Weakly haemolytic variants of Brachyspira hyodysenteriae newly emerged in Europe belong to a distinct subclade with unique genetic properties.

Brachyspira (B.) hyodysenteriae is widespread globally, and can cause mucohaemorrhagic colitis (swine dysentery, SD) with severe economic impact in infected herds. Typical strains of B. hyodysenteriae are strongly haemolytic on blood agar, and the haemolytic activity is believed to contribute to virulence in vivo. However, recently there have been reports of atypical weakly haemolytic isolates of B. hyodysenteriae (whBh). In this study, 34 European whBh and 82 strongly haemolytic isolates were subjected to comparative genomic analysis. A phylogenetic tree constructed using core single nucleotide polymorphisms showed that the whBh formed a distinct sub-clade. All eight genes previously associated with haemolysis in B. hyodysenteriae were present in the whBh. No consistent patterns of amino acid substitutions for all whBh were found in these genes. In contrast, a genome region containing six coding sequences (CDSs) had consistent nucleotide sequence differences between strongly and whBh isolates. Two CDSs were predicted to encode ABC transporter proteins, and a TolC family protein, which may have a role in the export of haemolysins from B. hyodysenteriae. Another difference in this region was the presence of three CDSs in whBh that are pseudogenes in strongly haemolytic isolates. One of the intact CDSs from whBh encoded a predicted PadR-like transcriptional repressor that may play a role in repression of haemolysis functions. In summary, a sub-clade of whBh isolates has emerged in Europe, and several genomic differences, that potentially explain the weakly haemolytic phenotype, were identified. These markers may provide targets for discriminatory molecular tests needed in SD surveillance.

Absence of a set of plasmid-encoded genes is predictive of reduced pathogenic potential in Brachyspira hyodysenteriae.

The gene content of 14 strains of the intestinal spirochaete Brachyspira hyodysenteriae was compared using a DNA microarray. A consistent difference occurred in a block of four genes on the ~36 Kb plasmid, with these being present in six virulent strains and absent in eight strains with reduced pathogenic potential. These genes encoded a predicted radical S-adenosylmethionine domain protein, a glycosyl transferase group 1-like protein, an NAD dependent epimerase and a dTDP-4-dehydrorhamnose 2-5 epimerase: they may be involved in rhamnose biosynthesis and glycosylation. The absence of these plasmid genes in B. hyodysenteriae isolates is predictive of reduced pathogenic potential.

Characterizing candidate decompression rates for hypobaric hypoxic stunning of pigs. Part 2: Pathological consequences.

Pigs are commonly stunned pre-slaughter by exposure to carbon dioxide (CO2), but this approach is associated with significant welfare concerns. Hypobaric hypoxia, achieved with gradual decompression (also known as Low Atmospheric Pressure Stunning or LAPS) may be an alternative, allowing the retention of welfare friendly handling approaches and group stunning. Although validated in poultry, the feasibility and welfare consequences of gradual decompression for pigs are unknown. Here, we characterize pathological changes in 60 pigs resulting from exposure to a range of candidate decompression curves (ranging from 40 to 100 ms-1 ascent equivalent, with two cycle durations 480 and 720 s). To protect welfare, we worked on unconscious, terminally anesthetized pigs which were subject to detailed post-mortem examinations by a specialized porcine veterinary pathologist. All pigs were killed as a result of exposure to decompression, irrespective of cycle rate or length. Pigs showed no external injuries during ante-mortem inspections. Exposing pigs to decompression and the unavoidable subsequent recompression resulted in generalized congestion of the carcass, organs and body cavities including the ears, oral cavity, conjunctivae and sclera, mucosa of other external orifices (anus and vulva), nasal planum, nasal cavities including nasal conchae, frontal sinuses, cranium, meninges, brain, larynx, trachea, lungs, heart, parietal pleura of the thoracic cavity, peritoneum of the abdominal cavity, stomach, small intestine, caecum, colon, liver, spleen and kidneys and representative joint cavities in the limbs (stifles and elbows). Various severities of hemorrhage were observed in the conjunctivae and sclera, mucosa of other external orifices (anus and vulva), nasal cavities including nasal conchae, frontal sinuses, cranium, meninges, brain, larynx, tracheal lumen, lungs, parietal pleura of the thoracic cavity, liver, spleen and kidneys and representative joint cavities in the limbs (stifles and elbows). In general, faster decompression rates produced higher scores, but in the conjunctivae, sclera and kidneys, faster decompression rates were associated with marginally lower congestion scores. There was considerable individual variation in pathological scores across all body regions. The congestion and hemorrhage observed could translate into welfare harms in conscious pigs undergoing this type of stunning, depending when in the cycle the damage is occurring, but no welfare related conclusions can be drawn from the responses of unconscious pigs. Since recompression is always required, its effects cannot be separated from decompression, however cessation of cardiac activity several minutes before recompression should have eliminated any haemodynamic effects relating to cardiac function and blood pressure. This study represents the first systematic attempt to identify candidate rate profiles to underpin future explorations of decompression as a stunning method for pigs. These pathological findings also inform discussions about the likely carcass quality implications of this novel stunning method.

