Occurrence and characterization of rmtB-harbouring Salmonella and Escherichia coli isolates from a pig farm in the UK.

OBJECTIVES: To characterize and elucidate the spread of amikacin-resistant Enterobacteriaceae isolates from environmental samples on a pig farm in the UK, following the previous identification of index Salmonella isolates harbouring the rmtB gene, a 16S rRNA methylase. METHODS: Environmental samples were collected during two visits to a pig farm in the UK. Isolates were recovered using selective media (amikacin 128 mg/L) followed by real-time PCR and WGS to analyse rmtB-carrying Salmonella and Escherichia coli isolates. RESULTS: Salmonella and E. coli isolates harbouring the rmtB gene were detected at both farm visits. All Salmonella isolates were found to be monophasic S. enterica serovar Typhimurium variant Copenhagen of ST34. rmtB-harbouring E. coli isolates were found to be one of three STs: ST4089, ST1684 and ST34. Long-read sequencing identified the rmtB gene to be chromosomally located in Salmonella isolates and on IncFII-type plasmids in E. coli isolates. The results showed the rmtB gene to be flanked by IS26 elements and several resistance genes. CONCLUSIONS: We report on the occurrence of rmtB-harbouring Enterobacteriaceae on a pig farm in the UK. rmtB confers resistance to multiple aminoglycosides and this work highlights the need for surveillance to assess dissemination and risk.

Surveillance Focus; Causes of mortality in feral wild boar in the Forest of Dean.

This focus article has been prepared by Cat Man, veterinary lead of the APHA Wildlife Expert Group, and Susanna Williamson, veterinary lead of the APHA Pig Expert Group.

Investigation into an outbreak of Border disease virus in pigs in England.

Border disease (BD) was first reported in 1959 in lambs from the border region of England and Wales. The causative virus (BD virus; BDV) has since been identified in several other ruminant species and pigs. The virus is prevalent in sheep flocks of UK, Europe and USA and has potential to inflict substantial economic losses. Natural BDV infection of pigs was first reported in the UK in 1992 from pigs with haemorrhagic lesions and more recently from healthy pigs in Spain and Japan. Here, a persistent problem of poor growth and anaemia in a small proportion of growing pigs on a mixed pig and sheep holding was investigated and tissues were tested in a pan viral microarray. The microarray detected BDV RNA in several tissues which was further confirmed by sequencing, specific BDV PCR and immunohistochemistry. Phylogenetically, the virus clustered with other BDVs in the sub-genotype 1b. This investigation highlights likely interspecies transmission of pestiviruses and their impact on pestivirus detection and eradication programs.

Large-scale genomic analysis of antimicrobial resistance in the zoonotic pathogen Streptococcus suis.

BACKGROUND: Antimicrobial resistance (AMR) is among the gravest threats to human health and food security worldwide. The use of antimicrobials in livestock production can lead to emergence of AMR, which can have direct effects on humans through spread of zoonotic disease. Pigs pose a particular risk as they are a source of zoonotic diseases and receive more antimicrobials than most other livestock. Here we use a large-scale genomic approach to characterise AMR in Streptococcus suis, a commensal found in most pigs, but which can also cause serious disease in both pigs and humans. RESULTS: We obtained replicated measures of Minimum Inhibitory Concentration (MIC) for 16 antibiotics, across a panel of 678 isolates, from the major pig-producing regions of the world. For several drugs, there was no natural separation into 'resistant' and 'susceptible', highlighting the need to treat MIC as a quantitative trait. We found differences in MICs between countries, consistent with their patterns of antimicrobial usage. AMR levels were high even for drugs not used to treat S. suis, with many multidrug-resistant isolates. Similar levels of resistance were found in pigs and humans from regions associated with zoonotic transmission. We next used whole genome sequences for each isolate to identify 43 candidate resistance determinants, 22 of which were novel in S. suis. The presence of these determinants explained most of the variation in MIC. But there were also interesting complications, including epistatic interactions, where known resistance alleles had no effect in some genetic backgrounds. Beta-lactam resistance involved many core genome variants of small effect, appearing in a characteristic order. CONCLUSIONS: We present a large dataset allowing the analysis of the multiple contributing factors to AMR in S. suis. The high levels of AMR in S. suis that we observe are reflected by antibiotic usage patterns but our results confirm the potential for genomic data to aid in the fight against AMR.

