2005; Watson et al., 2012) and other countries such as Belgium, China, France and The Netherlands (Smet et al., 2008; Costa et al., 2004; Costa et al., 2006; Costa et al., 2009; Dierikx et al., 2010; Liu et al., 2007; Meunier et al., 2006). Farm animals can harbour different types of ESBL genes, carried by various bacteria including E. coli, although the CTX-M types are currently usually the most prevalent ESBL gene type. CTX enzymes confer resistance to β-lactam antibiotics such as amoxicillin, but also to β-lactam antibiotics that are 3rd and 4th generation cephalosporins, such as cefotaxime, ceftazidime and cefquinome (Livermore et al., 2007), and hence the name “extended spectrum”. As cephalosporin antibiotics are important front-line antibiotics for use in humans, selection of resistant bacteria in animals to these antibiotics is of concern (Collignon et al., 2009). In a longitudinal study on three single-sited farrow-to-finisher ESBL positive pig farms in Denmark, 50% of pigs were positive for cefotaxime-resistant coliforms immediately after birth, 58% were positive just before weaning, 29% were positive during weaning and 12% were positive during finishing (Hansen et al., 2013). Whilst a longitudinal study of pigs has not been carried out in the UK, the Danish results are of interest as a comparison to our study. In a pilot study in Switzerland, 15.2% of 59 pigs were positive for ESBL bacteria, with mainly CTX-M 1 ESBL type, but also some group 9 CTX-M isolates (Geser et al., 2011).The presence

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of bacteria containing the CTX-M ESBL gene was also observed in 558 swine faecal samples in Korea (21.5%), with the most predominant CTX-M type identified as CTX-M-14 (n=82), followed by CTX-M-15 (n=16). Isolates producing CTX-M-3, CTX-M-27, CTX-M-55, and CTX-M-65 were also identified (Tamang et al., 2013). In a Spanish study, ESBL isolates were characterised as having SHV-12 ESBL genes (41% of isolates), CTX-M-1 ESBL genes (10% of isolates), CTX-M-9 ESBL genes (10% of isolates) and CTX-M-14 ESBL genes (10% of isolates) (Escudero et al., 2010). The Spanish study did not monitor for the prevalence of ESBL positive bacteria from pigs, but used isolates from a previous study where 36% (29/80) of pooled faecal samples from pigs gave rise to E. coli from agar containing 1 mg/L cefotaxime (Moreno et al., 2007). Studies have shown that ceftiofur and cefquinome can both select for CTX-M producing strains in pigs (Cavaco et al., 2008). Conversely, there is evidence that a voluntary ban on the use of cephalosporins in pigs in Denmark in 2010 had a positive impact on the occurrence of ESBL E. coli in both herds and pigs at slaughter (Agersø et al., 2013). Since 2008 approximately 20 ESBL E. coli isolates have been recovered from clinical diagnostic specimens from pigs in England and Wales as part of routine scanning surveillance at AHVLA (AHVLA unpublished observations). However, this is the first study in the UK in which the prevalence of ESBL bacteria from healthy pigs has been determined. Methods Study design The study design was consistent, where possible, with the technical specifications for the previous EU monitoring scheme for Salmonella in slaughter pigs (Commission Decision 2006/668/EC). The target sample size of 600 pigs was based on sample size calculations from the EU monitoring scheme for Salmonella in slaughter pigs, which used an estimated prevalence of 50% with an accuracy of 4% and 95% confidence (EFSA 2008a). Additional sample size calculations were undertaken prior to the present study which showed that by sampling approximately 600 pigs at slaughter it would be possible to determine (with 80% power and 95% confidence) a 20% overall reduction (or increase) in Salmonella prevalence or a 40% reduction/ increase in carcase contamination. In anticipation of non-responses or inadequate samples, a further 10% of pigs were scheduled for sampling. The study was managed and run by AHVLA in close collaboration with the Food Standards Agency (FSA). Abattoir recruitment and schedule of sampling Sampling was scheduled to take place between 14th January 2013 and 12th April 2013. The three month sampling period was determined by the funders. The study sought to include the high throughput abattoirs i.e. those representing 80 % of all slaughtered finishing pigs. Approved pig abattoirs in the UK were recruited to the study by the British Pig Executive (BPEX) and the FSA organisations in Great Britain and Northern Ireland. Sampling was weighted so that the number of carcases to sample in each of the selected abattoirs was proportional to the throughput of the abattoir. The sampling schedule was randomized so that the day of sampling and the carcase to be sampled on a given day was based on a random selection. The sampling day within each month was randomly chosen

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from the days the selected slaughterhouse was usually open. The individual carcase to be sampled was randomly chosen from the total number of carcases that the selected slaughterhouse processed daily. Reserve carcases were also scheduled in case the first selected carcase was not suitable for sampling/ could not be sampled. The procedure for selecting the random samples was set up in Microsoft Excel. The total number of carcases to be sampled was stratified by calendar month. Details on the sampling days are given in Appendix II. For example, Abattoir J was assigned 48 carcases during the study period – this was based on the slaughter kill throughput of the abattoir. The 48 carcases were stratified by calendar month and so they were assigned 16 carcases to sample per month. Within each month the sampling date for each of the 16 carcases was randomly assigned – as a result, the abattoir was sometimes scheduled to sample more than one carcase on a given day. The abattoir undertook sampling of the 48 carcases on 34 days. All randomisation procedures were carried out by the Epidemiology Surveillance and Risk Group (ESRG), AHVLA Weybridge. Sample collection Samples were collected by trained staff of the FSA in Great Britain and by the Veterinary Public Health Unit of the Department of Agriculture and Rural Development (DARD) in Northern Ireland. Nine samples were collected along the processing line from each selected carcase: one rectal swab immediately post-stun; two blood samples post-bleed; whole caecum, tonsils, whole heart and tongue at the evisceration point; and two carcase swabs at pre-chill. One carcase swab was taken on the left or right side of the carcase using one single sponge for all four sites described in Annex A of Standard ISO 17604 (hind limb, abdomen, mid-dorsal region, jowl). The second carcase swab was taken, using the same sites, but on the opposite side of the carcase. Each sample was separately packaged and labelled with a unique identifier. After collection, samples were chilled and transported by courier to AHVLA Bury as quickly as possible in an insulated shipping box specifically designed for the survey which maintained the temperature of the contents between +2oC and +8oCfor up to 72 hours. Samples were kept refrigerated until bacteriological examination. Data collection A standardized data collection form, labelled with the same unique identifier as the samples, was completed by trained personnel after sampling. The information collected related to the abattoir processes and sampled carcase, including slaughtering speed, singeing time, scalding temperature, the sampling date and time, carcase weight, and the name and address of the farm of origin. Eligibility criteria All samples taken were from carcasses deemed fit for consumption by the Competent Authority. The exclusion criteria were as follows: any carcase that was totally condemned; animals with a live weight of less than 50kg; animals that had undergone emergency slaughter; and animals kept in the UK for less than 3 months prior to slaughter were excluded from the study. If the selected carcase was excluded, a carcase provided on the reserve list was selected instead. Bacteriological examination was carried out within 24 hours of the samples arriving at AHVLA Bury and no later than 96 hours after the sample was collected. Any samples tested after this deadline were excluded from the analysis of Salmonella, Toxoplasma, PRRSv and Yersinia.

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Microbiology The isolation of Salmonella, Yersinia, Campylobacter and ESBL E. coli along with the AMR testing of C. coli isolates was performed at AHVLA Bury. The Salmonella serotyping and phage typing, speciation of Campylobacter and the definitive identification of ESBL isolates were all undertaken by the AHVLA Weybridge Bacteriology Department. Determination of the presence of PRRSv antibody was undertaken by the AHVLA Weybridge Virology Department whilst the detection and viral analysis by polymerase chain reaction (PCR) was carried out by AHVLA Penrith and AHVLA Weybridge, respectively. The presence of Hepatitis E antibody and RNA quantification was carried out by PHE. All Toxoplasma testing was undertaken at the Toxoplasma Reference Unit, Public Health Wales (PHW). Upon receipt of the samples at AHVLA Bury, sampling kits were unpacked and checked for completeness and all samples were logged onto the Regional Laboratory data system. Samples for onward distribution were re-packaged and sent by courier to PHE and PHW. The caecal content was milked and separated into four separate samples – one each for isolation of Salmonella, Campylobacter and E. coli and one for storage at AHVLA Weybridge for future HEV testing. Salmonella The Salmonella isolation method was a modification of that described in Annex D of ISO 6579:2002 ‘Detection of Salmonella spp. in animal faeces and in samples of the primary production stage’. A pre-enrichment culture was prepared in Buffered Peptone Water (BPW) and incubated for 16-20 hours. This was then sub-cultured into Selective Modified Semi-Solid Rappaport-Vassiliadis (MSRV) medium (with novobiocin at 0.001%) and incubated for up to 48 hours at 41.5oC. MSRV plates were examined at 24 hours for growth typical of Salmonella, suspect growths were sub cultured onto Brilliant Green agar (BGA) and Xylose lysine desoxycholate media (XLD). MSRV plates without growth were re incubated for a further 24 hours and the process repeated. BGA plates and XLD media were incubated for 18-24 hours at 37oC and examined for the presence of Salmonella like growth. Presumptive Salmonella colonies were confirmed using standard biochemical and serological procedures. All strains isolated and confirmed as Salmonella spp. were sent to AHVLA Weybridge for serotyping according to the White-Kauffmann-Le Minor scheme (Grimont and Weill, 2007). Isolates of Salmonella serovar Typhimurium and monophasic Typhimurium strains were also phage typed at Weybridge in compliance with the Public Health England, Colindale schemes (Anderson et al., 1977, Ward et al., 1987). Toxoplasma One blood sample (EDTA plasma), along with the whole heart and whole tongue, were re-packaged and sent to the Toxoplasma Reference Unit, Swansea for testing. The Sabin-Feldman Dye Test was used for serodiagnosis (Reiter-Owonaet al., 1999). The heart and tongue tissue from seropositive pigs have been stored for future molecular investigations using nucleic acid amplification testing (NAAT).

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Hepatitis E virus Anti-HEV seroprevalence and class-specific antibody Whole blood samples anti-coagulated with EDTA taken from pigs at time of slaughter were received at PHE Colindale during the course of the study. Plasma was retrieved after centrifugation and stored at -200C prior to testing for anti-HEV and for HEV RNA. For serology three assays were used. Firstly a commercially available assay made by Wantai, China was used to test for “total” anti-HEV using the HEV antibody ELISA kit (Fortress Diagnostics Ltd, Northern Ireland) and the results interpreted in accordance with the manufacturer’s protocol. The format is an immunometric assay and is likely to be very sensitive and able to detect anti-HEV from any species. It will however not differentiate between antibody classes. It has a small dynamic range and for quantification of antibody samples with Binding Ration (BR) ≥ 20 were tested at a 1:10 dilution and the resulting BRs adjusted by x10. A quantitative IgM class-specific assay was developed based upon the IgM capture principle which is extensively used for the diagnosis of many acute infections in humans, including HEV infection. Using all components of human kit except the solid phase which was replaced with one coated with commercially available antibody to swine µFc. In brief, a solid phase coated with µ-specific anti-pig antibody is allowed to capture swine IgM from plasma at an optimised fixed dilution, after incubation and washing the captured IgM is interrogated with horseradish peroxidise (HRPO) labelled HEV orf2 antigen. Reactivity in this assay is measured in binding ratios (BR) against normal anti-HEV negative plasma and can be expressed either as BR values or as arbitrary units against serial dilutions in normal pig plasma of a strong IgM positive control. This calibration curve was run in each assay. Samples with a BR greater than that given by a 1 in 33 dilution in normal pig plasma of the IgM pool (equivalent to >2.9 arbitrary pig IgM units per ml; au/ml) were considered to contain IgM antibody. Detection, quantification and characterisation of HEV RNA Following extraction of nucleic acid from plasma HEV RNA was detected and measured in a quantitative Taqman assay (Garson et al., 2012) of high specificity (better than 99.9% for repeatably quantifiable samples) and expressed in IU/ml by comparison against the WHO international standard. The lower limit of quantification was 100 IU/ml and the limit of detection, defined by Poisson titration, was 22 IU/ml. In six samples with sufficiently high levels of viraemia, HEV orf2 was amplified and sequenced by Sanger methodology as previously described (Ijaz et al., 2005). Samples giving reactions below the level of quantification were confirmed to contain HEV RNA through amplification in a second PCR using inner primers JVHEVF and JVHEVR (Garson et al., 2012; Jothikumar et al., 2006). Sequences of orf 2 from amplified plasma HEV RNA were assembled into phylogenetic trees as previously described (Ijaz et al., 2005) and compared against current UK human sequences and porcine sequences retrieved from GenBank. Yersinia spp. Yersinia enterocolitica was isolated by the cold enrichment method. At AHVLA Bury, a tonsil scrape was added to one universal of Phosphate Buffer Solution (PBS) and a carcase swab was rinsed in PBS to achieve approximately a 10% v/v suspension. In addition, 2ml of a

