Porcine intestinal glycosphingolipids recognized by Brachyspira hyodysenteriae.

Swine dysentery caused by Brachyspira hyodysenteriae is a disease present worldwide with an important economic impact on the farming business, resulting in an increased use of antibiotics. In the present study, we investigated the binding of B. hyodysenteriae to glycosphingolipids from porcine small intestinal epithelium in order to determine the glycosphingolipids involved in B. hyodysenteriae adhesion. Specific interactions between B. hyodysenteriae and two non-acid glycosphingolipids were obtained. These binding-active glycosphingolipids, were characterized by mass spectrometry as lactotetraosylceramide (Galß3GlcNAcß3Galß4Glcß1Cer) and the B5 glycosphingolipid (Galα3Galß4GlcNAcß3Galß4Glcß1Cer). Comparative binding studies using structurally related reference glycosphingolipids showed that B. hyodysenteriae binding to lactotetraosylceramide required an unsubstituted terminal Galß3GlcNAc sequence, while for binding to the B5 pentaosylceramide the terminal Galα3Galß4GlcNAc sequence is the minimum element recognized by the bacteria. Binding of Griffonia simplicifolia IB4 lectin to pig colon tissue sections from healthy control pig and B. hyodysenteriae infected pigs showed that in the healthy pigs the Galα3Gal epitope was mainly present in the lamina propria. In contrast, in four out of five pigs with swine dysentery there was an increased expression of Galα3Gal in the goblet cells and in the colonic crypts, where B. hyodysenteriae also was present. The one pig that had recovered by the time of necropsy had the Galα3Gal epitope only in the lamina propria. These data are consistent with a model where a transient increase in the carbohydrate sequence recognized by the bacteria occur in colonic mucins during B. hyodysenteriae infection, suggesting that the mucins may act as decoys contributing to clearance of the infection. These findings may lead to novel strategies for treatment of B. hyodysenteriae induced swine dysentery.

Improving the consistency of experimental swine dysentery inoculation strategies.

Swine dysentery (SD) caused by pathogenic Brachyspira spp. is an economic challenge for the swine industry. In research settings, experimental reproduction of swine dysentery typically relies on intragastric inoculation which has shown variable success. This project aimed to improve the consistency of the experimental inoculation protocol used for swine dysentery in our laboratory. Over six experiments, we evaluated the influence of group housing in inoculated pigs using a frozen-thawed broth culture of strongly hemolytic B. hyodysenteriae strain D19 (Trial A), compared the relative virulence of B. hyodysenteriae strains D19 and G44 (Trial B), compared inoculum volumes (50 mL vs 100 mL) for G44 and B. hampsonii 30446 (Trial C), and performed three independent trials evaluating intragastric inoculation using different oral inoculation methods: oral feed balls (Trial D), and oral syringe bolus of 100 mL (Trial E) or 300 mL (Trial F). Intragastric inoculation with a fresh broth culture of B. hyodysenteriae strain G44 resulted in a shorter incubation period and a higher proportionate duration of mucohemorrhagic diarrhea (MMHD) compared to D19. Intragastric inoculation with either 50 or 100 mL of B. hampsonii 30446 or B. hyodysenteriae (G44) were statistically equivalent. Oral inoculation with 100 mL or 300 mL also yielded similar results to intragastric inoculation but was more expensive due to the additional work and supplies associated with syringe training. Our future research will use intragastric inoculation with 100 mL of a fresh broth culture containing B. hyodysenteriae strain G44 as it yields a high incidence of mucohaemorrhagic diarrhea with a reasonable cost.

Whole-genome analyses reveal a novel prophage and cgSNPs-derived sublineages of Brachyspira hyodysenteriae ST196.