Antimicrobial susceptibility monitoring of Mycoplasma hyopneumoniae isolated from seven European countries during 2015-2016.

Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia, a chronic respiratory disease, causing significant economic losses. Results from the 2015-2016 MycoPath pan-European antimicrobial susceptibility monitoring survey of M. hyopneumoniae are presented. In total, 147 M. hyopneumoniae porcine isolates from Belgium, France, Germany, Great Britain, Hungary, Italy, and Spain were tested. One isolate per farm was retained from pigs that had not been recently treated with antimicrobial agents. The minimal inhibitory concentration (MIC) of 13 antimicrobial agents was determined in a central laboratory using a broth microdilution method, with Friis Medium, incubated at 35 ± 1 °C for 5-12 days. M. hyopneumoniae NCTC 10110 was used as Quality Control. MIC50/MIC90 (mg/L) values were: enrofloxacin 0.06/1; marbofloxacin 0.06/2; spiramycin 0.06/0.25; tulathromycin ≤0.001/0.004; gamithromycin 0.06/0.5; tylosin 0.016/0.06; tilmicosin 0.06/0.5; florfenicol 0.5/1; doxycycline 0.25/1; oxytetracycline 0.25/2; lincomycin 0.06/0.25; tiamulin 0.016/0.06 and valnemulin ≤0.001/0.004. Compared with the data from 2010 to 2012 MycoPath study (50 isolates), MIC50/90 results were similar and the majority were within ± two dilution steps, except for the MIC50 of oxytetracycline which is more than two dilution steps higher in the present study. Between-country comparisons show some differences in the MIC values for the fluoroquinolones, tulathromycin and tylosin, but the limited sample size per country precludes performing meaningful country comparisons for several countries. Standardized laboratory methods and interpretive criteria for MIC testing of veterinary mycoplasmas are clearly needed; there are currently no clinical breakpoints available to facilitate data interpretation and correlation of MICs with in vivo efficacy.

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.

The use of oral fluids to monitor key pathogens in porcine respiratory disease complex.

BACKGROUND: The usefulness of oral fluid (OF) sampling for surveillance of infections in pig populations is already accepted but its value as a tool to support investigations of porcine respiratory disease complex (PRDC) has been less well studied. This study set out to describe detection patterns of porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), swine influenza virus type A (SIV) and Mycoplasma hyopneumoniae (M. hyo) among farms showing differing severity of PRDC. The study included six wean-to-finish pig batches from farms with historical occurrence of respiratory disease. OF samples were collected from six pens every two weeks from the 5th to the 21st week of age and tested by real time PCR for presence of PRRSV, SIV and M. hyo and by quantitative real time PCR for PCV2. Data was evaluated alongside clinical and post-mortem observations, mortality rate, slaughter pathology, histopathology, and immunohistochemistry testing data for PCV2 antigen where available. RESULTS: PRRSV and M. hyo were detectable in OF but with inconsistency between pens at the same sampling time and within pens over sequential sampling times. Detection of SIV in clinical and subclinical cases showed good consistency between pens at the same sampling time point with detection possible for periods of 2-4 weeks. Quantitative testing of OF for PCV2 indicated different patterns and levels of detection between farms unaffected or affected by porcine circovirus diseases (PCVD). There was good correlation of PCR results for multiple samples collected from the same pen but no associations were found between prevalence of positive test results and pen location in the building or sex of pigs. CONCLUSIONS: Detection patterns for PRRSV, SIV and M. hyo supported the effectiveness of OF testing as an additional tool for diagnostic investigation of PRDC but emphasised the importance of sampling from multiple pens and on multiple occasions. Preliminary evidence supported the measurement of PCV2 load in pooled OF as a tool for prediction of clinical or subclinical PCVD at farm level.

Antimicrobial susceptibility monitoring of Mycoplasma hyopneumoniae and Mycoplasma bovis isolated in Europe.

Mycoplasma hyopneumoniae in pigs and Mycoplasma bovis in cattle are major pathogens affecting livestock across Europe and are the focus of the MycoPath pan-European antimicrobial susceptibility monitoring programme. Fifty M. hyopneumoniae isolates from Belgium, Spain and the United Kingdom (UK), and 156 M. bovis isolates from France, Hungary, Spain and the UK that met specific criteria were tested for antimicrobial susceptibility in a central laboratory by using a microbroth dilution method. Specific isolate criteria included recovery from animals not recently treated with antimicrobials, isolates from different locations within each country and retaining only one isolate per farm. MIC50/MIC90 values were 0.031/0.5, 0.031/0.5, 0.062/0.25, ≤0.001/0.004, 0.031/0.125, 0.25/0.5 and 0.062/0.25mg/L for enrofloxacin, marbofloxacin, spiramycin, tulathromycin, tylosin, florfenicol and oxytetracycline respectively against M. hyopneumoniae and 0.25/4, 1/4, 4/16, >64/ >64, 32/ >64, 2/4 and 4/64mg/L, respectively against M. bovis. MIC50/MIC90 values for tiamulin and valnemulin against M. hyopneumoniae were 0.016/0.062 and ≤0.001/ ≤0.001mg/L respectively. The MIC50/MIC90 values of danofloxacin and gamithromycin for M. bovis were 0.25/1 and >64/ >64mg/L respectively. The highest MIC90 values for M. hyopneumoniae were found in the UK at 1.0mg/L for enrofloxacin, marbofloxacin and florfenicol. In contrast, for M. bovis the lowest MIC90 value was 1.0mg/L, but ranged to >64mg/L. Specific laboratory standards and clinical breakpoints for veterinary Mycoplasma species are required as no independently validated clinical breakpoints are specified for veterinary Mycoplasma species, which makes data interpretation and correlation to in vivo efficacy difficult.