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control sample, spiked with 2 to 3 colonies of Y. enterocolitica (NCTC 10460 FD NO. 3067), was added to a universal of PBS (10% v/v) and processed in parallel with each batch of test samples. The samples were stored at 2-8oC and sub-cultured weekly; 0.1ml was subcultured onto Yersinia selective agar (Oxoid CIN MED PO0287A) for 3 successive weeks. The plates were incubated at 30oC and examined at 24 hours and 48 hours. Identification of Y. enterocolitica was confirmed by colony morphology and biochemical tests (API 20E, Biomerieux). Porcine reproductive and respiratory syndrome virus A pilot study on PRRSv was initially conducted under the Defra-funded Scanning Surveillance for Diseases of Pigs Project (ED1200) to estimate the prevalences of antibody and viral RNA and provide data for a proposal for BPEX to fund testing of the rest of the samples. Plasma samples were returned to AHVLA Bury from PHE and serology was carried out on a random selection of 50 plasma samples, after which PCR was also undertaken on the corresponding tonsil samples. Following the pilot study, serology was carried out on all the remaining samples, as detailed below. All available plasma samples remaining were tested for antibodies to PRRSv by ELISA at AHVLA. This was carried out using the IDEXX PRRS X3 enzyme-linked immunoassay according to the manufacturer’s instructions for the detection of antibodies to PRRSv. The manufacturer has confirmed that this kit is suitable for use in serum and plasma samples, however at this current time only serum samples have been fully validated by AHVLA Weybridge (this project will assist with validation of the test for plasma). From the validation work performed by the manufacturer, the diagnostic sensitivity and specificity estimates are 98.8% and 99.9%, respectively. Tonsil samples from pigs which tested positive or inconclusive in the PRRSv antibody ELISA were then tested in the diagnostic PRRSv real-time RT-PCR assay which targets conserved areas of the nucleocapsid (ORF7) gene in both genotype 1 and genotype 2 PRRS viruses as described by Frossard et al. (2012). Sequencing of the PRRSv ORF5 gene was subsequently performed on all tonsil samples testing positive in the diagnostic PRRSv PCR to characterize the viruses present as described in Frossard et al. (2013). PCR amplicons were purified using Beckman AMPure® solid phase reversible immobilisation (SPRI) technique. Cycle sequencing was performed using forward and reverse primers and ABI BigDye® chemistry at the end of which the dye terminators were removed using Beckman CleanSEQ® SPRI. Samples were sequenced on an ABI capillary electrophoresis DNA analyser and the raw data analysed by ABI SeqScape® software and compared to a library of British PRRSV strains. Sample results are reported as percentage similarity to Porcilis vaccine and up to three closest strain matches. Pigs were classified as PRRSv-positive if they tested positive in either the antibody ELISA and/or the diagnostic PCR. If they tested inconclusive in the antibody ELISA and negative in the diagnostic PCR, they were classified as PRRSv-negative. Pigs testing negative in the antibody ELISA were not tested by PCR and were classified as PRRSv-negative. Some pigs were tested by PCR only; those testing positive by PCR were designated as PRRSv-positive, if negative by PCR they were classified as “not known”.

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Thermophilic Campylobacter spp. Thermophilic Campylobacter spp. were isolated from 69.3% (95% CI 65.2–73.2) of pigs in the 2003 abattoir study with 90% of isolates identified as C. coli (Milnes et al., 2008). In 2006/07 the prevalence of Campylobacter was 79.5% (95% CI 76.0-83.0), similarly most isolates (81%) were confirmed as C. coli (FZ2023/ VFDS001 final report). The purpose of this study was to obtain 170 C. coli isolates for AMR testing and not to provide a comparable prevalence estimate on the occurrence of C. coli in pigs. A subset of 250 pigs was therefore randomly selected by the ESRG at AHVLA Weybridge, stratified by abattoir and scheduled month of sampling, based on an expected prevalence of C. coli of 70%. Isolation for Campylobacter was undertaken at AHVLA Bury. Briefly, the caecal content was streaked directly onto modified charcoal cefoperazone deoxycholate agar (mCCDA) (CM739 and supplement SR155, Oxoid, UK) and Prestons broth (Oxoid SR117/CM67). Cultures were incubated in a microaerobic atmosphere (84% N2/10% CO2/6% O2) at 41.5oC and examined for typical Campylobacter colonies after 24–48 hours, broths were subcultured onto CCDA at 48 hours and incubated as previously stated. Appropriate positive controls were inoculated onto mCCDA and incubated alongside. Colonies showing typical morphology for Campylobacter were taken from each mCCDA plate and subcultured onto 5% sheep blood agar (PB0123A, Oxoid). All suspect cultures were confirmed as thermophilic Campylobacter spp. by colony morphology, Gram stain, motility, catalase and oxidase activity and growth conditions in accordance with ISO 10272-1:2006. Campylobacter isolates were sent to AHVLA Weybridge for speciation by PCR. The PCR method used was based on the PCRs published by Best et al. (2003), Randall et al., (2010) and Mayr et al., (2009). This method is a multiplex PCR that allows the simultaneous identification of C. jejuni (mapA), C. coli (ceuE) and C. lari (gyrA). Briefly, a 1 µl-loop of growth from the subculture plates was picked and emulsified in 100 µl of distilled sterile water in a 0.5 ml microcentrifuge tube and heated to 100°C for 10 min to obtain boiled cell lysates. PCR reaction mixtures were prepared containing 12.5 µl of HotStarTaq mastermix (Qiagen, Crawley,UK), 2 µl of boiled cell lysate, 25 mM MgCL2, 20 µM of mapA, ceuE, gyrA1 and gyrA2 primer pairs (Sigma-Aldrich, Dorset, UK) and 5 µM of each ceuE, mapA and gyrA probe (Sigma-Aldrich). An internal amplification control (IAC) to detect the presence of inhibitory substances for the PCR reaction was included and a complementary probe as described by Randall et al. (2010). Therefore, 2 pmol of IAC and 5 µM of IAC probe (Sigma-Aldrich) were included and the complete reaction mixture was made up to 25 µl in nuclease free water. The PCR reaction was performed on the Stratagene MX3005p (Stratagene, La Jolla, USA), amplification consisted of an initial hold at 95°C for 15 minutes, then 40 cycles of 94°C for 30 seconds and annealing/extension at 60°C for 30 seconds and at 72°C for 30 seconds. Fluorescence was read at 60°C, and a cycle threshold of between 10 and 32 was viewed as a positive result for either the mapA probe (C. jejuni), the ceuE probe (C. coli) or the gyrA probe (C. lari). In the absence of a positive signal for either C. jejuni, C. coli, or C. lari, a positive signal for the IAC is necessary to validate a negative result.

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Antimicrobial resistance testing of the C. coli isolates was undertaken at AHVLA Bury. Dilution methods were performed according to methods as described by CLSI and EFSA (CLSI, 2006, EFSA, 2007). The antimicrobials tested in this study are given in Appendix III together with the epidemiological cut-off value used to determine the susceptibility of C. coli. The epidemiological cut-off values have been developed by the European Committee for Antimicrobial Susceptibility Testing (EUCAST) for the differentiation of wild-type isolates (completely susceptible isolates) from isolates showing any degree of resistance (Appendix III). The method and interpretation of the results are given in the EFSA Journal 2007. Extended Spectrum Beta Lactamase E. coli CHROMagar ECC was used for isolation of all E. coli whilst CHROMagar CTX (CHROMagar, France) and ESBL Brilliance (Oxoid) agar were used for isolation of presumptive ESBL E. coli. At AHVLA Bury, 1g caecal contents were added to 9ml of buffered peptone water (BPW). After overnight incubation at 37ºC, 10ul of the BPW was inoculated onto Chromagar ECC, Chromager CTX-M (Chromagar, France) and ESBL Brilliance (Oxoid) agars which were then incubated for 48 hours at 37ºC and examined at 24 and 48 hours for the presence of suspect ESBL E. coli. A single presumptive ESBL E. coli isolate from each ESBL medium (CHROMagar CTX and Oxoid ESBL Brilliance agar) was sub-cultured onto blood agar to check for purity and confirmed as E. coli using spot oxidase and indole tests. Where presumptive ESBL E. coli colonies were present on Chromagar CTX-M with differing colonial morphology, each colony type was subject to further investigation. Isolates were checked for cephalosporin/ clavulanate synergy (if positive indicates an ESBL phenotype) using the Mast disc diffusion ESBL synergy tests. ESBL phenotype E. coli colonies from CHROMagar CTX and from Oxoid ESBL Brilliance agar were tested at AHVLA Weybridge for the presence of the CTX-M, OXA, SHV and TEM genes by multiplex PCR (Fang et al., 2008). Isolates positive for CTX-M genes were amplified with PCR sequencing primers as described previously (Carattoli et al., 2008; Sabate et al., 2002) and sequenced to determine CTX-M sequence type. Isolates positive for SHV genes, but not CTX genes, were also sequenced using suitable primers to determine SHV gene sequence type. Statistical analysis The analysis was undertaken by ESRG, AHVLA Weybridge. Questionnaire data and laboratory test results were data entered or imported into Microsoft Access. Data were cleansed and analysed using Microsoft Excel and Stata v. 12 (StataCorp, USA). Data were checked for implausible and/ or inconsistent values. The prevalence for each infection was calculated using the survey command (svy) in Stata to take account of clustering of pigs within farms. Results for the different months of sampling were compared using chi-squared tests, while taking into account within-farm clustering. Agreement between sample type for Salmonella and Yersinia was examined using a kappa test. The interpretation used for Cohen’s kappa statistic was: poor agreement k≤0.20, fair agreement

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0.21≤k≥0.40, moderate agreement 0.41≤k≥0.60, substantial agreement 0.61≤k≥0.80, and good agreement k≥0.81. For C. coli, the proportion of isolates resistant to each antimicrobial was calculated and the resistance profiles identified in the isolates were examined. The number and combination of infectious agents identified in each pig were tabulated. Associations between some of the infections were also investigated using chi-square tests (while taking account of clustering of pigs within farms) where these co-infections were thought to be of particular epidemiological relevance; the co-infections investigated were (i) PRRS seropositivity and Salmonella carriage, (ii) Salmonella and Yersinia carriage and (iii) Salmonella/Yersinia and ESBL E. coli carriage. Results Overall Sampling was scheduled to take place between 14th January 2013 and 12th April 2013; 33 (5.2%) of the pigs were rescheduled for sampling which was undertaken in late April/ early May. Fourteen abattoirs participated in the study: 12 in England and two in Northern Ireland. The study included the largest 14 abattoirs of the 169 approved premises in the UK who between them processed over 80% of the 10 million finishing pigs slaughtered in 2013. This resulted in abattoirs in NI and England being selected as none of the largest abattoirs were located in Scotland or Wales. Overall, 654 pigs were scheduled for sampling during the study period. Samples were collected from 648 pigs, very few problems were identified during the study and for most (>96%) pigs all nine samples were taken. Two pigs were identified as originating from the Republic of Ireland and one pig had not been kept in the UK for 3 months prior to slaughter. As such, these three pigs were excluded from all of the results below. The laboratory testing of 19 pigs at AHVLA Bury was carried out more than 96 hours after sample collection. As such, these 22 pigs were excluded from the prevalence results for Salmonella, Toxoplasma, PRRSv and Yersinia. For AMR testing in Campylobacter and ESBL E. coli and for HEV, all 19 pigs were included. This decision was based on expert opinion as the delay in testing was not deemed to impact upon the results. In total, 5,605 samples were collected from the remaining 626 pigs; 625 rectal swabs, 1,250 plasma samples, 619 caecal samples, 620 tonsil samples, 621 tongue tissue, 622 heart tissue and 1,248 carcase swabs. The 626 pigs originated from 439 farms. Between 1 and 10 pigs were sampled from each farm. Most of the pigs sampled originated from farms in England (81.7%), followed by Northern Ireland (13.4%), Scotland (4.5%) and Wales (0.3%). The number of pigs sampled in each region was found to be proportionate to the UK pig population; 81.0% in England, 9.6% in Northern Ireland, 8.8% in Scotland and 0.58% in Wales (UK Zoonoses Report 2012). One third of the pigs were from farms in Yorkshire and the Humber (207 pigs, 33.1%) with a further 22.8% from the East of England (Table 1); both regions are associated with pig farming and are regions of dense pig populations. The number of pigs sampled by region was found to be broadly proportionate to the regional UK pig population (Table 1). Over half (53.5%) of the pigs sampled were from finishing only farms, a further 20.3% were reported

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from finishing and breeding farms with the remainder (26.2%) reported as not known (Table 2). The majority of the pigs were less than 12 months old (89.5%) with 9.0% reported as over 12 months. Most of the pigs (77.6%) were kept in controlled housing or all-in-all-out systems. Only 3.5% of the pigs were reported as born outdoors and reared/ kept in controlled housing since weaning and a further 0.5% were kept fully outdoors which is lower than expected, however the housing information was not known for 18.4%. The age of the pigs was tabulated against the production system recorded on the data collection form (Table 2). This shows that most of pigs aged 12 months or older were recorded as Breeders or Finishers and none were classified as Finishing only pigs. Salmonella A total of 619 caeca, 624 carcase swabs and 625 rectal swabs, from 626 pigs, were tested for Salmonella. All three samples were collected from 618 (98.7%) of the pigs whilst two samples were collected from six (1.0%) pigs and only one sample from two (0.3%) pigs. In total, 291 of the 626 pigs tested positive for Salmonella in at least one sample. Of the three sample types tested, the highest number of positive samples were taken from the caecum (189/619) followed by the rectal swab (150/625), with considerably fewer of the carcase swabs testing positive (60/624). After accounting for within-farm clustering, the prevalence of Salmonella in the caecal samples was 30.5% (95% CI 26.5-34.6), the prevalence in the rectal swab samples was 24.0% (95% CI 20.5-27.5) and the prevalence in the carcase swab samples was 9.6% (95% CI 7.3-11.9). Salmonella carriage was determined by testing caecal contents whereas carcase contamination was measured by testing carcase swabs. A rectal swab sample was also taken to determine whether this was a good indicator of caecal carriage. Of the 618 pigs for which both intestinal samples were collected, 11.7% tested positive in both the caecal and rectal swab sample. There was a fair agreement between caecal contents and rectal swabs (kappa value of 0.21) (Table 3). As expected, there was poor agreement between carcase swabs and caecal contents (and carcase swabs and rectal swabs), as indicated by a kappa value ≤0.12. The agreement between sample types was also tested for each of the serovars S. Typhimurium, S. 4,5,12:i:-, S. 4,12:i:- and S. Derby in turn. The kappa statistics ranged from 0.16 (S. 4,12:i-) to 0.37 (S. Derby) (Tables 4a-4d). Salmonella carriage as determined by caecal sampling varied by abattoir from 11.3% to 46.8%, whereas carcase contamination ranged from 0% to 21%. The prevalence ratio of caecal carriage: carcase contamination by abattoir was examined which ranged from 0.0 to 1.17 with an average of 0.31. For all but two abattoirs the prevalence of caecal carriage was higher than the carcase contamination. It should be noted however that some of the prevalence data are based on small sample sizes and the method of comparison is crude, however it highlights potential differences between abattoirs. Salmonella positivity in the caecal contents was examined by age (Table 5; Figure 1); prevalence varied from 25.9% in pigs aged less than 6 months up to 40.7% in pigs aged over 12 months. Salmonella positivity in the carcase swab samples was also found to increase slightly with age from 7.3% in pigs aged less than 6 months up to 10.9% in pigs aged over 12