Whole-Genome Sequencing of Brachyspira hyodysenteriae Isolates From England and Wales Reveals Similarities to European Isolates and Mutations Associated With Reduced Sensitivity to Antimicrobials.

Brachyspira hyodysenteriae is the principal cause of swine dysentery, a disease that threatens economic productivity of pigs in many countries as it can spread readily within and between farms, and only a small number of antimicrobials are authorized for treatment of pigs. In this study, we performed whole-genome sequencing (WGS) of 81 B. hyodysenteriae archived at the Animal and Plant Health Agency (APHA) from diagnostic submissions and herd monitoring in England and Wales between 2004 and 2015. The resulting genome sequences were analyzed alongside 34 genomes we previously published. Multi-locus sequence typing (MLST) showed a diverse population with 32 sequence types (STs) among the 115 APHA isolates, 25 of them identified only in England; while also confirming that the dominant European clonal complexes, CC8 and CC52, were common in the United Kingdom. A core-genome SNP tree typically clustered the isolates by ST, with isolates from some STs detected only within a specific region in England, although others were more widespread, suggesting transmission between different regions. Also, some STs were more conserved in their core genome than others, despite these isolates being from different holdings, regions and years. Minimum inhibitory concentrations to commonly used antimicrobials (Tiamulin, Valnemulin, Doxycycline, Lincomycin, Tylosin, Tylvalosin) were determined for 82 of the genome-sequenced isolates; genomic analysis revealed mutations generally correlated well with the corresponding resistance phenotype. There was a major swine dysentery intervention program in 2009-2010, and antimicrobial survival curves showed a significant reduction in sensitivity to tiamulin and valnemulin in isolates collected in and after 2010, compared to earlier isolates. This correlated with a significant increase in post-2009 isolates harboring the pleuromutilin resistance gene tva(A), which if present, may facilitate higher levels of resistance. The reduction in susceptibility of Brachyspira from diagnostic submissions to pleuromutilins, emphasizes the need for prudent treatment, control and eradication strategies.

Brachyspira hyodysenteriae isolate multilocus sequence type dashboard.

This focus article has been prepared by Susanna Williamson, Emma Stubberfield and Anna Brzozowska of the APHA and Jill Thomson of SRUC - Veterinary Consulting Services, Edinburgh.

Fibrocartilaginous Embolic Encephalopathy in a Pig.

A 1-year-old boar was investigated after presenting with acute onset collapse and obtundance. No significant gross lesions were observed at post-mortem examination. Histopathological investigation revealed a severe bilateral and multifocal necrotizing encephalopathy with an amorphous material, which obstructed neuroparenchymal vessels in the metencephalon and mesencephalon. Alcian blue staining identified the material as of cartilaginous origin and a diagnosis of cerebral fibrocartilaginous embolism was established. No gross evidence of vertebral disc disease was detected and the origin of the embolic material was not found. Although cerebral fibrocartilaginous embolism has been reported in a human, and rarely in animals, it has not been reported previously in the pig.

Genomic and Immunogenic Protein Diversity of Erysipelothrix rhusiopathiae Isolated From Pigs in Great Britain: Implications for Vaccine Protection.