BACKGROUND: Brachyspira (B.) hyodysenteriae is a fastidious anaerobe spirochete that can cause swine dysentery, a severe mucohaemorragic colitis that affects pig production and animal welfare worldwide. In Switzerland, the population of B. hyodysenteriae is characterized by the predominance of macrolide-lincosamide-resistant B. hyodysenteriae isolates of sequence type (ST) ST196, prompting us to obtain deeper insights into the genomic structure and variability of ST196 using pangenome and whole genome variant analyses. RESULTS: The draft genome of 14 B. hyodysenteriae isolates of ST196, sampled during a 7-year period from geographically distant pig herds, was obtained by whole-genome sequencing (WGS) and compared to the complete genome of the B. hyodysenteriae isolate Bh743-7 of ST196 used as reference. Variability results revealed the existence of 30 to 52 single nucleotide polymorphisms (SNPs), resulting in eight sublineages of ST196. The pangenome analysis led to the identification of a novel prophage, pphBhCH20, of the Siphoviridae family in a single isolate of ST196, which suggests that horizontal gene transfer events may drive changes in genomic structure. CONCLUSIONS: This study contributes to the catalogue of publicly available genomes and provides relevant bioinformatic tools and information for further comparative genomic analyses for B. hyodysenteriae. It reveals that Swiss B. hyodysenteriae isolates of the same ST may have evolved independently over time by point mutations and acquisition of larger genetic elements. In line with this, the third type of mobile genetic element described so far in B. hyodysenteriae, the novel prophage pphBhCH20, has been identified in a single isolate of B. hyodysenteriae of ST196.

Brachyspira Species Avidity to Colonic Mucins from Pigs with and without Brachyspira hyodysenteriae Infection Is Species Specific and Varies between Strains.

Brachyspira hyodysenteriae is commonly associated with swine dysentery (SD), a disease that has an economic impact on the swine industry. B. hyodysenteriae infection results in changes to the colonic mucus niche with massive mucus induction, which substantially increases the number of B. hyodysenteriae binding sites in the mucus. We previously determined that a B. hyodysenteriae strain binds to colon mucins in a manner that differs between pigs and mucin types. Here, we investigated if adhesion to mucins is a trait observed across a broad set of B. hyodysenteriae strains and isolates and furthermore at a genus level (B. innocens, B. pilosicoli, B. murdochii, B. hampsonii, and B. intermedia strains). Our results show that binding to mucins appears to be specific to B. hyodysenteriae, and within this species, the binding ability to mucins varies between strains/isolates, increases for mucins from pigs with SD, and is associated with sialic acid epitopes on mucins. Infection with B. hyodysenteriae strain 8dII results in mucin glycosylation changes in the colon, including a shift in sialic acid-containing structures. Thus, we demonstrate through hierarchical cluster analysis and orthogonal projections to latent structures discriminant analysis (OPLS-DA) models of the relative abundances of sialic acid-containing glycans that sialic acid-containing structures in the mucin O-glycome are good predictors of B. hyodysenteriae strain 8dII infection in pigs. The results emphasize the role of sialic acids in governing B. hyodysenteriae interactions with its host, which may open perspectives for therapeutic strategies.

Differential expression of hemolysin genes in weakly and strongly hemolytic Brachyspira hyodysenteriae strains.

BACKGROUND: Swine dysentery (SD) is a diarrheal disease in fattening pigs that is caused by the strongly hemolytic species Brachyspira (B.) hyodysenteriae, B. hampsonii and B. suanatina. As weakly hemolytic Brachyspira spp. are considered less virulent or even non-pathogenic, the hemolysin is regarded as an important factor in the pathogenesis of SD. Four hemolysin genes (tlyA, tlyB, tlyC, and hlyA) and four putative hemolysin genes (hemolysin, hemolysin activation protein, hemolysin III, and hemolysin channel protein) have been reported, but their role in strong hemolysis is not entirely clear. Our study aimed to assess the transcriptional activity of eight (putative) hemolysin genes in a strongly hemolytic (B204) and a weakly hemolytic (G423) B. hyodysenteriae strain during non-hemolytic and hemolytic growth stages. RESULTS: Strongly and weakly hemolytic B. hyodysenteriae strains caused hemolysis on blood agar at different growth stages, namely during log phase (B204) and stationary/death phase (G423). During the lag, early log, late log (stationary phase in G423) and death phase (time points 1-4) strains differed in their hemolysin gene transcription patterns. At time point 1, transcription of the putative hemolysin gene was higher in B204 than in G423. At time point 2, tlyA and tlyC were upregulated in B204 during hemolysis. TlyB and hlyA were upregulated in both strains at all time points, but higher transcription rates were observed in the weakly hemolytic strain G423. The transcription activity of the hemolysin channel protein gene was quite similar in both strains, whereas the hemolysin activation protein gene was upregulated in the non-hemolytic stage of B204 at time point 4. Sequence analysis revealed deletions, insertions and single nucleotide polymorphisms in the G423 hlyA promoter, although without altering the transcription activity of this gene. CONCLUSION: Our data indicate a combined activity of TlyA and TlyC as the most probable underlying mechanism of strong hemolysis in B. hyodysenteriae. Further studies should verify if the expression of tlyA is upregulated by the putative hemolysin gene. Depending on their immunogenic potential TlyA and TlyC may serve as possible vaccine candidates, especially since vaccines for an effective control of swine dysentery are currently not available.