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months although again this variation was not statistically significant (Table 6; p=0.79; Figure 2).The proportion of pigs that tested positive for Salmonella in the caecal content sample was not found to vary significantly between the different months of sampling (p=0.43). A total of 20 different serovars were identified from the 291 positive pigs, as well as two rough strains (Table 7). The most commonly isolated were monophasic Typhimurium variants S. 4,5,12:i:- (found in 21.0% of the positive pigs) and S. 4,12:i:- (19.2% of positive pigs), as well as S. Typhimurium (18.6% of positive pigs), S. Derby (16.2% of positive pigs) and S. Bovismorbificans (14.1% of positive pigs). These were the five most commonly isolated serovars in each of the sample types. S. Choleraesuis was isolated from three pigs. No pigs were found to be infected with S. Enteritidis, S. Hadar, S. Infantis or S. Virchow. Thirty-six pigs (12.4% of positive pigs) tested positive for two different serovars in two sample types. The definitive phage types (DTs) for the isolates of S. Typhimurium, S. 4,5,12:i:- and S. 4,12:i:- are shown in Table 8. For all three serovars, DT193 was most commonly isolated, being found in roughly two-thirds of the pigs with monophasic strains of S. Typhimurium and a third of pigs with S. Typhimurium. Phage type U288 was also relatively common among pigs infected with S. Typhimurium, having been isolated from almost a quarter of pigs with this serovar. Toxoplasma Plasma samples from 626 pigs were tested for antibodies to Toxoplasma. For six pigs, it was not possible to test all dilutions of blood required by the Dye Test. In these samples, there was a fine mesh of fibres interfering with the ability to see Toxoplasma tachyzoites. These fibres could not be removed by benchtop centrifugation. It is possible that they were due to partial clotting; at the abattoir, blood samples were collected in a larger vessel then decanted into the EDTA tubes and this may have taken sufficient time in some cases to allow the clotting process to commence. For three of the pigs, the samples were unreadable only at the most concentrated level of blood used, and for the other three pigs the samples were unreadable at the two highest blood concentrations. Whilst no antibodies were detected at the dilutions tested, we cannot unequivocally exclude the possibility that lower levels of antibody might have been detected at lower dilutions. Therefore, these six pigs were excluded from the analysis. Of the remaining 620 pigs, 46 were seropositive giving a seroprevalence of 7.4% (95% CI 5.3-9.5) after accounting for clustering within farms. The seropositivity of Toxoplasma varied from 5.5% in pigs aged less than 6 months, to 6.6% in those aged between 6 and 12 months, to 11.1% in pigs aged 12 months or older but the difference was not statistically significant (p=0.42;Table 9; Figure 3). Hepatitis E virus Plasma samples from 640 pigs were tested for markers of hepatitis E virus (HEV) infection at PHE. In the Wantai immunometric assay test optical density (S)/cut-off (CO) ratios for plasma samples and dilutions ranged between 0.02 and 318. A total of 594 pigs were seropositive by manufacturers’ criteria (S/CO ≥ 1), giving a seroprevalence of 92.8% (95% CI 90.7-95.0) after adjusting for clustering of pigs within farms. It was possible to categorise

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the Wantai total antibody seropositive samples into those that were reactive for IgM and those which were not. A total of 327 (55.1%) samples were considered to contain IgM and to represent recent acute infection (Total antibody positive, IgM ≥ 2.9Pau/ml). The remaining 267 (44.9%) which were unreactive for IgM were considered to represent past infection (Table 10). Hepatitis E viral RNA was detected in 37 of the 640 plasma samples, seven of which contained no detectable total anti-HEV antibody in the screening assay (Table 10), the remaining 30 were all seropositive. Of the seven with no detectable total anti-HEV antibody, three had low level IgM (3.4, 3.6 and 4.9 au/ml). Twenty viraemic samples contained markers of recent infection (Total antibody positive and IgM reactive≥ 2.9 au/ml) and 10 markers of past infection (Total antibody positive only). Broadly speaking the samples that contained HEV RNA could be divided in to those samples in which the plasma HEV RNA load could be quantified as equal to or greater than 22 IU/ml (twelve samples) and those that lay below the limit of quantification (25 samples). The prevalence of current infection, defined by the presence of detectable plasma HEV RNA, adjusting for clustering within farms, was 5.8% (95% CI 3.9-7.6). There was no evidence that the prevalence of active infection varied significantly between NUTS1 regions (p=0.11). It was possible to undertake molecular characterisation studies in six pig samples which contained sufficient HEV RNA for sequencing. The data showed the sequences to belong to G3 clustering within the UK human G3 group 1 sequences. In total, 601 pigs were either seropositive and/ or had HEV RNA detected, so the prevalence of current and/ or past infection was 93.9% (95% CI 92.0-95.8) after adjusting for within-farm clustering. Yersinia spp. Overall, 624 carcase swabs and 620 tonsil samples, from 624 pigs, were tested for the presence of Yersinia. One third (204/620; 32.9%) of the tonsil samples tested positive for Yersinia compared with only 1.9% (12/of 624) of the carcase swabs. For tonsil samples, the prevalence was 32.9% (95% CI 28.8-37.0), after accounting for clustering within farms, and for carcase swabs the prevalence was 1.9% (95% CI 0.8-3.0). Of the 620 pigs for which both sample types were collected, 10 (1.6%) pigs tested positive in both samples with the remaining 196 (31.6%) pigs testing positive in only one sample. The kappa test confirmed the poor agreement between the sample types (kappa statistic=0.06) with, unsurprisingly, very strong evidence that the tonsils identified significantly more positive pigs than the carcase swabs (p≤0.001). The proportion of pigs that tested positive for Yersinia in the tonsils was not found to vary significantly between the different months of sampling (p=0.22). The majority of the positive pigs (87.3%) and carcases (91.7%) were infected with Y. enterocolitica (Table 11). A further 21 (10.3%) of the positive pigs were infected with Y. pseudotuberculosis. After accounting for within-farm clustering, the prevalence of Y. enterocolitica carriage was 28.7% (95% CI 24.8-32.7) whilst the prevalence on carcases was 1.8% (95% CI 0.7-2.8). The prevalence of Y. pseudotuberculosis carriage was 3.4% (95% CI 2.0-4.8). There was no apparent clustering of the less common Yersinia species (Y.

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frederiksenii/ intermedia, Y. kristensenii and Y. pseudotuberculosis) within a particular geographic region. Roughly a quarter of the pigs aged <6 months and >12 months were found to carry Yersinia in the tonsils compared to roughly a third of those aged 6-12 months (Table 12; p=0.22; Figure 4). All of the positive carcase swabs were from pigs aged 6-12 months. Porcine reproductive and respiratory syndrome virus Pilot study Fifty matching plasma and tonsil samples were tested for PRRSv by both antibody ELISA and diagnostic PRRSv PCR, respectively. Three of these pigs have since been excluded from the analysis as they did not meet the inclusion criteria. In the remaining forty seven pigs, sample-to-positive (S/P) ratios in the ELISA test ranged from -0.08 to 4.7. Twenty two (46.8%) pigs were seropositive (S/P>0.4) and three (6.4%) pigs had inconclusive serology results (S/P ratios between 0.2 and 0.4). Only one pig tested positive for PRRSv by PCR; this pig was also seropositive (Table 13). Full dataset Including those tested in the pilot study, which were not tested a second time, a total of 621 plasma samples were tested for antibodies to PRRSv. Sample-to-positive (S/P) ratios ranged between -0.1 and 4.7. A total of 362 pigs were seropositive for PRRSv (S/P>0.4), so after accounting for clustering of pigs within farms the seroprevalence was 58.3% (95% CI 53.1-63.4). Eleven (1.8%) other pigs had inconclusive ELISA results (S/P ratios between 0.2 and 0.4). Following the pilot study, only the tonsils from pigs with either positive or inconclusive serology results were tested by PCR. PCR results for ELISA-negative pigs tested during the pilot study have therefore been excluded from subsequent analyses and considered as “Not tested” in Table 14. The tonsils of four other pigs for which plasma samples, and hence ELISA results, were missing were also tested by PCR. Of the 372 tonsil samples tested, 31 were positive for PRRS viral RNA. After accounting for within-farm clustering, the prevalence of PRRSv RNA-positive tonsils was thus 8.3% (95%CI 5.5-11.2). All of these pigs had PRRSv RNA of genotype 1 and had also tested seropositive (Table 14). The four pigs for which plasma samples were missing tested negative by PCR but their PRRSv status is not known as previous infection cannot be ruled out without serology. Seropositivity was found to vary significantly between age groups (p=0.002) with the highest level found in pigs aged less than six months (68.5%; 95% CI 54.5-82.5) and lowest in those aged >12 months (32.1% (95% CI 15.0-49.3)) (Table 15; Figure 5). The prevalence of PRRSv RNA-positive tonsils was similar across the three age groups (Table 16; Figure 6). Combining the ELISA and PCR results, a total of 362 out of 625 pigs tested positive so the overall prevalence of current and/ or past infection was 57.9% (95% CI 52.8-63.1); the denominator includes the four (0.6%) pigs for which ELISA results were not available and whose status is hence not known.

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Sequencing of the ORF5 PRRSv gene was undertaken on 29 of the 31 tonsil samples from which PRRSviral RNA was detected. Two PCR-positive tonsils were not suitable for sequencing as there was insufficient viral nucleic acid in the samples. Six samples did not yield useable sequence data, which is not unexpected as the sequencing assay is not as sensitive as the diagnostic RT-PCR test. Table 17 details PRRSv sequence results for the 23 successfully sequenced viruses and indicates their origin by geographic region. One of the 23 pigs was tested after the 96 hour time limit, however for the purpose of this report the sequence from this pig has been included in the sequencing results. The table also identifies both the historic PRRSv strains in the database showing closest homology and the PRRSv strains in the OZ0150 study showing closest homology to each OZ0150 virus sequence. The phylogenetic tree (figure 1) illustrates the genetic diversity of the PRRSv ORF5 genes from the 23 samples in this study in comparison to the vaccine virus and previously sequenced viruses from GB pigs. Co-infections Six hundred and ten pigs were tested for all of the following organisms: Salmonella carriage (in the caecum), Yersinia carriage (in the tonsils), Toxoplasma (seropositivity), HEV (seropositivity and/ or RNA presence) and PRRSv (seropositivity and/ or RNA presence). Of these 610 pigs, only 14 pigs (2.3%) tested negative for all of these infectious agents (Table 18). One pig had evidence of having been infected with all five of these infectious agents (Table 18). The combinations of infections that were found in these 610 pigs are shown in Table 19. A slightly higher proportion of the PRRSv-seropositive pigs were found to carry Salmonella in the caecum (32.7%) than in the PRRSv-seronegative pigs (27.3%) but this difference was not statistically significant (p=0.19; Table 20). The association was also not significant when this was restricted to just include carriage of Salmonella Typhimurium or monophasic strains of Salmonella Typhimurium (p=0.78). PRRSv-seropositive pigs were also no more likely to carry Yersinia than PRRSv-seronegative pigs (p=0.67; Table 21). Of the 615 pigs that were tested for both Salmonella carriage (in the caecum) and Yersinia carriage (in the tonsils), 10.4% were infected with both of these organisms. There was no evidence of a statistically significant association between the two infections (p=0.78) (Table 22). There was also no evidence that pigs carrying either Salmonella or Yersinia were more likely to also carry ESBL E. coli than pigs which were not carrying either of those infections (p=0.75) (Table 23).