Erysipelas, caused by the bacterium Erysipelothrix rhusiopathiae, is re-emerging in swine and poultry production systems worldwide. While the global genomic diversity of this species has been characterized, how much of this genomic and functional diversity is maintained at smaller scales is unclear. Specifically, while several key immunogenic surface proteins have been identified for E. rhusiopathiae, little is known about their presence among field strains and their divergence from vaccines, which could result in vaccine failure. Here, a comparative genomics approach was taken to determine the diversity of E. rhusiopathiae strains in pigs in Great Britain over nearly three decades, as well as to assess the field strains' divergence from the vaccine strain most commonly used in British pigs. In addition, the presence/absence and variability of 13 previously described immunogenic surface proteins was determined, including SpaA which is considered a key immunogen. We found a high diversity of E. rhusiopathiae strains in British pigs, similar to the situation described in European poultry but in contrast to swine production systems in Asia. Of the four clades of E. rhusiopathiae found globally, three were represented among British pig isolates, with Clade 2 being the most common. All British pig isolates had one amino acid difference in the immunoprotective domain of the SpaA protein compared to the vaccine strain. However, we were able to confirm using in silico structural protein analyses that this difference is unlikely to compromise vaccine protection. Of 12 other known immunogenic surface proteins of E. rhusiopathiae examined, 11 were found to be present in all British pig isolates and the vaccine strain, but with highly variable degrees of conservation at the amino acid sequence level, ranging from 0.3 to 27% variant positions. Moreover, the phylogenetic incongruence of these proteins suggests that horizontal transfer of genes encoding for antigens is commonplace for this bacterium. We hypothesize that the sequence variants in these proteins could be responsible for differences in the efficacy of the immune response. Our results provide the necessary basis for testing this hypothesis through in vitro and in vivo studies.

Small-scale pig producers and disease surveillance: key threats.

A series of meetings for small-scale pig producers raised awareness of surveillance for pig diseases in Great Britain and highlighted different types of disease threat. This focus article summarises some key messages from those meetings and two of the threats discussed.

First detection of ivermectin resistance in oesophagostomum dentatum in pigs.

In 2016 suspected reduced ivermectin (IVM) efficacy in Oesophagostomum species in pigs was reported in England. Following this initial report, APHA raised awareness amongst private pig veterinary practitioners of the need to monitor the efficacy of the worm control on pig units. In 2017 another veterinary practitioner highlighted a potential in-field lack of IVM efficacy in treating Oesophagostomum species in sows on another breeder-finisher unit. In this trial, the efficacy of IVM against Oesophagostomum species worms has been investigated to determine whether suspected reduced efficacy (52% reduction in mean faecal egg count 14 days post ivermectin administration) on a mixed indoor and outdoor breeder-finisher pig farm in England reflected true IVM resistance under controlled experimental conditions. On days 0 and 40 of the trial, twenty helminth-naive pigs were artificially infected per os with 5000 Oesophagostomum L3 obtained from the farm under investigation. The pigs were allocated to treatment or control groups (n = 10 per group). Treatment group pigs received IVM (0.3 mg kg body weight) by sub-cutaneous injection as per manufacturer's instructions on day 44. Control group animals were left untreated. Faecal worm egg counts were monitored throughout the trial from day 15 post infection to determine time to patency. On day 50 all pigs were euthanased to assess the worm burdens. Resistance to IVM was confirmed in Oesophagostomum dentatum based on the results of a faecal egg count reduction test (FECRT) and a controlled efficacy test (CET). Efficacy based on mean reduction in faecal egg count of IVM-treated pigs compared to untreated control pigs was 86%. Mean reduction in IVM-treated pig worm burdens was 5% against an adult worm population and 94% against an L3/L4 population. The apparent discrepancy between FECRT and CET efficacy results appears to be due to egg development and/or oviposition suppression in IVM-treated female worms. The detection of IVM resistance in Oesophagostomum species worms for the first time in UK pigs is particularly important considering the global situation where resistance to pyrantel, levamisole and benzimidazole anthelmintics in Oesophagostomum species in pigs have already been reported. The results also provide an opportunity to discuss the wider issue of anthelmintic usage and efficacy on pig farms and highlight the need for wider surveillance for the occurrence of anthelmintic resistance in pigs.

Syndromic surveillance by veterinary practitioners: a pilot study in the pig sector.

Traditional indicator-based livestock surveillance has been focused on case definitions, definitive diagnoses and laboratory confirmation. The use of syndromic disease surveillance would increase the population base from which animal health data are captured and facilitate earlier detection of new and re-emerging threats to animal health. Veterinary practitioners could potentially play a vital role in such activities. In a pilot study, specialist private veterinary practitioners (PVP) working in the English pig industry were asked to collect and transfer background data and disease incident reports for pig farms visited during the study period. Baseline data from 110 pig farms were received, along with 68 disease incident reports. Reports took an average of approximately 25 minutes to complete. Feedback from the PVPs indicated that they saw value in syndromic surveillance. Maintenance of anonymity in the outputs would be essential, as would timely access for the PVPs to relevant information on syndromic trends. Further guidance and standardisation would also be required. Syndromic surveillance by PVPs is possible for the pig industry. It has potential to fill current gaps in the collection of animal health data, as long as the engagement and participation of data providers can be obtained and maintained.