Role of Sialic Acid in Brachyspira hyodysenteriae Adhesion to Pig Colonic Mucins.

Infection with Brachyspira hyodysenteriae results in mucoid hemorrhagic diarrhea. This pathogen is associated with the colonic mucus layer, mainly composed of mucins. Infection regulates mucin O-glycosylation in the colon and increases mucin secretion as well as B. hyodysenteriae binding sites on mucins. Here, we analyzed potential mucin epitopes for B. hyodysenteriae adhesion in the colon, as well as the effect of colonic mucins on bacterial growth. Associations between B. hyodysenteriae binding to pig colonic mucins and mucin glycan data showed that B. hyodysenteriae binding was associated with the presence of N-glycolylneuraminic acid (NeuGc) on mucins. The role of sialic acid in B. hyodysenteriae adhesion was analyzed after the removal of sialic acid residues on the mucins by enzymatic treatment with sialidase A, which decreased bacterial binding to the mucins. The effect of pig colonic mucins on B. hyodysenteriae growth was determined in carbohydrate-free medium. B. hyodysenteriae growth increased in the presence of mucins from two out of five infected pigs, suggesting utilization of mucins as a carbon source for growth. Additionally, bacterial growth was enhanced by free sialic acid and N-acetylglucosamine. The results highlight a role of sialic acid as an adhesion epitope for B. hyodysenteriae interaction with colonic mucins. Furthermore, the mucin response and glycosylation changes exerted in the colon during B. hyodysenteriae infection result in a potentially favorable environment for pathogen growth in the intestinal mucus layer.

The tva(A) Gene from Brachyspira hyodysenteriae Confers Decreased Susceptibility to Pleuromutilins and Streptogramin A in Escherichia coli.

The tva(A) gene suspected to confer resistance to pleuromutilins in Brachyspira hyodysenteriae was tested for functionality in Escherichia coli AG100A and Staphylococcus aureus RN4220. Expression of the cloned tva(A) gene conferred decreased susceptibility to pleuromutilin (P) and streptogramin A (SA) antibiotics in E. coli and had a minor effect in S. aureus The finding provides evidence of the direct association of tva(A) with the PSA resistance phenotype.

Decreased electrogenic anionic secretory response in the porcine colon following in vivo challenge with Brachyspira spp. supports an altered mucin environment.