AMR of Campylobacter coli Pigs have been included in the analysis for Campylobacter even when the caecum was tested more than 96 hours after collection as the aim was to identify isolates for susceptibility testing rather than to obtain a robust prevalence estimate; however, the three pigs which were from farms in the Republic of Ireland or which were not kept in the UK for the three months before slaughter have been excluded. In total, the caeca from 215 pigs were tested for the presence of Campylobacter. Campylobacter was recovered from the caeca of 182 pigs, giving a prevalence of 84.7% (95% CI 79.9-89.4) after adjusting for within-farm clustering. The majority (84.1%; 153/182) of

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these pigs were infected with C. coli, although 14 (7.7%) pigs tested positive for C. jejuni, four (2.2%) had mixed C. coli and C. jejuni, and two (1.1%) had C. lari. Six cultures were negative by PCR for these three Campylobacter species; it is possible that they had another Campylobacter species, but this was not determined as the focus was to identify isolates of C. coli for AMR testing. A further three cultures sent to AHVLA Weybridge for speciation failed to grow but these were also not investigated further. Hence, the final number of C. coli isolates available for AMR testing was 153. Each of the 153 C. coli isolates were tested for their in vitro susceptibility to seven antimicrobials. Table 24 displays the number of isolates susceptible to each of these antimicrobials. Isolates showing any degree of resistance (i.e. non-wild-type isolates) are referred to as resistant in this report. The greatest levels of resistance were observed against tetracyclines (77.8%) and streptomycin (66.0%). There was also a moderate level of resistance against erythromycin (27.5%), nalidixic acid (17.0%) and ciprofloxacin (12.4%). No resistance was observed against either chloramphenicol or gentamicin. The MIC values obtained for the 153 C. coli isolates ranged from 2 to 16 for chloramphenicol, 0.06 to 4 for ciprofloxacin, 0.5 to 32 for erythromycin, 0.12 to 1 for gentamicin, 2 to 64 for nalidixic acid, 1 to 16 for streptomycin and 0.25 to 16 for tetracycline. Only nine (5.9%) of the C. coli isolates were sensitive to all seven antimicrobials (Table 25). The maximum number of antimicrobials against which any isolate expressed resistance was five (Table 25). The resistance profiles identified in the isolates are shown in Table 26. Extended Spectrum Beta Lactamase E. coli Pigs were included in the analysis for ESBL E. coli regardless of whether or not the caecum was tested within 96 hours of collection; however, the three pigs which were from farms in the Republic of Ireland or which were not kept in the UK for the three months before slaughter were excluded. Hence, the caeca from a total of 637 pigs were tested for ESBL E. coli. On selective ESBL agars, presumptive ESBL E. coli were isolated from 149 (23.4%) of these pigs and sent to AHVLA Weybridge for confirmation of ESBL genes present. E. coli isolated from non-selective media were not examined further at this stage. In total, presumptive ESBL E. coli with CTX-M enzyme genes were recovered from 140 pigs (22.0%), E. coli with TEM enzyme genes from 55 pigs (8.6%), E. coli with SHV-12 enzyme genes from fourteen pigs (2.2%) and E. coli with OXA enzyme genes from eight pigs (1.3%). The combinations of genes identified from each pig are shown in Table 28. However, it should be noted that whilst all CTX-M genes in bacterial isolates will confer resistance in the bacteria to extended spectrum β-lactam antibiotics, such as the third and fourth generation cephalosporins, only some specific OXA, SHV (such as SHV-12) and TEM genes are “ESBL genes”. Thus, all CTX-M bacteria will be ESBLs, but not necessarily bacteria that contain OXA or SHV or TEM genes. The CTX-M sequence type was confirmed for 139 of the 140 CTX-M E. coli positive pigs (one strain was not sequenced due to sequencing failure for this strain). Usually, only one CTX-M isolate from each pig was tested. However, CTX-M sequence types 1 and 27 were both identified from one pig for which more than one isolate was tested. As isolates were obtained from both CHROMagenic ESBL agars, there were a few limited instances where an

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isolate was tested from both media types from one pig, but this was the only example where this gave rise to more than one CTX-M type in one pig. CTX-M sequence type 1 was identified in 85.0% of the CTX-M positive pigs but sequence types 3, 14, 15, 27, 32 and 55 were also identified (Table 27). The overall prevalence of CTX-M ESBL E. coli in the 637 pigs, after accounting for clustering within farms, was 22.0% (95% CI 17.8-26.1). The SHV sequence type was determined for E. coli isolates from pigs that were CTX-M negative but SHV positive (isolates from Brilliance agar only); all of these were found to be SHV sequence type 12 and are thus also ESBL E. coli, since SHV-12 is known to be an ESBL type SHV gene (Teshager et al., 2000). The OXA and TEM E. coli were not further examined and both (when they were detected) occurred with a known CTX-M or SHV ESBL enzyme (Table 28), which was presumed to have been the gene conferring the ESBL E. coli phenotype. As all 149 pigs found to give rise to presumptive ESBL E. coli on the selective agars were confirmed to carry ESBL E. coli, the overall prevalence was 23.4% (95% CI 19.2-27.6). Discussion This is the first UK-wide study of Toxoplasma, HEV, PRRSv and ESBL E. coli in UK pigs at slaughter. The abattoirs participating in the survey processed ≥80% of the UK pig slaughter throughput; this coverage combined with the randomized sampling approach provides a robust and representative estimates of prevalence. There are a number of issues to consider when interpreting the data presented in this report. The sampling schedule (the day of sampling and the carcase to be sampled) was randomised, hence for some abattoirs more than one carcase was sampled on a given day which could have resulted in pigs being sampled from the same farm on the same day. However this only occurred in two instances and would suggest limited clustering of pigs. In addition, all of the prevalence and seroprevalence data presented were adjusted to take into account within-farm clustering. Epidemiological data was collected alongside the samples. However the study was not designed to examine co-infections and lacks power to do so. Therefore interpretation of the co-infection data should be made with caution. Salmonella Levels of current Salmonella carriage, as monitored by testing caecal contents and rectal swabs, were high at 30.5% and 24.0% respectively; this is considerably higher than the 2007 average EU prevalence, based on lymph node testing (10.3%) (EFSA, 2008a). Furthermore, Salmonella carriage, determined by caecal testing, is significantly higher than the 2007 caecal results where 21.9% of pigs were found to be positive (95% CI 18.7–25.3). The results therefore indicate that the carriage of Salmonella by 1 in 5 pigs remains and therefore efforts will continue to be required to prevent contamination of carcases particularly in light of future EU plans for a reduction in Salmonella contamination of pig meat. In this study rectal swabs were included to determine Salmonella carriage compared with caecal

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sampling (as a possible test for future studies). Although there was fair agreement between caecal and rectal sampling, the latter was less sensitive. The prevalence of Salmonella carriage by age varied from 25.9% in pigs aged less than 6 months up to 40.7% in pigs aged over 12 months. One may expect the pigs aged less than 12 months to be representative of finishing pigs and those aged 12 months or older to be breeders; in the EU baseline surveys of finishing pigs and holdings with breeding pigs UK Salmonella prevalence was 40.4% and 46.3% respectively (however it should be noted that the breeding pig survey was carried out at a holding level rather than at the abattoir). Sampling was only undertaken in this study during January to April, as such the impact of excluding sampling during May to December should be considered. There is little evidence of a major effect of seasonality on the carriage of Salmonella in UK pigs (Marrier, personal communication; Milnes et al., 2008). In the 2003 GB abattoir study by Milnes et al. (2008), although the variation in seasonal prevalence was not statistically significant, Salmonella prevalence was found to be lowest in spring (20.0%) and highest in autumn (25.2%). Hence, the prevalence observed in this study may have been slightly higher if we had continued monitoring throughout the year rather than during January to April only. But, given that a strong seasonal effect has not been demonstrated it is unlikely that the prevalence would be significantly different if pigs had been sampled across a 12 month period compared to that observed in our study. The contamination of pig carcases during the slaughter process was monitored in this study. Pigs may be infected with Salmonella on the farm of origin or following infection when being transported or in the lairage. Pigs for slaughter may then have their skin contaminated in the lairage or at any point through the processing line as a result of the leakage or spreading of faeces or intestinal contents during processing. The proportion of Salmonella contaminated carcases was lower than the Salmonella prevalence in caecal contents. The levels of Salmonella carcase contamination varies between abattoirs which suggests that the processing, in particular, decontamination by scalding and singeing, as well as general hygiene is variably applied. The contamination rate of carcases in UK pigs was significantly higher in 2007 compared with this study (15.1% versus 9.6%) (EFSA Journal, 2008; Marier et al., 2008). At the EU level, pig carcasses were most likely to become contaminated with Salmonella during April to September 2007 (EFSA, 2008b). Hence the reduction in Salmonella carcase contamination observed in this study may be an artefact of study design, having only sampled during lower risk months, or it may reflect a real decrease in carcase contamination. The most common serovars isolated in this study were S. 4,5,12:i:- followed by S. 4,12:i:-, S. Typhimurium and S. Derby; accounting for 75% of isolates. This is in contrast to the previous baseline survey where S. Typhimurium (57.9%), S. Derby (26.3%) and S. Reading (5.4%) were the most common serovars (UK results) (Marier et al., 2008). Salmonella 4,[5],12:i:- is considered to be an important monophasic variant of S. Typhimurium, and has been increasingly reported from pigs and people in many EU and third countries. The caecal prevalence of this serovar was 0.8% in 2007 (versus 10.5% in this study). The rapid emergence and dissemination of monophasic Typhimurium DT193 since 2006 has been

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reflected in surveillance data from clinical disease cases in weaned pigs and in human data from sporadic and outbreak-related illnesses (EFSA Journal, 2010). The definitive phage type DT193 was most commonly isolated, similar to the 2003 and 2007 studies, representing 33% of isolations (96 of 291 positive pigs) (Table 8). It is also the most commonly identified definitive type of S. Typhimurium causing disease in pigs (identified via diagnostic pig submissions to AHVLA). A high level (6.5% prevalence) of S. Bovismorbificans was identified in this study, accounting for 14.1% of positive pigs. This compares to 0.78% prevalence in the 2007 survey (1.93% of positive pigs). S. Bovismorbificans has caused a number of foodborne outbreaks in Europe in recent years and can be associated with severe symptoms (MMWR, 2012; Rimhanen-Finne et al., 2011). The isolation of S. Choleraesuis is also of concern for pig health since this serovar causes clinical disease in pigs (Cheng-Hsun et al., 2004). This serovar was not isolated in the 2007 study. Source attribution to assess the public-health impact of pork consumption was carried out as part of a Quantitative Microbiological Risk Assessment undertaken by AHVLA in consortium with National Food Institute, Technical University of Denmark (DTU) and Dutch National Institute for Public Health and the Environment (RIVM) (QMRA, 2009). The authors found that meat products, particularly beef and pork, were important sources of human S. Typhimurium infections and an estimated 10-20% of human Salmonella infections in the EU may be attributable to pigs and pork. A further study on source attribution, (Pires et al., 2011), estimated that pigs were responsible for 29.6% of human salmonellosis in the EU at that time (the second largest contributor behind laying hens) and that the pig reservoir was responsible for the majority of S. Typhimurium infections in humans. More recently, EFSA have estimated that over 50% of Salmonella infections in the EU may be attributable to pigs and pork, the change having partly come about because of a reduction in Salmonella in poultry following the introduction of National Control Programmes for Salmonella control in several sectors of the poultry industry (EFSA Journal, 2012). The control of Salmonella in pig herds is complex and will need a multi-factorial approach to reduce contamination throughout the food chain. Results from this study indicate a rise in Salmonella carriage, compared with the 2007 baseline survey, but a potential reduction in carcase contamination. Thus, whilst there is a reduction in risk to public health because of the reduction in contamination along the processing line, the supply of potentially infected pigs continues. Consequently, there is a continued reliance on procedures aimed at reducing the risk of cross-contamination, whilst the need remains to reduce the likelihood of introduction of Salmonella into the processing line in the first place through the carriage of Salmonella in pigs being supplied to the abattoir. Toxoplasma The seroprevalence of Toxoplasma gondii in this study was 7.4% (95% CI 5.3-9.5). As recognised in the ACMSF Toxoplasma risk profile, previous seroprevalence data for UK-reared pigs is sparse. Nevertheless, this figure is comparable with those published several

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decades ago in which 4-12% of UK pigs tested positive using the Dye Test (Rawal, 1959; McColm et al., 1981; Jackson et al., 1987). Comparisons with more recent serological surveys conducted in other countries are problematical due to differences in sampling procedures, serological testing methods and choices of threshold for positive cut-off values (ACMSF, 2011). Nevertheless the estimate from this study falls within the range of recent seroprevalence estimates from other European countries: 2.6% in the Netherlands (Van der Giessen et al., 2007), 4.2% in Latvia (Deksne and Kirjušina, 2013), 4.7% in Ireland (Halová et al., 2013), 15.6% in Portugal (de Sousa et al., 2006), 16.3% in Italy (Villari et al., 2009), 16.6% in Spain (García-Bocanegra et al., 2010), 23.3% in Switzerland (Berger-Schoch et al., 2011) and 26% in the Czech Republic (Bártová and Sedlák, 2011). In addition to the methodological differences previously mentioned, the wide variation observed between countries may be attributable to differing husbandry systems and climatic conditions, which affect exposure of the pigs to the parasite (ACMSF, 2011). Seroprevalence had decreased in several European countries from the 1990s due to increasingly intensive management systems (Dubey, 2009, EFSA, 2007). However, as consumer demand for outdoor-reared pork meat is increasing, the prevalence of Toxoplasma may show a parallel increasing trend again due to greater access of pigs to environmental sources of infection (ACMSF, 2011, Dubey, 2009). Outdoor farming currently accounts for around 40% of commercial pig breeding herds in the UK. In this survey, only one of the Toxoplasma-positive pigs was recorded as being born outdoors but the information concerning the production system was relatively poorly completed so it was not possible to accurately assess any potential association with seroprevalence. Nevertheless, this survey provides a useful baseline against which to measure future trends in seroprevalence as husbandry practices evolve. Seropositivity in the human population has been found to vary geographically within the UK, with the highest levels thought to be in Northern Ireland and the lowest in England and Scotland; within GB, seropositivity is generally highest in the west (ACMSF, 2011). Porcine seroprevalence might also be expected to vary between regions due to differences in local husbandry practices and geographical or climatic features; all factors that may affect oocyst survival and dispersal. However, no clear spatial heterogeneity was identified in these results. In this study, pigs were sampled during January to May hence the possible impact of seasonality should be considered. Most of the pigs sampled in this study would have been born in late summer/ early autumn and this may have a bearing on their exposure and sero-status. It is difficult to gauge the precise public health implications of the findings for a number of reasons. Firstly, the correlation between seropositivity and the number of viable cysts of T. gondii in edible tissue has not yet been fully elucidated (EFSA 2007, ACMSF 2011). In addition, the relative contribution of the foodborne route of transmission to the overall human disease burden, as well as the contribution of different food vehicles, is unknown (EFSA 2007, ACMSF 2011). Thus, whilst the seroprevalence identified in this survey is considerably lower than that found in a recent survey of sheep in Great Britain, in which 74% of animals tested seropositive (Hutchinson et al., 2011), the significance of this difference to UK consumers is unclear.