Pathotyping the Zoonotic Pathogen Streptococcus suis: Novel Genetic Markers To Differentiate Invasive Disease-Associated Isolates from Non-Disease-Associated Isolates from England and Wales.

Streptococcus suis is one of the most important zoonotic bacterial pathogens of pigs, causing significant economic losses to the global swine industry. S. suis is also a very successful colonizer of mucosal surfaces, and commensal strains can be found in almost all pig populations worldwide, making detection of the S. suis species in asymptomatic carrier herds of little practical value in predicting the likelihood of future clinical relevance. The value of future molecular tools for surveillance and preventative health management lies in the detection of strains that genetically have increased potential to cause disease in presently healthy animals. Here we describe the use of genome-wide association studies to identify genetic markers associated with the observed clinical phenotypes (i) invasive disease and (ii) asymptomatic carriage on the palatine tonsils of pigs on UK farms. Subsequently, we designed a multiplex PCR to target three genetic markers that differentiated 115 S. suis isolates into disease-associated and non-disease-associated groups, that performed with a sensitivity of 0.91, a specificity of 0.79, a negative predictive value of 0.91, and a positive predictive value of 0.79 in comparison to observed clinical phenotypes. We describe evaluation of our pathotyping tool, using an out-of-sample collection of 50 previously uncharacterized S. suis isolates, in comparison to existing methods used to characterize and subtype S. suis isolates. In doing so, we show our pathotyping approach to be a competitive method to characterize S. suis isolates recovered from pigs on UK farms and one that can easily be updated to incorporate global strain collections.

Review of pig health and welfare surveillance data sources in England and Wales.

The capability to set baselines and monitor trends of health and welfare conditions is an important requirement for livestock industries in order to maintain economic competitiveness and sustainability. Monitoring schemes evaluate the relative importance of conditions so that: appropriate actions can be determined, prioritised and implemented; new and (re)emerging conditions can be promptly detected and the effectiveness of any actions can be measured. In 2011, the national pig levy board published a strategy document highlighting health and welfare conditions of importance to the pig industry that were to be targeted for control. In this study, existing schemes that could be used to monitor or set baselines for these conditions in pigs were reviewed, in order to evaluate their suitability for this purpose, using a standardised surveillance evaluation framework (SERVAL). The schemes included: government-funded surveillance of endemic and exotic disease and pig welfare; industry surveillance of endemic diseases; regional schemes for improving pig health; national accreditation schemes and information collected by retailers, private veterinary practices and private laboratories. The evaluation of each scheme highlights its capability to monitor any of the targeted conditions. This study identifies the biases, strengths and gaps in each scheme and provides discussion of opportunities for future development.

Evidence of Illegitimate Recombination Between Two Pasteurellaceae Plasmids Resulting in a Novel Multi-Resistance Replicon, pM3362MDR, in Actinobacillus pleuropneumoniae.

Evidence of plasmids carrying the tetracycline resistance gene, tet(B), was found in the previously reported whole genome sequences of 14 United Kingdom, and 4 Brazilian, isolates of Actinobacillus pleuropneumoniae. Isolation and sequencing of selected plasmids, combined with comparative sequence analysis, indicated that the four Brazilian isolates all harbor plasmids that are nearly identical to pB1001, a plasmid previously found in Pasteurella multocida isolates from Spain. Of the United Kingdom isolates, 13/14 harbor plasmids that are (almost) identical to pTetHS016 from Haemophilus parasuis. The remaining United Kingdom isolate, MIDG3362, harbors a 12666 bp plasmid that shares extensive regions of similarity with pOV from P. multocida (which carries blaROB-1 , sul2, and strAB genes), as well as with pTetHS016. The newly identified multi-resistance plasmid, pM3362MDR, appears to have arisen through illegitimate recombination of pTetHS016 into the stop codon of the truncated strB gene in a pOV-like plasmid. All of the tet(B)-carrying plasmids studied were capable of replicating in Escherichia coli, and predicted origins of replication were identified. A putative origin of transfer (oriT) sequence with similar secondary structure and a nic-site almost identical to that of RP4 was also identified in these plasmids, however, attempts to mobilize them from an RP4-encoding E. coli donor strain were not successful, indicating that specific conjugation machinery may be required.