Patterns of antimicrobial resistance in Streptococcus suis isolates from pigs with or without streptococcal disease in England between 2009 and 2014.

Antimicrobial resistance in Streptococcus suis, a global zoonotic pathogen of pigs, has been mostly studied only in diseased animals using surveys that have not evaluated changes over time. We compared patterns of resistance between S. suis isolates from clinical cases of disease (CC) and non-clinical case (NCC) pigs in England, collected over two discrete periods, 2009-2011 and 2013-2014. Minimum inhibitory concentrations (MIC) of 17 antimicrobials (nine classes) were determined on 405 S. suis isolates categorised by sampling period and disease association to assess changes in resistance over time and association with disease. First, isolates were characterized as resistant or susceptible using published clinical breakpoints. Second, epidemiological cut-offs (ECOFF) were derived from MIC values, and isolates classified as wild type (WT) below the ECOFF and non-wild type (NWT) above the ECOFF. Finally, isolate subsets were analysed for shifts in MIC distribution. NCC isolates were more resistant than CC isolates to cephalosporins, penams, pleuromutilins, potentiated sulphonamides and tetracyclines in both study periods. Resistance levels among CC isolates increased in 2013-2014 relative to 2009-2011 for antimicrobials including aminoglycosides, cephalosporins, fluoroquinolones, pleuromutilins, potentiated sulphonamides and tetracyclines. The prevalence of isolates categorised as NWT for five or more classes of antimicrobials was greater among NCC than CC isolates for both time periods, and increased with time. This study used standardised methods to identify significant shifts in antimicrobial resistance phenotypes of S. suis isolated from pigs in England, not only over time but also between isolates from known clinical cases or disease-free pigs.

Immunomagnetic separation of the intestinal spirochaetes Brachyspira pilosicoli and Brachyspira hyodysenteriae from porcine faeces.

Porcine intestinal spirochaetes are fastidious anaerobic organisms and, as a consequence, it has been necessary to develop various protocols to enhance their isolation from or detection in faeces. Immunomagnetic separation (IMS) is a method developed recently to improve separation of target cells from mixed cell suspensions. The purpose of the present study was to compare the relative sensitivity of IMS for isolation of Brachyspira pilosicoli and Brachyspira hyodysenteriae with current routine diagnostic methods (culture on selective media and PCR) for detection of these micro-organisms in pig faeces. Neither direct nor indirect IMS methods enhanced the sensitivity of detection of either organism when performed with the recommended washings during sample processing. Performance of the IMS procedure without washing gave sensitivity at levels similar to direct culture onto selective medium. Further development of IMS techniques is required to improve isolation rates of Brachyspira species from faecal samples.

Expression by Lawsonia intracellularis of type III secretion system components during infection.

Contact-dependent secretion systems, such as the type III secretion system (T3SS), have been shown to play significant roles in the pathogenicity of many gram-negative bacterial pathogens. Lawsonia intracellularis is a novel, obligate intracellular gram-negative bacterium, which has been identified as the etiological agent of proliferative enteropathies in numerous animal species. Analysis of the genome sequence of the L. intracellularis strain PHE/MN1-00 has revealed the presence of a T3SS secretion system in this bacterium. In this study we aimed to determine whether this important virulence mechanism is also present in L. intracellularis strain LR189/5/83. Using a PCR-based approach, we verified the presence of a genomic region encoding a T3SS. Specifically, a gene highly homologous to the yscN energiser component of the prototypic T3SS of Yersinia spp. was identified and termed lscN. Two further open reading frames (ORFs) contiguous with lscN were also identified: lscO and lscQ, which are also homologues of ORFs within the T3SS of Yersinia spp. To establish whether this T3SS may be functional, expression was monitored directly by RT-PCR and indirectly by detection of serological responses in vaccinated and infected animals. Transcripts for lscN and lscQ were detected and purified rLscQ was recognized by antiserum from infected pigs, indicating expression in vivo during infection. By analogy to other bacteria, this T3SS may be crucial for intracellular development and is likely to play a significant role in the virulence of this unusual pathogen.