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The data collected in this study goes some way to addressing one of the key recommendations of the ACMSF report (ACMSF 2011). The results provide a nationally representative baseline seroprevalence against which future survey results and the effectiveness of control measures can be monitored. However, a number of other data gaps remain which will be imperative to explore before the scale of the risk posed by pork and pork products can be accurately inferred. Hepatitis E virus This is the first survey of HEV in slaughter pigs in the UK. In a previous study on one UK pig herd, HEV seroprevalence (anti-HEV antibody) was roughly estimated to be 85%, although this was obtained from the testing of only 40 pigs (Banks et al., 2004). The identity of the swine sequences for a 98 base pair fragment of one virus from this herd and a second virus from a different herd when compared to a single human isolate of HEV was 90.8% (Banks et al., 2004). The data produced from this current survey has shown that nine out of ten pigs have been infected with HEV by the time they enter the food chain and reflects similar findings in other countries (Leblanc et al., 2010; Wacheck et al., 2013). The correlation between HEV antibody and RNA suggested that at the time of slaughter, 267 (44.9%) pigs had evidence of a past infection, whilst 327 (55.1%) had evidence of a recently cleared infection. HEV RNA was detected in the plasma of 37 pigs, indicating the presence of viraemia compatible with a current HEV infection. Additional molecular characterisation studies indicated the virus found in the pig samples belonged to G3 and displayed genetic similarities to some group 1 HEV strains from human cases acquired outside the UK. Monitoring of HEV in England and Wales shows HEV in current cases of hepatitis E to fall into two phylogenetically distinct and separate genetic groups. Analysis of the phylogenetic tree by year has demonstrated a clear trend for clustering in time with the majority of sequences from 2003‐2009 clustering in group 1 whilst increasingly sequences from 2010‐2012 clustered in group 2 indicating the possibility of two coincidental endemic infections causing sequential epidemics currently occurring in England and Wales (Ijaz S et al., in press). Analysis of data from human cases diagnosed in 2012 shows 35% to have a group 1 virus and 65% to have a group 2 virus infection. Current data from studies in English blood donors indicate that over the past year the proportion of G3 viruses in group 2 has increased significantly and the prevalence of viraemia at the time of donation is 1 in 2800 equating to a 0.1-0.2% annual attack rate in England and Wales (personal communication, NHSBT/PHE study group). The viruses sequenced from six plasma samples in pigs included in this survey fall into the group 1 virus phylogeny. Group 2 infections were not seen in UK pigs in this study suggesting that the majority of HEV infections in the UK human population are unlike/may not have originated from UK domestically-produced pig meat. However, we accept that this analysis is based on only a small number of samples. Human plasma containing HEV RNA is infectious indicating that RNA detection is a marker for viraemia though in the human situation coincidence of anti-HEV with viraemia appears to reduce infectivity of the plasma. Thus, the detection of plasma HEV RNA in pigs is similarly likely to represent viraemia at the time of slaughter it is plausible that the tissue load associated with this viraemia might be infectious per orem in humans as has been reported previously (Colson et al., 2010; Tei et al., 2003; Masuda et al., 2005).

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If G3 group 2 viruses found in people in the UK are imported in food, then it is possible that a proportion of the human G3 group 1 viruses may also be imported as well. Though HEV G3 is relatively benign as a human pathogen, consideration has to be given to this widespread zoonosis and its control. Exhorting consumers to cook food adequately would be an option. Given the transmissibility of porcine HEV infection (de Deus et al., 2008; Casas et al., 2009) in swine, elimination of HEV is also unlikely and the remaining option would seem to be containment and adopting a strategy to reduce the likelihood of being viraemic at slaughter. Immunisation or early exposure leading to clearance of virus by the time of slaughter would be options. Ultimately though, information regarding the origin and co-infection(s) of each pig will be critical to analytical interpretation of the data and will lead to a better understanding of the dynamics of HEV infection in our national swine herd. Yersinia spp. The prevalence of Y. enterocolitica carriage was significantly higher in this study compared with the 2003 UK abattoir survey [28.7% (95% CI 24.8-32.7) versus 10.2% (95% CI 8.9–11.5)] (Milnes et al., 2008) and is higher than Y. enterocolitica carriage reported in other studies (Liang et al. 2012; O’Sullivan et al., 2011) however the studies are not directly comparable. In this study tonsil samples were tested for Yersinia spp. compared to caecal samples in the 2003 survey. Higher rates of carriage were found in the 2003 survey during December to May, and also from January to March in a study by Bhaduri et al. (2005) which includes the sampling timeframe for this study. Therefore the increase seen may be, in part, an artefact of the study design; if sampling had been carried out throughout the year, lower isolation rates may have been observed thus reducing the overall prevalence. The apparent rise in the prevalence of Yersinia should be treated with caution given the lack of a comparable method across the studies. Y. pseudotuberculosis was identified in 10.3% of the positive pigs (3.4% prevalence overall); in a previous study in England by Ortiz Martinez et al. (2010) 18% of the pigs were found to carry Y. pseudotuberculosis. This is the first time a UK-wide study, representative of the UK pig population, has been undertaken to assess the contamination of carcases with Yersinia. Although over one quarter of the pigs were found to be carrying Y. entercolitica, very few carcases (≤ 2%) were contaminated with this organism. Given that more than one quarter of pigs in this study were found to carry Y. entercolitica it is encouraging that so few carcases were found to be contaminated with the organism indicating that the processes applied at the abattoir to reduce contamination of the carcases are having a positive effect. This is similar to the prevalence of carcase contamination observed in pigs at slaughter in Italy (Bonardi et al., 2013). In Sweden, pathogenic Y.enterocolitica was detected by PCR in 16% of the carcase swab samples (Lindblad at al., 2007). PCR was not undertaken on the carcase samples in this study and so a direct comparison cannot be made however one would expect the prevalence by PCR to be higher than traditional culture methods (as seen in the study by Bhaduri et al., 2005 and Liang et al., 2012). The method used in this study was based on a UKAS accredited procedure for the isolation of Yersina enterocolitica in faeces. A study by Van Damme et al. (2013) found that carcase swab samples collected under official monitoring in Belgium and tested by the ISO 10273:2003 method resulted in an underestimate of contamination with Yersinia enterocolitica. Of 254 samples taken, only eight out of the 29 Yersinia-positive carcasses were detected using this method. Although

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the Van Damme study is not directly comparable to ours, if future sampling is undertaken it would be beneficial to compare sampling methodology to optimise detection. Y. enterocolitica is divided into six biotypes (1A, 1B, 2, 3, 4, and 5) and numerous serotypes. Most Y. enterocolitica types associated with human infections belong to bioserotypes 1B/O:8, 2/O:9, 3/O:3, 4/O:3, and 2/O:5,27. In a previous study of English pigs at slaughter, the most common biotypes of Y. enterocolitica were 2/O:9 (33%) and 2/O:5 (26%) (Ortiz Martinez et al., 2010). Biotyping of the isolates was not undertaken in this study because of the low prevalence and therefore hazard on the carcasses, so the predominant type and range of biotypes cannot be reported. A number of farm management practices have been associated with the carriage of Y. enterocolitica in pigs (Vilar et al., 2013; Virtanen et al., 2011). A number of variables associated with the farm and abattoir were collected alongside the samples in this study. Thus a limited risk factor analysis to explore possible associations with Yersinia carriage could be undertaken. Approximately one quarter of slaughter pigs were found to be infected with Y. enterocolitica. However, the controls in place in abattoirs are effective in preventing widespread contamination of carcases. Porcine reproductive and respiratory syndrome virus The seroprevalence of PRRSv observed in this study was 58.3% (95% CI 53.1-63.4). This is similar to the GB herd seroprevalence of 56% reported from non-randomised, non-structured testing of 356 pig herds between 2001 and 2003 (Richardson, 2004). Velasova et al.(2012) reported a lower prevalence estimated from a cross-sectional study of English pig farms during 2008-09. That study also differed from this one in that it was assessing the prevalence at herd level and also only classified vaccinated herds as PRRSv-positive if there was evidence of virus from PCR testing of growing pigs. Evans et al (2008) also reported a seroprevalence at herd-level of 39.8% from a cross-sectional study of 103 pig herds in 2003-2004 with an additional 26.2% classified as vaccinated. The seroprevalence assessed in this study is national pig level measured at the point of slaughter which has not been measured previously in England and Wales. The structured wide geographic coverage of this UK survey, combined with the randomised sampling approach, provides a representative estimate of the seroprevalence of PRRSv in UK pigs at slaughter. However, a limitation of this estimate is that antibodies made in response to vaccination cannot be distinguished from those made in response to field PRRSv infection and the seroprevalence is therefore likely to be an over-estimate of the true seroprevalence to field PRRSv only. No data was available on the PRRSv vaccination status of the sampled pigs. In GB, vaccination of rearing pigs is not inexpensive and is generally undertaken in rearing pigs only when there is a strong expectation of challenge by field virus during the rearing period. Pig veterinarians estimate that between 10% and 55% of growing pigs are currently vaccinated for PRRSv in East Anglia (unpublished observations) and reports from 110 commercial pig units elsewhere in GB in 2013 indicated that approximately 11% were vaccinating growing pigs for PRRSv (BPEX, Health and Welfare monitoring project). Interestingly, the seroprevalence in older pigs (>12 months) of breeding age, where PRRSv vaccination is more widely practised, was significantly lower than in younger pigs in this study. The presence of PRRSv RNA in the

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tonsils of a proportion of seropositive pigs confirms field virus challenge in a proportion of the seropositive pigs. The carcase dead weights were recorded for all pigs less than six months old except one, and were at least 50kg. This indicates that the sampled pigs were too old for the PRRSv ELISA to be detecting maternal antibody and the results reflect an active immune response in the sampled pig to PRRS virus (field or vaccine). The PCR testing detected PRRS viral RNA in 8.56% of PRRSv seropositive pigs (31/362). PRRS virus usually persists in tonsils for several weeks post-infection and has been shown to be viable for up to 130 days by virus isolation in experimental conditions. While only viral nucleic acid (RNA) is detected by PCR rather than infectious virus particles, this is a standard approach to PRRS virus detection (OIE 2012). Detection of viral RNA is indicative of the presence of whole virus, given the inherent instability of viral RNA in animal tissues, and while this is not evidence that the virus is viable, it is suggestive of such. No PCR-positive tonsils were from pigs in the early stages of primary infection as the PCR-positive tonsils were from pigs with antibody to PRRSv; the ELISA can detect antibody to PRRSv from around 9-10 days post-infection. However, it is possible that the PRRSv RNA detected in the tonsils could derive from exposure of seropositive pigs to virus during transport or lairage if pigs were kept overnight before slaughter. Virus-positive cells have been found in tonsils as early as 12 hours post-infection (Rossow et al., 1996), and while this data is derived from experimental infection of gnotobiotic pigs, where infection would be particularly successful, it indicates that there is the possibility that the RNA detected in the tonsils could be derived from very recent re-exposure to virus rather than persisting from infection on farm. As PRRS vaccination of growing pigs is usually carried out at or near weaning, it is very unlikely that the PCR-positive tonsils identified in this study derive from recently vaccinated animals. As tonsils from most seronegative pigs were not tested by PCR, it is possible that detection of a few PRRSv-positive pigs in the early stages of infection were missed, although PRRSv RNA was not detected in any of the 28 tonsils from seronegative pigs in the pilot study Importantly, all PCR positive samples were of genotype 1 (European) PRRSv which is endemic in the UK and none were genotype 2 PRRSv, which is exotic to the UK. This provides further evidence of freedom from genotype 2 PRRSv (Frossard et al., 2012 and 2013). Assessing the presence of PRRS viral RNA in the tonsils as well as evidence of exposure during the growing period (antibody) has revealed that a proportion of healthy pigs may have been infectious at slaughter. Contaminated vehicles are a recognised means of transmission of PRRSv to uninfected units or of recirculation/reintroduction of infection onto units which are already infected and attempting control. It is also a means of transmitting different PRRSv strains between units. Detection of PRRS viral RNA in at least 1 in 20 slaughter pigs overall (tonsils of antibody negative pigs were not tested for viral RNA), and over 8% of seropositive pigs, emphasises the potential risk of transmission and underlines the need for good biosecurity and rigorous cleaning and disinfection during and after transport to slaughter. These are messages which the industry will wish to promote and are also key messages for reducing the risk of spread of other contagious pathogens including those causing notifiable disease.