Molecular epidemiology of isolates with multiple mcr plasmids from a pig farm in Great Britain: the effects of colistin withdrawal in the short and long term.

Background: The environment, including farms, might act as a reservoir for mobile colistin resistance (mcr) genes, which has led to calls for reduction of usage in livestock of colistin, an antibiotic of last resort for humans. Objectives: To establish the molecular epidemiology of mcr Enterobacteriaceae from faeces of two cohorts of pigs, where one group had initially been treated with colistin and the other not, over a 5 month period following stoppage of colistin usage on a farm in Great Britain; faecal samples were also taken at ∼20 months. Methods: mcr-1 Enterobacteriaceae were isolated from positive faeces and was WGS performed; conjugation was performed on selected Escherichia coli and colistin MICs were determined. Results: E. coli of diverse ST harbouring mcr-1 and multiple resistance genes were isolated over 5 months from both cohorts. Two STs, from treated cohorts, contained both mcr-1 and mcr-3 plasmids, with some isolates also harbouring multiple copies of mcr-1 on different plasmids. The mcr-1 plasmids grouped into four Inc types (X4, pO111, I2 and HI2), with mcr-3 found in IncP. Multiple copies of mcr plasmids did not have a noticeable effect on colistin MIC, but they could be transferred simultaneously to a Salmonella host in vitro. Neither mcr-1 nor mcr-3 was detected in samples collected ∼20 months after colistin cessation. Conclusions: We report for the first known time on the presence in Great Britain of mcr-3 from MDR Enterobacteriaceae, which might concurrently harbour multiple copies of mcr-1 on different plasmids. However, control measures, including stoppage of colistin, can successfully mitigate long-term on-farm persistence.

Identification of a New Antimicrobial Resistance Gene Provides Fresh Insights Into Pleuromutilin Resistance in Brachyspira hyodysenteriae, Aetiological Agent of Swine Dysentery.

Brachyspira hyodysenteriae is the aetiological agent of swine dysentery, a globally distributed disease that causes profound economic loss, impedes the free trade and movement of animals, and has significant impact on pig health. Infection is generally treated with antibiotics of which pleuromutilins, such as tiamulin, are widely used for this purpose, but reports of resistance worldwide threaten continued effective control. In Brachyspira hyodysenteriae pleuromutilin resistance has been associated with mutations in chromosomal genes encoding ribosome-associated functions, however the dynamics of resistance acquisition are poorly understood, compromising stewardship efforts to preserve pleuromutilin effectiveness. In this study we undertook whole genome sequencing (WGS) and phenotypic susceptibility testing of 34 UK field isolates and 3 control strains to investigate pleuromutilin resistance in Brachyspira hyodysenteriae. Genome-wide association studies identified a new pleuromutilin resistance gene, tva(A) (tiamulin valnemulin antibiotic resistance), encoding a predicted ABC-F transporter. In vitro culture of isolates in the presence of inhibitory or sub-inhibitory concentrations of tiamulin showed that tva(A) confers reduced pleuromutilin susceptibility that does not lead to clinical resistance but facilitates the development of higher-level resistance via mutations in genes encoding ribosome-associated functions. Genome sequencing of antibiotic-exposed isolates identified both new and previously described mutations in chromosomal genes associated with reduced pleuromutilin susceptibility, including the 23S rRNA gene and rplC, which encodes the L3 ribosomal protein. Interesting three antibiotic-exposed isolates harboured mutations in fusA, encoding Elongation Factor G, a gene not previously associated with pleuromutilin resistance. A longitudinal molecular epidemiological examination of two episodes of swine dysentery at the same farm indicated that tva(A) contributed to development of tiamulin resistance in vivo in a manner consistent with that seen experimentally in vitro. The in vitro studies further showed that tva(A) broadened the mutant selection window and raised the mutant prevention concentration above reported in vivo antibiotic concentrations obtained when administered at certain doses. We show how the identification and characterisation of tva(A), a new marker for pleuromutilin resistance, provides evidence to inform treatment regimes and reduce the development of resistance to this class of highly important antimicrobial agents.