Brachyspira spp. cause diarrheal disease in multiple animal species by colonization of the colon, resulting in colitis, mucus induction, and disrupted ion transport. Unique to spirochete pathogenesis is the immense production of mucus, resulting in a niche mucin environment likely favoring spirochete colonization. Mucin rheological properties are heavily influenced by anionic secretion, and loss of secretory function has been implicated in diseases such as cystic fibrosis. Here, the effects on the agonist-induced electrogenic anionic secretory response by infectious colonic spirochete bacteria Brachyspira hyodysenteriae and Brachyspira hampsonii were assessed in the proximal, apex, and distal sections of colon in Ussing chambers. Activation of secretion via isoproterenol, carbachol, and forskolin/3-isobutyl-1-methylxanthine demonstrated a significantly decreased change in short-circuit current ( Isc) in Brachyspira-infected pigs in all sections. Tissue resistances did not account for this difference, rather, it was attributed to a decrease in anionic secretion as indicated by a decrease in bumetanide inhibitable Isc. Quantitative RT-PCR and Western blot analyses determined that the major anionic channels of the epithelium were downregulated in diarrheic pigs paired with altered mucin gene expression. The investigated cytokines were not responsible for the downregulation of anion channel gene transcripts. Although IL-1α was upregulated in all segments, it did not alter cystic fibrosis transmembrane conductance regulator (CFTR) mRNA expression in Caco-2 monolayers. However, a whole cell Brachyspira hampsonii lysate significantly reduced CFTR mRNA expression in Caco-2 monolayers. Together, these findings indicate that these two Brachyspira spp. may directly cause a decreased anionic secretory response in the porcine colon, supporting an altered mucin environment likely favoring spirochete colonization. NEW & NOTEWORTHY This research demonstrates for the first time that the niche mucin environment produced by two infectious spirochete spp. is supported by a decrease in the electrogenic anionic secretory response throughout the porcine colon. Our findings suggest that the host's cytokine response is not likely responsible for the decrease in anionic secretory function. Rather, it appears that Brachyspira spp. directly impede ion channel transcription and translation, potentially altering colonic mucin rheological properties, which may favor spirochete colonization.

An atypical weakly haemolytic strain of Brachyspira hyodysenteriae is avirulent and can be used to protect pigs from developing swine dysentery.

The anaerobic intestinal spirochaete Brachyspira hyodysenteriae colonises the large intestine of pigs and causes swine dysentery (SD), a severe mucohaemorrhagic colitis. SD occurs worldwide, and control is hampered by a lack of vaccines and increasing antimicrobial resistance. B. hyodysenteriae strains typically produce strong beta-haemolysis on blood agar, and the haemolytic activity is thought to contribute to the pathogenesis of SD. Recently, weakly haemolytic variants of B. hyodysenteriae have been identified in Europe and Australia, and weakly haemolytic strain D28 from Belgium failed to cause disease when used experimentally to infect pigs. Moreover, pigs colonised with D28 and then challenged with virulent strongly haemolytic strain B204 showed a delay of 2-4 days in developing SD compared to pigs not exposed to D28. The current study aimed to determine whether Australian weakly haemolytic B. hyodysenteriae strain MU1, which is genetically distinct from D28, could cause disease and whether exposure to it protected pigs from subsequent challenge with strongly haemolytic virulent strains. Three experimental infection studies were undertaken in which no diseases occurred in 34 pigs inoculated with MU1, although mild superficial lesions were found in the colon in 2 pigs in one experiment. In two experiments, significantly fewer pigs exposed to MU1 and then challenged with strongly haemolytic virulent strains of B. hyodysenteriae developed SD compared to control pigs not previously exposed to MU1 (p = 0.009 and p = 0.0006). These data indicate that MU1 lacks virulence and has potential to be used to help protect pigs from SD.

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.

The role of transportation in the spread of Brachyspira hyodysenteriae in fattening farms.