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The detection of PRRS viral RNA in slaughter pigs from this survey has also helped indicate that tonsils from slaughter pigs may be a useful sampling methodology for future PRRSv surveys and project work. Sequencing of one PRRSv gene in the samples from PCR-positive tonsils was undertaken to add to information on PRRSv diversity which, from AHVLA surveillance data, is already known to be increasing. Of the samples successfully sequenced from 23 pigs, three showed greater than 98.5% similarity of their ORF5 gene to that of the Porcilis PRRSv vaccine. Their ORF5 gene homologies were 99.2%, 99.8% and 100% to vaccine virus. These are termed ‘vaccine-like’ and represent 13% of the sequenced PRRSv PCR-positive samples. While only one gene was sequenced and could be compared in this study, the data suggests that these may derive from vaccine and studies elsewhere using the same genetic region have detected vaccine virus in both vaccinated and non-vaccinated pigs (Grosse Beilage et al.,2009). Similar vaccine-like viruses have been identified in GB in recent years, although at a lower rate (Frossard et al., 2013), possibly because most isolates sequenced in recent years are from disease outbreaks whereas these were detected in clinically normal pigs. The other 20 isolates showed between 88.4% and 98.3% similarity to PRRSv vaccine. The degree of genetic difference of a field PRRS virus from the vaccine strain cannot, alone, predict the degree of protection that would be afforded by the vaccine to infection by the field virus. In general terms, as genetic diversity increases, the probability that corresponding antigenic differences could affect immunity also increases. The diversity of PRRS viruses in the OZ0150 samples reflects the continuing increase in diversity of PRRSv which has been detected in recent years in other British submissions (all genotype 1), with no distinct novel clades being found. PRRSv strains are generally considered to be closely related if the homology is 98.5% or more. It is difficult to state a homology level which indicates that strains are significantly different, especially as this analysis is based on just one gene of the virus. Some of the viruses are sufficiently similar to one another to be potentially linked epidemiologically and several of these were detected in pigs from different geographic regions. AMR in Campylobacter coli Campylobacter was isolated from 84.7% (95% CI 79.9-89.4) of the pigs tested in the study, which is significantly higher than in the 2003 abattoir study (69.3%, 95% CI 65.2–73.2) (Milnes et al., 2008); however, the methodology differs between the studies. This study was not designed to provide a prevalence estimate, but rather to maximise the isolates available for subsequent susceptibility testing. As expected, the vast majority of the positive pigs were infected with C. coli, although infections with both C. jejuni and C. lari were also identified. These three Campylobacter species are most commonly associated with human infections (EFSA and ECDC, 2013). The number of C. coli isolates tested for antimicrobial resistance fell slightly short of the 170 required by the EU legislation but additional porcine C. coli isolates will be sourced from the stored caecal samples and tested in order to reach the target for submission to the EU. Of the C. coli isolates tested 77.8% were resistant to tetracyclines and 66.0% to streptomycin. Previous abattoir surveys used a different (breakpoint) method and

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susceptibility criteria to assess susceptibility. The breakpoint concentrations used in the previous abattoir studies are shown at Table 2. Resistance levels for these antimicrobials have been found to vary considerably throughout Europe but comparably high tetracycline and/ or streptomycin resistance has been recorded for C. coli isolates from pigs in several other countries (Danmap, 2012; de Jong, 2012; EFSA and ECDC, 2013; Gallay et al., 2007; Payot 2004; van Looveren et al., 2001). This may be related to the frequent as well as historical use of these compounds in veterinary medicine (VMD, 2010). Erythromycin is one of the first-line treatments for human campylobacteriosis cases when treatment is necessary. Macrolide resistance tends to be comparatively high among C. coli from pigs in Europe relative to other food animal species (EFSA and ECDC, 2013; Gibreel and Taylor, 2006). Previous surveys in other European countries have reported widely varying resistance levels (Aarestrup et al., 1997; Danmap, 2012; De Jong et al., 2012; Gallay et al., 2007; Payot 2004; Pezzoti et al., 2003; van Looveren et al., 2001) but the average among swine C. coli isolates tested in the EU in 2011 is comparable with the value obtained in this study of 24.5% (EFSA and ECDC, 2013). In quinolones, mutations in the topoisomerase enzymes (gyrA or parC) are frequently responsible for resistance (EFSA and ECDC, 2013). This usually confers resistance to both quinolones and fluoroquinolones in Campylobacter so the resistance levels for nalidixic acid and ciprofloxacin are typically similar. In this survey, 17.0% of the isolates were resistant to nalidixic acid and 12.4% were resistant to ciprofloxacin; the seven isolates resistant to the former but not the latter will be re-tested for confirmation. The resistance levels observed were, again, similar to those in the two previous abattoir surveys (10% and 16%, respectively, for ciprofloxacin, and 17% and 27%, respectively, for nalidixic acid); the previous surveys applied the same breakpoint although the methodology differed. Resistance in the UK thus appears to be at a similar or slightly lower level than in many other European countries, where ≥30% resistance has frequently been reported (Aarestrup et al., 1997, de Jong et al., 2012, EFSA and ECDC, 2013, Payot 2004, Pezzoti et al., 2003, van Looveren et al., 2001). Fluoroquinolones are also used for the treatment of campylobacter infections in human medicine, and although resistance to nalidixic acid and ciprofloxacin was observed in this study, it is encouraging to note that it does not appear to have increased since the abattoir survey a decade ago. No resistance was observed against either chloramphenicol or gentamicin among the isolates tested. In the abattoir surveys of 1999/2000 and 2003, chloramphenicol resistance was identified in 3% and 46% of C. coli isolates from pigs, respectively, although the high level observed in the latter survey is probably an artefact of the breakpoint used at that time, which lies within the normal susceptible population. Gentamicin resistance within the EU is generally low (Aarestrup et al., 1997; Danmap, 2012; EFSA and ECDC, 2013; de Jong, 2012; van Looveren et al., 2001). The considerable variation in resistance levels observed for the different antimicrobials within the EU may be largely attributable to variations in usage practices. Whilst most human Campylobacter infections are self-limiting, some require treatment with antibiotics. Infections with resistant Campylobacter strains have been shown to be associated with an increased risk of invasive illness or death (Helmset al., 2005; Melbak,

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2005), although invasive campylobacter infection is rare. In this survey, only nine C. coli isolates were sensitive to all antimicrobials tested, whilst more than a quarter were resistant to three or more antimicrobials. Eight isolates expressed co-resistance to ciprofloxacin, erythromycin and tetracycline, a combination which includes two of the antimicrobials of key interest in the treatment of campylobacteriosis. In the joint scientific opinion from ECDC, EFSA and EMA, resistance to quinolones (including fluoroquinolones) and macrolides in Campylobacter was regarded as of high relevance for public health based on the possible human health consequences (EFSA, 2009b). Whilst it is reassuring to note the relative stability in resistance levels to ciprofloxacin since the previous abattoir surveys and the degree to which the results from the UK are comparable to other EU member states, the continuing high levels observed for some antimicrobials remains a concern, particularly given the continuing significant rise in human Campylobacter infections in both the UK and Europe as a whole (EFSA and ECDC, 2013). As most MSs appear to have adopted the guidance issued by EFSA in 2007 regarding AMR monitoring in Campylobacter (EFSA, 2007; EFSA and ECDC, 2013), increasingly harmonised and comparable data should become available over coming years which will facilitate a better assessment of trends and between-country comparisons. Extended Spectrum Beta Lactamase E. coli Previous studies focusing on ESBLs in pigs have shown varying prevalence and ESBL types in different countries. In Switzerland, a prevalence of ESBL bacteria of 15.2% was recorded for ESBLs in faecal samples, although only 59 pigs were examined (Geser et al., 2011), whilst in a larger study in Korea, 21.5% of 558 swine faecal samples were positive for CTX-M E. coli (Tamang et al., 2013). In a longitudinal study of 3 farms in Denmark, the percentages of ESBL-coliform positive pigs varied from 12% for finishing pigs to 58% just before weaning (Hansen et al., 2013). In our study, 23.5% of 637 pigs gave rise to an ESBL phenotype isolate on agar, of which 22.0% were confirmed to be CTX-M E. coli, and 2.2% were confirmed to be SHV-12 E. coli (some pig samples were positive for both CTX and SHV-12). This is similar to the prevalence observed in the Korean study (Tamang et al., 2013), but slightly higher than the small study in Switzerland (Geser et al., 2011), whilst both higher and lower than the percentages of ESBLs in the different animal groups in the longitudinal study in Denmark (Hansen et al., 2013). In this study, the most common CTX-M type was CTX-M 1 and this was also observed in the study in Switzerland (Geser et al., 2011). Conversely, in Korea the most common CTX-M type in pigs was CTX-M 14 (Tamang et al., 2013), which comprised only 2.1% of CTX-M ESBL positive pigs in the UK. Other CTX-M types seen in isolates from pigs in Korea were CTX-M-3, CTX-M-15, CTX-M-27, CTX-M-55, and CTX-M-65 (Tamang at el., 2013). All of these CTX-M types were also seen in isolates from pigs in this study with the exception of CTX-M 65. A few pigs in our study were also positive for SHV-12, which has been reported as a major ESBL type in pigs from Spain (Escudero et al., 2010). In Europe, the most common CTX-M type found in chickens also tends to be CTX-M 1 (Randall et al., 2011, Dierikx et al., 2010, Meunier et al., 2006). However the main CTX-M sequence types seen in humans in the UK are CTX-M-15/-3 and, much less frequently, CTX-M-14/-9 (Tarrant et al., 2007).

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The use of two agars for isolation of ESBLs improved the isolation rate and made the study more robust. In particular, the SHV-12 strains were only isolated from the ESBL Brilliance agar (although these did grow on CHROMagar CTX, but as very small colonies at 24 hours), whilst the CHROMagar CTX resulted in the isolation of CTX-M E. coli from a higher number of pigs than CTX-M isolates from the ESBL Brilliance agar. In conclusion, this study has shown that 22% of pigs in the UK harbour mainly CTX-M 1 ESBL E. coli, and this is similar to the percentages of pigs colonised with ESBLs in some other countries. CTX-M-1 is the enzyme most frequently detected in scanning surveillance of clinical diagnostic material submitted to AHVLA from both pigs and poultry. In the UK, CTX-M 1 is not one of the ESBL types commonly associated with human ESBL E. coli infections. Conclusion This study has filled a number of data gaps and provided a snapshot and distribution of a number of potentially zoonotic organisms, organisms of pig health concern and antimicrobial resistance patterns. It will enable government and industry policy makers to clarify and prioritise work towards controlling these pathogens and reducing the risks from antimicrobial resistance along the pig meat supply chain. It will also enable the UK to provide evidence on antimicrobial resistance in pigs to the EU Commission as part of the mandatory annual returns on antimicrobial resistance in animals and humans. This study came about through a co-ordinated approach between government and industry that was initiated by Defra and was jointly funded. The result has been the delivery of a highly cost-effective study and collection of a large set of highly relevant data. Additional work on HEV is planned on the stored caecal contents and other testing of plasma and tonsil samples for evidence of swine influenza and PED infections is being considered. Through the extension and strengthening of links across government and Industry, this study has also enabled a more collaborative and broad approach to addressing public and animal health concerns in relation to pigs in the UK. Future planned work Caecal content samples have been stored at -70oC at AHVLA Weybridge for future HEV testing by AHVLA. A subset of randomly selected E. coli isolates has also been stored for future AMR testing. Tonsil samples and plasma samples have been stored for testing by AHVLA for antibodies to swine influenza and porcine epidemic diarrhoea (PED) viruses respectively (subject to industry approval and funding). In addition, the heart and tongue tissue from Toxoplasma seropositive pigs have been stored for future molecular investigations using nucleic acid amplification testing (NAAT). Further analysis of the data including a review of co-infections and multivariate analyses is planned, subject to funding, to determine risk factors associated with the infections. Acknowledgements This project was funded by Defra, the Food Standards Agency (FSA), the British Pig Executive (BPEX – a division of the Agriculture and Horticulture Development Board), the

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Veterinary Medicines Directorate (VMD), Public Health England (formerly the Health Protection Agency) and Public Health Wales. The PRRSv pilot study was funded from Defra-funded project ED1200 Scanning Surveillance for Diseases in Pigs in England and Wales. The authors thank Industry for supporting this work, the abattoirs for participating in this study, FSA Operations and DARD for collecting the samples and AHVLA colleagues for their contribution to the study. Project Team & colleagues contributing to the final report: Laura Powell (Project Leader and Epidemiologist, AHVLA Weybridge) Tanya Cheney (Deputy and Epidemiologist, AHVLA Weybridge) Rob Davies (Salmonella consultant, AHVLA Weybridge) Chris Teale (AMR consultant, AHVLA Weybridge) Luke Randall (ESBL E. coli, AHVLA Weybridge) Laboratory Services Department (AHVLA Bury) John Rodgers (Campylobacter speciation, AHVLA Weybridge) Fabrizio Lemma (ESBL E. coli, AHVLA Weybridge) Mary O’Mara (AHVLA Weybridge) Fen Palmer-Mole (AHVLA Weybridge) Andrew White (AHVLA Weybridge) Jane Errington (AHVLA Penrith) Jeremy Hawthorn (AHVLA Weybridge) Susanna Williamson (AHVLA Bury) Jean-Pierre Frossard (AHVLA Weybridge) PHE (Richard Tedder; Samreen Ijaz; Judith Heaney) PHW (Ed Guy) References Advisory Committee on the Microbiological Safety of Food (ACMSF) (2011). Risk profile in relation to Toxoplasma in the food chain. Available: http://www.food.gov.uk/multimedia/pdfs/consultation/criskproToxoplasmafoodchain.pdf. Accessed 05/01/11. Aarestrup FM, Nielsen EM, Madsen M, and Engberg J (1997) Antimicrobial Susceptibility Patterns of Thermophilic Campylobacter spp. from humans, Pigs, Cattle, and Broilers in Denmark Antimicrobial Agents and Chemotherapy 41(10): 2244-2250. AggarwalR, Naik S (2009) Epidemiology of hepatitis E: current status. J Gastroenterol Hepatol 24 (9):1484-93. Agersø Y, Aarestrup FM 2013. Voluntary ban on cephalosporin use in Danish pig production has effectively reduced extended-spectrum cephalosporinase-producing Escherichia coli in slaughter pigs. J Antimicrob Chemother 2013; 68: 569–572. Anderson ES and others (1977).Bacteriophage-typing designations of Salmonella Typhimurium. The Journal of Hygiene 78, 297-300.

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APPENDIX I

Objectives – as detailed in the EVID2

A study of Salmonella, Toxoplasma, Hepatitis E virus, Yersinia, Porcine Reproductive and Respiratory Syndrome virus,ESBL E. coli, Campylobacter and AMR in UK pigs at slaughter was carried out by AHVLA on behalf of the funders - Defra, FSA, BPEX, PHE and VMD.

The aims of the study were met by undertaking the following objectives:

No. Objective Met (Yes/ No)

1. To contribute to the planning and introduction of the study for Salmonella, Toxoplasma, Hepatitis E, Yersinia, ESBL E. coli, Campylobacter and AMR in UK pigs at slaughter.

Yes

2. To help construct a sampling frame of pig carcasses and abattoirs in the UK based on information supplied by FSA.

Yes

3. To randomly allocate the samples of pig carcasses from the sampling frame for each abattoir participating in the survey.