Emergence of Klebsiella pneumoniae subspecies pneumoniae as a cause of septicaemia in pigs in England.

Between 2011 and 2014 outbreaks of septicaemia due to Klebsiella pneumoniae subspecies pneumoniae (Kpp) were diagnosed on thirteen English pig farms. The most consistent features were rapid deaths of pigs from ten-days-old to weaning, seasonal occurrence (May to September), affected farms being outdoor breeding herds and the location of all but one of the outbreaks in the East Anglia region in Eastern England. Molecular characterisation of the outbreak Kpp isolates showed that by multilocus sequencing all were sequence type 25 (ST25) of K2 capsular type with a combination of a 4.3kb plasmid (pKPMC25), three phage sequences and the rmpA virulence gene. No archived Kpp isolates of porcine origin pre-dating 2011 were identified as ST25. In 2013 there was the first detection of an outbreak Kpp isolate showing antimicrobial resistance to six antibiotics. Human infection with Kpp ST25 has not been reported in the UK.

Characterisation of porcine circovirus type 2 in porcine circovirus disease cases in England and Wales.

Confirmed cases of porcine circovirus disease (PCVD) in Great Britain have shown a steady decline since the availability of porcine circovirus type 2 (PCV2) vaccines. However, PCVD is still occasionally diagnosed. The authors carried out a genotyping study to characterise PCV2 associated with confirmed PCVD cases in England and Wales from 2011 to January 2016 (n=65). A partial fragment of PCV2 genome encompassing ORF2 was amplified and sequenced from 45 cases of PCVD. The majority of sequences were genotype PCV2b but four sequences were PCV2d. The significance of the emergence of PCV2d in England and elsewhere in the world is not yet known, although it does appear to represent an ongoing global genotype shift.

Serotypes and Spa types of Erysipelothrix rhusiopathiae isolates from British pigs (1987 to 2015).

Erysipelothrix spp. cause a range of clinical signs in pigs and at least 28 different Erysipelothrix spp. serotypes have been identified. In this study, 128 isolates of Erysipelothrix spp. from pigs in Great Britain from 1987 to 2015 were characterised by serotyping and multiplex real time PCR assays targeting the surface protective antigen (Spa) and the main genotypes (Erysipelothrix rhusiopathiae, Erysipelothrix tonsillarum and Erysipelothrix spp. strain 2). All 128 British isolates were characterised as E. rhusiopathiae and were classified as serotypes 1a (n=21), 1b (n=17), 2 (n=75), 5 (n=2), 9 (n=2), 10 (n=2), 11 (n=4) and 15 (n=1), while four isolates were untypeable. All isolates were positive for the spa A gene. Serotypes 1a, 1b and 2 constituted 88.3% of the isolates; current serotype 2 based vaccines should protect against these isolates.

"Pathotyping" Multiplex PCR Assay for Haemophilus parasuis: a Tool for Prediction of Virulence.

Haemophilus parasuis is a diverse bacterial species that is found in the upper respiratory tracts of pigs and can also cause Glässer's disease and pneumonia. A previous pangenome study of H. parasuis identified 48 genes that were associated with clinical disease. Here, we describe the development of a generalized linear model (termed a pathotyping model) to predict the potential virulence of isolates of H. parasuis based on a subset of 10 genes from the pangenome. A multiplex PCR (mPCR) was constructed based on these genes, the results of which were entered into the pathotyping model to yield a prediction of virulence. This new diagnostic mPCR was tested on 143 field isolates of H. parasuis that had previously been whole-genome sequenced and a further 84 isolates from the United Kingdom from cases of H. parasuis-related disease in pigs collected between 2013 and 2014. The combination of the mPCR and the pathotyping model predicted the virulence of an isolate with 78% accuracy for the original isolate collection and 90% for the additional isolate collection, providing an overall accuracy of 83% (81% sensitivity and 93% specificity) compared with that of the "current standard" of detailed clinical metadata. This new pathotyping assay has the potential to aid surveillance and disease control in addition to serotyping data.