BACKGROUND: Direct and indirect contact among animals and holdings are important in the spread of Brachyspira hyodysenteriae. The objective of this study was to investigate the role of slaughterhouse vehicles in spreading B. hyodysenteriae between unconnected farms. RESULTS: Multilocus sequence typing (MLST) and Multiple Locus Variable number tandem repeat Analysis (MLVA) were used to characterize B. hyodysenteriae strains isolated from trucks. Before cleaning, 976 batches of finishing pigs transported by 174 trucks from 540 herds were sampled. After cleaning, 763 of the 976 batches were also sampled. Sixty-one of 976 and 4 of 763 environmental swabs collected from trucks before and after cleaning and disinfection operations, respectively, were positive for B. hyodysenteriae. The 65 isolates in this study originated from 48 farms. Trucks were classified into five categories based on the number of visited farms as follows: category 1: 1-5 farms, category 2: 6-10 farms, category 3: 11-15 farms, category 4: 16-20 farms, category 5: >21 farms. Although the largest number of vehicles examined belonged to category 1, the highest percentage of vehicles positive for B. hyodysenteriae was observed in categories 3, 4 and 5. Specifically, 90.9% of trucks belonging to category 5 were positive for B. hyodysenteriae, followed by categories 4 and 3 with 85.7% and 83.3%, respectively. The results of MLST and MLVA suggest that trucks transporting pigs from a high number of farms also play a critical role in spreading different B. hyodysenteriae genetic profiles. STVT 83-3, which seems to be the current dominant type in Italy, was identified in 56.25% of genotyped isolates. The genetic diversity of isolated strains from trucks was high, particularly, in truck categories 3, 4 and 5. This result confirmed that MLST and MLVA can support the study of epidemiological links between different B. hyodysenteriae farm strains. CONCLUSIONS: This study highlights the potential role of shipments in B. hyodysenteriae spread. Moreover, it emphasizes the importance of strict vehicle hygiene practices for biosecurity programmes.

Brachyspira hyodysenteriae Infection Regulates Mucin Glycosylation Synthesis Inducing an Increased Expression of Core-2 O-Glycans in Porcine Colon.

Brachyspira hyodysenteriae causes swine dysentery (SD), leading to global financial losses to the pig industry. Infection with this pathogen results in an increase in B. hyodysenteriae binding sites on mucins, along with increased colonic mucin secretion. We predict that B. hyodysenteriae modifies the glycosylation pattern of the porcine intestinal mucus layer to optimize its host niche. We characterized the swine colonic mucin O-glycome and identified the differences in glycosylation between B. hyodysenteriae-infected and noninfected pigs. O-Glycans were chemically released from soluble and insoluble mucins isolated from five infected and five healthy colon tissues and analyzed using porous graphitized carbon liquid chromatography tandem mass spectrometry. In total, 94 O-glycans were identified, with healthy pigs having higher interindividual variation, although a larger array of glycan structures was present in infected pigs. This implied that infection induced loss of individual variation and that specific infection-related glycans were induced. The dominating structures shifted from core-4-type O-glycans in noninfected pigs toward core-2-type O-glycans in infected animals, which correlated with increased levels of the C2GnT glycosyl transferase. Overall, glycan chains from infected pigs were shorter and had a higher abundance of structures that were neutral or predominantly contained NeuGc instead of NeuAc, whereas they had a lower abundance of structures that were fucosylated, acidic, or sulfated than those from noninfected pigs. Therefore, we conclude that B. hyodysenteriae plays a major role in regulating colonic mucin glycosylation in pigs during SD. The changes in mucin O-glycosylation thus resulted in a glycan fingerprint in porcine colonic mucus that may provide increased exposure of epitopes important for host-pathogen interactions. The results from this study provide potential therapeutic targets and a platform for investigations of B. hyodysenteriae interactions with the host via mucin glycans.

An avirulent Brachyspira hyodysenteriae strain elicits intestinal IgA and slows down spread of swine dysentery.

Swine dysentery caused by Brachyspira hyodysenteriae, results in substantial economic losses in swine producing countries worldwide. Although a number of different vaccine approaches have been explored with regard to this disease, they show limitations and none of them have reached the market. We here determine the vaccine potential of a weakly haemolytic B. hyodysenteriae strain. The virulence of this strain was assessed in experimental infection trials and its protection against swine dysentery was quantified in a vaccination-challenge experiment using a seeder infection model. Systemic IgG production and local IgA production were monitored in serum and faeces respectively. Across all trials, pigs that were colonized by virulent, strongly haemolytic B. hyodysenteriae strains consistently developed swine dysentery, in contrast to none of the pigs colonized by the weakly haemolytic B. hyodysenteriae vaccine strain. In the seeder vaccination trial nearly all immunised animals developed swine dysentery on subsequent challenge with a virulent strain, but the speed of spread of swine dysentery and faecal score were significantly reduced in animals immunised with the weakly haemolytic strain compared to sham-immunised animals. The IgA response of immunised animals upon challenge with a virulent B. hyodysenteriae strain significantly correlated to a later onset of disease. The correlation between local IgA production and protection induced by a weakly haemolytic B. hyodysenteriae strain provides leads for future vaccine development against swine dysentery.