Yes

4. To work with funders to design and produce a questionnaire to gather epidemiological data on each sampled slaughter pig.

Yes

5. To design and produce a form to accompany samples to the laboratory.

Yes

6. To develop sampling protocols for the survey and to provide information to FSA for use in training for samplers.

Yes

7. To make up the sampling kits and send to the abattoirs as required.

Yes

8. To culture samples for Salmonella, undertake serotyping and phage typing on a subset of Salmonella isolates for UK.

Yes

9. To send plasma samples to the Toxoplasma Reference Unit in Swansea for testing for Toxoplasma. Tissue samples (heart and tongue) will also be sent with the plasma samples (tissue type to be confirmed by FSA Operations).

Yes

10. To send EDTA plasma sample to the Health Protection Agency, Colindale for HEV testing.

Yes

11. To process and archive caecal samples for future HEV testing.

Yes

12. To undertake testing of Yersinia, ESBL E. coli, Campylobacter and AMR.

Yes

13. To develop a database to manage the survey and hold the survey data.

Yes

14. To enter the epidemiological and testing data onto the database and to follow up any missing data or other

Yes

46

identified problems with the data.

15. To analyse the prevalence results for each disease/ organism and to produce a report on the findings of the baseline survey. (Toxoplasma results and HEV results to be sent to AHVLA by the Toxoplasma Reference Unit and HPA respectively for inclusion in the UK report).

Yes

16. To participate in meetings with Defra, FSA, BPEX, HPA and VMD as required to update them on progress with the project.

Yes

17. To submit a report on the prevalence of Salmonella, Toxoplasma, Hepatitis E virus, Yersinia, Porcine Reproductive and Respiratory Syndrome virus andESBL E. coli and AMR in C. coli in UK pigs at slaughter.

Yes

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APPENDIX II Number of pigs sampled and sampling days by abattoir (n=626)

Abattoir Number of pigs included in all

analyses

Number of sampling days

Minimum number of

pigs sampled per

day

Maximum number of

pigs sampled per

day

Median number of

pigs sampled per day

A 39 32 1 2 1

B 66 43 1 5 1

C 54 25 1 6 2

D 28 26 1 2 1

E 47 36 1 2 1

F 96 45 1 3 2

G 53 43 1 2 1

H 62 48 1 2 1

I 15 15 1 1 1

J 48 34 1 2 1

K 17 16 1 2 1

L 31 31 1 1 1

M 11 11 1 1 1

N 59 43 1 3 1

Total 626

Number of pigs sampled by day of the week (n=626)

Day of sampling Number of pigs % of pigs

Monday 121 19%

Tuesday 165 26%

Wednesday 136 22%

Thursday 133 21%

Friday 71 11%

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APPENDIX III ECOFFs and breakpoint concentrations used for Campylobacter spp. susceptibility testing in the current and previous abattoir surveys

Antimicrobial ECOFF mg/L R> used in study

Breakpoint concentration (mg/l) used in 1999/2000

and 2003 studies.

Ampicillin Not tested 32 Chloramphenicol Not in the

recommendations but the ECOFF is >16

8

Erythromycin 16 4 Trimethoprim Not tested 2 Kanamycin Not tested 16 Tetracycline 2 8 Tetracycline - 128 Nalidixic acid Not in the

recommendations but the ECOFF is >16

16

Furazolidone Not tested 8 Ciprofloxacin 1 1 Gentamicin 2 Not tested Streptomycin 4 Not tested

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Tables & Figures: Overall Table1: Number of pigs sampled by region (NUTS1 code) and population by region (n=626)

NUTS1 region Number of pigs –

2013 study % of pigs

Population -Number of

pigs* % of pigs

North East England 13 2.1% 89,052 2.00

North West England 19 3.0% 137,426 3.09

Yorkshire and the Humber 207 33.1% 1,223,649 27.52

East Midlands 62 9.9% 344,924 7.76

West Midlands 10 1.6% 187,851 4.22

East of England 143 22.8% 1,031,991 23.21

South East 12 1.9% 202,446 4.55

South West 46 7.3% 386,416 8.69

Wales 2 0.3% 25,809 0.58

Scotland 28 4.5% 389,995 8.77

Northern Ireland 84 13.4% 426,900 9.60

* Figures for Wales, Scotland and Northern Ireland are for 2012 (data source - UK Zoonoses Report) Figures for GB are from the 2010 Agricultural Survey

Table 2: Number of pigs sampled by age and production system Production system

Age Total

<6 months 6-12 months >12 months Not known

Breeding and finishing

4 81 42 0 127

Finishing only 19 310 0 6 335

Not known 32 114 14 4 164

Total 55 505 56 10 626

Salmonella Table 3: Agreement of Salmonella result between sample types (overall)

Sample type Rectal swab Carcase swab

Positive Negative Positive Negative

Caecal contents

Positive 72 117 25 164

Negative 78 351 33 397

Agreement: 68.5% Kappa: 0.21 Agreement: 68.2% Kappa: 0.07

Carcase swab

Positive 25 35

Negative 125 438

Agreement: 74.3% Kappa: 0.12

Table 4a: Agreement of Salmonella result between sample types (S. Typhimurium only)

Sample type Rectal swab Carcase swab

50

Positive Negative Positive Negative

Caecal contents

Positive 9 27 2 34

Negative 11 571 6 577

Agreement: 93.9% Kappa: 0.29 Agreement: 93.5% Kappa: 0.07

Carcase swab

Positive 1 8

Negative 19 595

Agreement: 95.7% Kappa: 0.05

Table 4b: Agreement of Salmonella result between sample types (S.4,12:i:- only)

Sample type Rectal swab Carcase swab

Positive Negative Positive Negative

Caecal contents

Positive 5 28 3 30

Negative 13 572 13 573

Agreement: 93.4% Kappa: 0.16 Agreement: 93.1% Kappa: 0.09

Carcase swab

Positive 3 13

Negative 15 592

Agreement: 95.5% Kappa: 0.15

Table 4c: Agreement of Salmonella result between sample types (S.4.5,12:i:- only)

Sample type Rectal swab Carcase swab

Positive Negative Positive Negative

Caecal contents

Positive 8 24 2 30

Negative 21 565 9 578

Agreement: 92.7% Kappa: 0.22 Agreement: 93.7% Kappa: 0.07

Carcase swab

Positive 3 9

Negative 26 585

Agreement: 94.4% Kappa: 0.12

Table 4d: Agreement of Salmonella result between sample types (S. Derby only)

Sample type Rectal swab Carcase swab

Positive Negative Positive Negative

Caecal contents

Positive 11 16 4 23

Negative 17 574 4 588

Agreement: 94.7% Kappa: 0.37 Agreement: 95.6% Kappa: 0.21

Carcase swab

Positive 4 4

Negative 24 591

Agreement: 95.5% Kappa:0.21

Table 5: Salmonella prevalence in the caecal contents by age (n=609) Age Number

tested Number positive

Prevalence

<6 months 54 14 25.9% (95% CI 13.6-38.3)

51

6-12 months 501 149 29.7% (95% CI 25.1-34.4)

>12 months 54 22 40.7% (95% CI 26.9-54.6)

p=0.21(p=0.09with test for trend) Table 6: Salmonella prevalence in the carcase swabs by age (n=614)

Age Number

tested

Number

positive

Prevalence

<6 months 55 4 7.3% (95% CI 0.0-14.5)

6-12 months 504 49 9.7 % (95% CI 7.2-12.3)

>12 months 55 6 10.9 % (95% CI 2.6-19.2)

p=0.79 (p=0.52 with test for trend)

52

53

Table 7: Salmonella serovars identified in the study

Serovar

Caecal contents Carcase swab Rectal swab Overall

No. positive samples

% of positive samples (n=189)

% of samples tested

(n=619)

No. positive samples

% of positive samples (n=60)

% of samples tested

(n=624)

No. positive samples

% of positive samples (n=150)

% of samples tested

(n=625)

No. positive

pigs

% of positive

pigs (n=291)

% of pigs tested

(n=626)

4,12:i:- 33 17.5% 5.3% 16 26.7% 2.6% 18 12.0% 2.9% 56 19.2% 8.9%

4,5,12:i:- 32 16.9% 5.2% 12 20.0% 1.9% 29 19.3% 4.6% 61 21.0% 9.7%

Agona 2 1.1% 0.3% - - - - - - 2 0.7% 0.3%

Bovismorbificans 20 10.6% 3.2% 5 8.3% 0.8% 23 15.3% 3.7% 41 14.1% 6.5%

Choleraesuis var Kunzendorf

- - - 1 1.7% 0.2% 3 2.0% 0.5% 3 1.0% 0.5%

Derby 27 14.3% 4.4% 8 13.3% 1.3% 28 18.7% 4.5% 47 16.2% 7.5%

Dublin - - - - - - 1 0.7% 0.2% 1 0.3% 0.2%

Goldcoast 3 1.6% 0.5% 1 1.7% 0.2% 4 2.7% 0.6% 6 2.1% 1.0%

Hindmarsh 1 0.5% 0.2% - - - - - - 1 0.3% 0.2%

Kedougou 6 3.2% 1.0% - - - 5 3.3% 0.8% 10 3.4% 1.6%

Livingstone - - - - - - 1 0.7% 0.2% 1 0.3% 0.2%

London 5 2.6% 0.8% - - - 3 2.0% 0.5% 7 2.4% 1.1%

Mbandaka 1 0.5% 0.2% - - - - - - 1 0.3% 0.2%

Newport - - - - - - 1 0.7% 0.2% 1 0.3% 0.2%

Ohio 1 0.5% 0.2% - - - - - - 1 0.3% 0.2%

Panama 6 3.2% 1.0% 1 1.7% 0.2% 4 2.7% 0.6% 9 3.1% 1.4%

Reading 8 4.2% 1.3% 1 1.7% 0.2% 3 2.0% 0.5% 9 3.1% 1.4%

Rissen 3 1.6% 0.5% 2 3.3% 0.3% 4 2.7% 0.6% 7 2.4% 1.1%

Stanley 5 2.6% 0.8% 2 3.3% 0.3% 3 2.0% 0.5% 7 2.4% 1.1%

Typhimurium 36 19.0% 5.8% 9 15.0% 1.4% 20 13.3% 3.2% 54 18.6% 8.6%

O_ROUGH:I:- - - - 1 1.7% 0.2% - - - 1 0.3% 0.2%

54

O_ROUGH:R:1,5 - - - 1 1.7% 0.2% - - - 1 0.3% 0.2%

Total 189 60 150 327*

* 36 pigs tested positive for 2 different serovars Table 8: Salmonella phage types identified in the study

Serovar Phage type

Caecal contents Carcase swab Rectal swab Overall

No. positive samples

% of positive samples (n=189)

% of samples tested

(n=619)

No. positive samples

% of positive samples (n=60)

% of samples tested

(n=624)

No. positive samples

% of positive samples (n=150)

% of samples tested

(n=625)

No. positive

pigs*

% of positive

pigs (n=291)

% of pigs tested

(n=626)

4,1

2:i:

-

120 6 3.2% 1.0% 1 1.7% 0.2% 1 0.7% 0.2% 8 2.7% 1.3%

193 24 12.7% 3.9% 9 15.0% 1.4% 13 8.7% 2.1% 38 13.1% 6.1%

208 - - - - - - 1 0.7% 0.2% 1 0.3% 0.2%

UNTY 3 1.6% 0.5% 6 10.0% 1.0% 3 2.0% 0.5% 12 4.1% 1.9%

4,5

,12

:i:-

104b 1 0.5% 0.2% 1 1.7% 0.2% 2 1.3% 0.3% 4 1.4% 0.6%

120 4 2.1% 0.6% - - - 2 1.3% 0.3% 6 2.1% 1.0%

193 21 11.1% 3.4% 6 10.0% 1.0% 21 14.0% 3.4% 40 13.7% 6.4%

U302 1 0.5% 0.2% - - - - - - 1 0.3% 0.2%

U311 - - - 1 1.7% 0.2% - - - 1 0.3% 0.2%

U323 3 1.6% 0.5% 4 6.7% 0.6% 3 2.0% 0.5% 7 2.4% 1.1%

NOPT 1 0.5% 0.2% - - - - - - 1 0.3% 0.2%

UNTY 1 0.5% 0.2% - - - 1 0.7% 0.2% 2 0.7% 0.3%

Tyh

imu

riu

m

32 2 1.1% 0.3% - - - - - - 2 0.7% 0.3%

104 2 1.1% 0.3% - - - - - - 2 0.7% 0.3%

104b 2 1.1% 0.3% - - - - - - 2 0.7% 0.3%

55

120 4 2.1% 0.6% - - - - - - 4 1.4% 0.6%

193 11 5.8% 1.8% 2 3.3% 0.3% 7 4.7% 1.1% 18 6.2% 2.9%

208 2 1.1% 0.3% 1 1.7% 0.2% 2 1.3% 0.3% 5 1.7% 0.8%

U288 6 3.2% 1.0% 2 3.3% 0.3% 6 4.0% 1.0% 12 4.1% 1.9%

U302 3 1.6% 0.5% 2 3.3% 0.3% 3 2.0% 0.5% 7 2.4% 1.1%

UNTY 4 2.1% 0.6% 2 3.3% 0.3% 2 1.3% 0.3% 8 2.7% 1.3%

* 3 pigs had more than one S. 4,12:i:- definitive phage type, 1 pig had more than one S. 4,5,12:i:- definitive phage type and 6 pigs had more than one S. Typhimurium definitive phage type

56

57

Toxoplasma Table 9: Toxoplasma seroprevalence by age (n=610)

p=0.42 (p=0.25 with test for trend) Hepatitis E virus Table 10: Agreement between HEV ELISA and RNA results (n=640)

Anti-HEV Plasma HEV RNA Age (months)

Total Ab

IgM No. No. +ve

Range Infection <6 6-12 >12

- 3 low evel IgM*

46 7 45-96 iu/ml (+4 BLQ)

early acute 0 7 0

+ + 327 20 27-5.95e+05 (+11 BLQ)

acute 1 18 1

+ - 267 10 All BLQ recovery ** 0 6 3 * 3.4, 3.6 and 4.9 au/ml ** one age not recorded BLQ – Below Limit of Quantification

Age Number tested

Number positive

Prevalence

<6 months 55 3 5.5% (95% CI 0.0-11.6)

6-12 months 501 33 6.6% (95% CI 4.3-8.9)

>12 months 54 6 11.1% (95% CI 2.1-20.1)

58

59

Yersinia spp. Table 11: Yersinia species identified in the study

Species

Carcase swab Tonsils Overall

No. positive samples

% of positive samples (n=12)

% of samples tested

(n=624)

No. positive samples

% of positive samples (n=204)

% of samples tested

(n=620)

No. positive

pigs

% of positive pigs (n=206)

% of pigs tested

(n=624)

Y. enterocolitica 11 91.7% 1.8% 178 87.3% 28.7% 179 86.9% 28.7%

Y. frederiksenii/ intermedia

1 8.3% 0.16% 1 0.49% 0.16% 2 0.97% 0.32%

Y. kristensenii - - - 4 2.0% 0.65% 4 1.9% 0.64%

Y. pseudotuberculosis - - - 21 10.3% 3.4% 21 10.2% 3.4%

Total 12 204 206

60

61

Table 12: Yersinia prevalence in the tonsils by age (n=610) Age Number

tested Number positive

Prevalence

<6 months 54 14 25.9% (95% CI 14.8-37.0)

6-12 months 501 171 34.1% (95%CI 29.6-38.7)

>12 months 55 14 25.5% (95% CI 12.9-38.0)

p=0.22(p=0.95 with test for trend) PRRSv Table 13: PRRSv ELISA and PCR results from the pilot study (n=47)

Testing method ELISA

Positive Inconclusive Negative

PCR Positive 1 0 0

Negative 21 3 22

Table 14: PRRSv ELISA and PCR results from the full study (including pigs tested in the pilot study) (n=626)

Testing method ELISA

Positive Inconclusive Negative Not tested

PCR

Positive 31 0 0 0

Negative 326 11 0 4

Not tested 5 0 248* 1

*Includes ELISA negative pigs tested by PCR during the pilot study Table 15: PRRSv seroprevalence by age (n=611) Age Number

tested Number positive

Prevalence

<6 months 54 37 68.5% (95% CI 54.5-82.5)

6-12 months 501 300 59.9% (95% CI 54.3-65.5)

>12 months 56 18 32.1% (95% CI 15.0-49.3)

p=0.002(p=0.0001 withtest for trend ) Table 16: PRRSv PCR results by age

Age Number tested Number positive % positive

<6 months 34 4 11.8%

6-12 months 312 25 8.0%

>12 months 19 2 10.5%

N.B.The 2 positives in the oldest age category were recorded as 12-18 months old.

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Table 17: PRRSv sequencing results: similarities to Porcilis vaccine and other porcine samples and geographic region of origin PRRSv similarities and geographic origin Sample number Pig ID % similarity to Porcilis

Significant similarity to other OZ0150 samples

Significant similarity to other historic samples County of origin

SA14-059315 160 96.5% Pig ID 252 (99.5%), Pig ID 260 (98.7%) 7619_(14-P0182-02-12) (99.0%)

East Anglia

SA14-059322 167 88.4% East Riding and North Lincolnshire

SA14-059407 252 96.0% Pig ID 160 (99.5%) East Riding and North Lincolnshire

SA14-059415 260 96.2% Pig ID 160 (98.7%) 7619_(14-P0182-02-12) (98.8%) East Anglia

SA14-059476 321 92.1% 218_(14-P0141-03-05) (97.7%) Northern Ireland

SA14-059570 411 90.4% East Anglia

SA14-059598 439 90.2% Pig ID 346 (99.5%) North Yorkshire

SA14-059604 445 94.9% Pig ID 160 (97.7%) East Anglia

SA14-059662 502 90.2%

125_(15-P0128-05-11) (98.0%), 17511_(15-P0097-08-11) (98.0%), 11969_(15-P0026-04-12) (98.0%)

North Yorkshire

SA14-059755 593 98.3% Pig ID 619 (98.5%) 14033_(15-P0271-08-12) (98.0%) East Anglia

SA14-059760 598 89.9%

4143_(14-P0437-01-12) (98.7%), 4142_(14-P0437-01-12) (98.3%), 5419_(14-P0772-12-12) (98.3%)

East Riding and North Lincolnshire

SA14-059781 619 99.8% Pig ID 593 (98.5%) Gloucestershire, Wiltshire and Bristol/Bath

SA14-059801 637 91.2% Pig ID 497 (99.2%) Dorset and Somerset

SA14-059501 346 90.4% Pig ID 439 (99.5%) East Riding and North Lincolnshire

SA14-059719 557 100.0% East Riding and North Lincolnshire

SA14-059736 574 91.3% Lincolnshire

63

SA14-059746 584 89.4% East Riding and North Lincolnshire

SA14-059767 605 93.9% Pig ID 648 (99.2%) East Riding and North Lincolnshire

SA14-059812 648 94.7% Pig ID 605 (99.2%) Northern Ireland

SA14-059563 404 89.4% Lincolnshire

SA14-059816 652 94.2% Northern Ireland

SA14-053360 26 99.2% East Anglia

SA14-059657 497 90.9% Pig ID 637 (99.2%) Dorset and Somerset

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Figure 1: Phylogenetic Analysis of OZ0150 PRRSv ORF5 Nucleotide Sequences

Alignment of the sequences was performed with the ClustalW algorithm, and phylogenetic analyses (neighbour-joining method with bootstrap test) were conducted using MEGA software version 3.1. The scale bar represents one nucleotide change per hundred. The virus sequences obtained from the tonsil samples in this project are compared to those from 322 other British samples, 18 Irish samples, and the Porcilis® PRRS vaccine.

65

Co infections

Table 18: Number of infectious agents identified in each of the 610 pigs that were tested against the full panel of Salmonella (carriage in the caecum), Yersinia (carriage in the tonsils), Toxoplasma (seropositivity), HEV (seropositivity and/or RNA presence) and PRRSv (seropositivity and/or RNA presence)

Number of infectious agents

No. pigs % of pigs (n=610)

0 14 2.3%

1 117 19.2%

2 242 39.7%

3 190 31.2%

4 46 7.5%

5 1 0.2%

Table 19: Co-infections identified in the 610 pigs that were tested against each of Salmonella (carriage in the caecum), Yersinia (carriage in the tonsils), Toxoplasma (seropositivity), HEV (seropositivity and/or RNA presence) and PRRSv (seropositivity and/or RNA presence)

Co-infection No. pigs % of pigs (n=610)

HEV PRRSv 138 22.62%

HEV 104 17.05%

HEV PRRSv Salmonella 71 11.64%

HEV PRRSv Yersinia 70 11.48%

HEV Yersinia 52 8.52%

HEV PRRSv Salmonella Yersinia 34 5.57%

HEV Salmonella 34 5.57%

HEV Salmonella Yersinia 24 3.93%

HEV PRRSv Toxoplasma 12 1.97%

HEV Toxoplasma 7 1.15%

PRRSv 7 1.15%

HEV PRRSv Salmonella Toxoplasma 6 0.98%

HEV Salmonella Toxoplasma 6 0.98%

HEV Toxoplasma Yersinia 6 0.98%

HEV PRRSv Yersinia Toxoplasma 4 0.66%

PRRSv Salmonella 4 0.66%

PRRSv Yersinia 4 0.66%

66

Salmonella 3 0.49%

HEV Salmonella Yersinia Toxoplasma 2 0.33%

Salmonella Yersinia 2 0.33%

Yersinia 2 0.33%

HEV PRRSv Salmonella Toxoplasma Yersinia 1 0.16%

PRRSv Salmonella Yersinia 1 0.16%

PRRSv Toxoplasma 1 0.16%

Toxoplasma 1 0.16%

Table 20: Association between PRRSv seropositivity and Salmonella carriage (in the caecum)

Number tested for Salmonella carriage

in caecum

Number (%) positive

PRRS Seropositive 358 117 (32.7%)

Seronegative/ Inconclusive

256 70 (27.3%)

p=0.19

Table 21: Association between PRRSv seropositivity and Yersinia carriage (in the tonsils)

Number tested for Yersinia carriage in

tonsils

Number (%) positive

PRRS Seropositive 359 115 (32.0%)

Seronegative/ Inconclusive

257 87 (33.9%)

p=0.67

Table 22: Association between Salmonella carriage (in the caecum) and Yersinia carriage (in the tonsils)

Number tested for Yersinia

Number (%) positive

Salmonella Positive 189 64 (33.9%)

Negative 426 139 (32.6%)

p=0.78

Table 23: Association between Salmonella/Yersinia carriage and ESBL E. coli

Number tested for ESBL E. coli

Number (%) positive

Salmonella/ Yersinia carriage

Positive 327 78 (23.9%)

Negative 291 66 (22.7%)

p=0.75

67

Campylobacter and AMR Table 24: Resistance levels observed in the C. coli isolates (n=153)

Antimicrobial Number resistant % resistant

Chloramphenicol 0 0.0%

Ciprofloxacin 19 12.4%

Erythromycin 42 27.5%

Gentamicin 0 0.0%

Nalidixic acid 26 17.0%

Streptomycin 101 66.0%

Tetracycline 119 77.8%

Table 25: Number of antimicrobials against which each C. coli isolate expressed resistance (n=153)

Resistance count Number of isolates

resistant % of isolates

0 9 5.9%

1 45 29.4%

2 57 37.3%

3 27 17.6%

4 8 5.2%

5 7 4.6%

Table 26: Resistance patterns identified in the C. coli isolates (n=153)

Resistance pattern Number of isolates % of isolates

S T 44 28.8%

T 26 17.0%

ERY S T 19 12.4%

S 17 11.1%

ERY T 9 5.9%

CIP ERY NA S T 7 4.6%

CIP NA S T 5 3.3%

CIP NA T 3 2.0%

CIP NA S 2 1.3%

ERY S 2 1.3%

ERY NA S T 2 1.3%

NA S T 2 1.3%

CIP NA 1 0.7%

CIP ERY NA T 1 0.7%

ERY 1 0.7%

ERY NA T 1 0.7%

NA 1 0.7%

68

NA S 1 0.7%

CIP: ciprofloxacin; ERY: erythromycin; NA: nalidixic acid; S: streptomycin; T: tetracycline ESBL E. coli Table 27: CTX-M sequence types identified in the study

CTX-M sequence type

No. pigs % of all pigs tested

(n=637) % of CTX-M ESBL E. coli-

positive pigs (n=140)

1 119 18.7% 85.0%

3 1 0.2% 0.7%

14 3 0.5% 2.1%

15 9 1.4% 6.4%

27 3 0.5% 2.1%

32 3 0.5% 2.1%

55 2 0.3% 1.4%

ND 1 0.2% 0.7%

Table 28: Genes identified in the presumptive ESBL E. coli-positive pigs (n=149)

ESBL Genes identified in pigs a

Other Beta-Lactamase

Genes Present b

Number of pigs c

% of all pigs tested (n=637)

% of ESBL positive pigs (n=149)

CTX total All results 140 22.00 93.96

CTX alone 111 17.43 74.50

CTX TEM 46 7.22 30.87

CTX TEM, OXA 5 0.78 3.36

CTX OXA 3 0.47 2.01

CTX-M d SHV-12 5 0.78 3.36

SHV-12 total All results 14 2.20 9.40

SHV-12 alone 8 1.26 5.37

SHV-12 TEM 6 0.94 4.03

SHV-12 OXA 0 0 0

SHV-12 d CTX 5 0.78 3.36

a – None of the SHV-12 genes were co-located in the same E. coli isolate as a CTX-M gene, so if present from the same pig, this represented two different isolates in that pig. Conversely, all of the OXA genes were co-located in the same E. coli isolate with either a CTX-M gene or an SHV-12 gene and in many cases a TEM gene also. The TEM

69

genes were also always co-located in the same E. coli isolate with either a CTX-M or SHV-12 gene, in addition to an OXA gene in some cases. b OXA and TEM genes were not tested for sequence type and as such it was not know if these genes conferred ESBL type resistance. These genes were never the only gene present in an isolate with a confirmed ESBL phenotype (e.g. CTX-M or SHV-12 gene always present in such isolates also). c - In many cases several E. coli were isolated from one pig, and in some cases these were CTX-M alone and CTX-M with TEM or another gene other than SHV. As such, the total gene combinations is higher than the total number of pigs positive for either CTX or SHV-12. d - CTX-M and SHV-12 gene on different isolate in the same pig.

70

Figures: Figure 1: Salmonella prevalence in the caecal contents by age (n=609)

Figure 2: Salmonella prevalence in the carcase swabs by age (n=614)

0 10 20 30 40 50 60

>12 months

6-12 months

<6 months

Age

Prevalence (%)

0 5 10 15 20

>12 months

6-12 months

<6 months

Age

Prevalence (%)

71

Figure 3: Toxoplasma seroprevalence by age (n=610)

Figure 4: Yersinia prevalence in the tonsils by age (n=610)

0 5 10 15 20 25

>12 months

6-12 months

<6 months

Age

Prevalence (%)

0 10 20 30 40 50

>12 months

6-12 months

<6 months

Age

Prevalence (%)

72

Figure 5: PRRSv seroprevalence by age (n=611)

Figure 6: PRRSv PCR results by age

0 20 40 60 80 100

>12 months

6-12 months

<6 months

Age

Prevalence (%)

0 5 10 15 20 25 30

>12 months

6-12 months

<6 months

Age

Prevalence (%)