Characterization of Brachyspira hyodysenteriae isolates from Italy by multilocus sequence typing and multiple locus variable number tandem repeat analysis.

AIMS: To evaluate and compare the capabilities of multilocus sequence typing (MLST) and multiple locus variable number tandem repeat analysis (MLVA) techniques to characterize Brachyspira hyodysenteriae isolates and to investigate the relationship between pleuromutilin resistance and genetic variability. METHODS AND RESULTS: MLST genotyping was performed on 180 B. hyodysenteriae isolates, and the results were evaluated considering profiles from 108 other strains previously reported in the database. In total, 37 sequence types were obtained. The MLVA approach completely characterized 172 strains and grouped the isolates into 22 different profiles. The combination of MLST and MLVA showed a slight increase in the discriminatory power, identifying 33 joint profiles. An antibiotic resistance analysis showed a reduction in the susceptibility to pleuromutilins over time, and a weak association between susceptibility to valnemulin and inclusion in clonal complex 4. CONCLUSION: MLST and MLVA are reliable methods for characterizing B. hyodysenteriae strains and they have comparable discriminatory power. SIGNIFICANCE AND IMPACT OF THE STUDY: The genotyping of B. hyodysenteriae isolates and a database of all the genetic profiles collected during the diagnostic activities could support traditional epidemiological investigations in identifying infection sources and routes of transmission among herds, and in developing more effective control measures.

An optimized swine dysentery murine model to characterize shedding and clinical disease associated with "Brachyspira hampsonii" infection.

BACKGROUND: The development of a mouse model as an in vivo pathogenicity screening tool for Brachyspira spp. has advanced the study of these economically important pathogens in recent years. However, none of the murine models published to date have been used to characterize the clinical signs of disease in mice, instead focusing on pathology following oral inoculation with various Brachyspira spp. The experiments described herein explore modifications of published models to characterize faecal consistency, faecal shedding and pathology in mice challenged with "Brachyspira hampsonii" clade II (Bhamp). METHODS AND RESULTS: In Experiment 1, 24 CF-1 mice were randomly allocated to one of three inoculation groups: sham (Ctrl), Bhamp, or B. hyodysenteriae (Bhyo; positive control). Half of each group was fed normal mouse chow (RMH) while the other received a low-zinc diet (TD85420). In Experiment 2, eight CF-1 mice and nine C3H/HeN mice were divided into Ctrl or Bhamp inoculation groups, and all fed TD85420. In Experiment 1, mice fed TD85420 demonstrated more severe mucoid faeces (P = 0.001; Kruskal Wallis) and faecal shedding for a significantly greater number of days (P = 0.005; Kruskal Wallis). Mean faecal scores of Bhamp inoculated mice trended higher than Ctrl (P = 0.06; Wilcoxon rank-sum) as did those of Bhyo mice (P = 0.0; Wilcoxon rank-sum). In Experiment 2, mean faecal scores of inoculated CF-1 mice were significantly greater than in C3H mice (P = 0.049; Kruskal Wallis) but no group differences in faecal shedding were observed. In both experiments, mice clustered based on the severity of colonic and caecal histopathology but high lesion scores were not always concurrent with high fecal scores. CONCLUSION: In our laboratory, CF-1 mice and the lower-zinc TD85420 diet provide a superior murine challenge model of "Brachyspira hampsonii" clade II.

Surveillance for nervous necrosis virus-specific antibodies in barramundi Lates calcarifer in Australian hatcheries.

We conducted single point-in-time and repeated cross-sectional studies of the prevalence of antibodies against nervous necrosis virus (NNV) in populations of adult barramundi Lates calcarifer in Australia. Serum samples collected between 2002 and 2012 were analyzed with indirect ELISA (n = 468). Most of the samples were sourced from broodstock with unknown exposure history, and these were compared with reference populations with confirmed history of exposure to NNV. Non-lethally collected gonad fluid samples from economically valuable barramundi broodstock (n = 164) were tested for the presence of NNV using RT-quantitative PCR at the time of blood sampling to compare infectivity with serostatus, but no virus was detected. NNV-specific immunoreactivity in broodstock was significantly lower than that for immunized and persistently infected populations. Seroprevalence increased over time in broodstock sampled longitudinally, probably reflecting repeated exposure to NNV in a region where the virus was endemic. The seroprevalence for the broodstock was 23.8% over the entire sample period while a cross-sectional survey conducted in 2012 found a seroprevalence of 34.5% with no significant difference between populations based on the geographic region or the history of occurrence of viral nervous necrosis (VNN) disease in the progeny in the respective hatcheries. Although serological surveillance was useful for studying the history of exposure of barramundi to NNV, the lack of association between serostatus in broodstock and the subsequent occurrence of VNN disease in their progeny indicates that ELISA tests for anti-NNV antibodies are not suitable for the purpose of preventing vertical transmission of NNV in barramundi.

Variation in hemolytic activity of Brachyspira hyodysenteriae strains from pigs.

Brachyspira hyodysenteriae is the primary cause of swine dysentery, which is responsible for major economic losses to the pig industry worldwide. The hemolytic activity of 10 B. hyodysenteriae strains isolated from stools of pigs with mild to mucohemorrhagic diarrhea was compared and seven hemolysis associated genes were sequenced. Hemolysis induced by these strains varied from strong to near absent. One weakly hemolytic B. hyodysenteriae strain showed sequence changes in five hemolysis associated genes (tlyA, tlyB, hemolysin III, hemolysin activation protein and hemolysin III channel protein) resulting in amino acid substitutions. The occurrence of weakly hemolytic strains identifiable as B. hyodysenteriae should be taken into account in swine dysentery diagnostics. The presence of these strains may affect herd dysentery status, with great impact on a farms trading opportunities.

Antimicrobial susceptibility of Brachyspira hyodysenteriae in Switzerland.

INTRODUCTION: Brachyspira (B.) hyodysenteriae is the causative agent of swine dysentery (SD), a severe mucohaemorrhagic diarrheal disease in pigs worldwide. So far, the antimicrobial susceptibility patterns of B. hyodysenteriae in Switzerland have not been investigated. Therefore, a panel of 30 porcine B. hyodysenteriae isolates were tested against 6 antimicrobial agents by using the VetMIC Brachy panel, a broth microdilution test. Tiamulin and valnemulin showed high antimicrobial activity inhibiting all isolates at low concentrations. The susceptibility testing of doxycycline revealed values from ≤0.25 µg/ ml (47%) to 2 µg/ml (10%). The MIC values of lincomycin ranged between ≤0.5 µg/ml (30%) and 32 µg/ml (43%). For tylosin, 57% of the isolates could not be inhibited at the highest concentration of ≥128 µg/ml. The MIC values for tylvalosin were between ≤0.25 µg/ml (10%) and 8 µg/ml (20%). These findings reveal Switzerland's favourable situation compared to other European countries. Above all, tiamulin and valnemulin are still effective antimicrobial agents and can be further used for the treatment of SD.

INTRODUCTION: Brachyspira (B.) hyodysenteriae est l'agent de la dysenterie porcine, une affection diarrhéique muco-hémorragique grave des porcs connue dans le monde entier. Jusqu'à ce jour, la sensibilité aux antibiotiques B. hyodysenteriae n'avait pas été étudiée en Suisse. C'est pour cela qu'on a examiné, au moyen du test de micro dilution VetMIC Brachy panel, un choix de 30 isolats porcins de B. hyodysenteriae quant à leur sensibilité face à 6 substances antimicrobiennes. La Tiamuline et la Valnémuline ont montré une activité antimicrobienne élevée, bloquant tous les isolats à de faibles concentrations. Les tests de sensibilité vis-à-vis de la Doxycilline ont donné des valeurs comprises entre ≤0.25 µg/ml (47%) et 2 µg/ml (10%). Les valeurs de CMI de la Lincomycine variaient entre ≤0.5 µg/ml (30%) et 32 µg/ml (43%). Avec la Tylosine, 57% des isolats n'ont pas pu être bloqués avec la concentration la plus élevée de ≥128 µg/ml. Les valeurs de CMI pour la Tylvalosine se situaient entre ≤0.25 µg/ml (10%) et 8 µg/ml (20%). Ces résultats montrent que la situation suisse est favorable en regard d'autres pays européens. La Tiamuline et la Valnémuline en particulier restent des substances antimicrobiennes efficaces qui peuvent continuer à être utiliser pour lutter contre la dysenterie porcine.

The levels of Brachyspira hyodysenteriae binding to porcine colonic mucins differ between individuals, and binding is increased to mucins from infected pigs with de novo MUC5AC synthesis.

Brachyspira hyodysenteriae colonizes the pig colon, resulting in mucohemorrhagic diarrhea and growth retardation. Fecal mucus is a characteristic feature of swine dysentery; therefore, we investigated how the mucin environment changes in the colon during infection with B. hyodysenteriae and how these changes affect this bacterium's interaction with mucins. We isolated and characterized mucins, the main component of mucus, from the colon of experimentally inoculated and control pigs and investigated B. hyodysenteriae binding to these mucins. Fluorescence microscopy revealed a massive mucus induction and disorganized mucus structure in the colon of pigs with swine dysentery. Quantitative PCR (qPCR) and antibody detection demonstrated that the mucus composition of pigs with swine dysentery was characterized by de novo expression of MUC5AC and increased expression of MUC2 in the colon. Mucins from the colon of inoculated and control pigs were isolated by two steps of isopycnic density gradient centrifugation. The mucin densities of control and inoculated pigs were similar, whereas the mucin quantity was 5-fold higher during infection. The level of B. hyodysenteriae binding to mucins differed between pigs, and there was increased binding to soluble mucins isolated from pigs with swine dysentery. The ability of B. hyodysenteriae to bind, measured in relation to the total mucin contents of mucus in sick versus healthy pigs, increased 7-fold during infection. Together, the results indicate that B. hyodysenteriae binds to carbohydrate structures on the mucins as these differ between individuals. Furthermore, B. hyodysenteriae infection induces changes to the mucus niche which substantially increase the amount of B. hyodysenteriae binding sites in the mucus.

Prevalence and risk factors for Lawsonia intracellularis, Brachyspira hyodysenteriae and Salmonella spp. in finishing pigs in Polish farrow-to-finish swine herds.

The aim of the study was to estimate the herd-level, within-herd prevalence, the frequency of mixed infections and risk factors for L. intracellularis, B. hyodysenteriae and Salmonella spp. in selected farrow-to-finish Polish pig herds. A total of 254 pooled fecal samples were collected from 9 to 24 week-old pigs in 70 herds. Real time PCR for detection of L. intracellularis and B. hyodysenteriae was performed. For Salmonella spp. bacteriological examination was performed. The herd-level prevalences of L. intracellularis, B. hyodysenteriae and Salmonella spp. among examined herds were 65.7%, 1.4% and 8.6%, respectively. The within-herd prevalences (in positive herds) for L. intracellularis, B. hyodysenteriae and Salmonella spp. were 51.5%, 75.0% and 30.4%, respectively. All herds with diarrhea observed during sampling were infected with L. intracellularis and 60% of herds with no diarrhea at the moment of sampling were infected with L. intracellularis (p=0.035). In herds with more than 200 sows the prevalence of Salmonella spp. was significantly higher compared to herds with less than 200 sows (p=0.027). In herds where all-in/all-out (AIAO) was respected, prevalence of L. intracellularis was significantly lower than in herds where this rule was not kept (p=0.024). Obtained results confirm that L. intracellularis is the major cause of bacterial diarrhea in finishing pigs. The present study identified AIAO and herd size as a risk factor, at the herd level, for L. intracellularis and Salmonella spp., respectively.