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

In vitro sensitivity of poultry Brachyspira isolates to essential oil components and in vivo reduction of Brachyspira intermedia in rearing pullets with cinnamaldehyde feed supplementation

Cecal enteritis due to Brachyspira infections tends to be chronic in laying hens. Limited availability of antimicrobial drugs for use in laying hens emphasizes the need for alternative control measures. A broth microdilution method was used to determine the antimicrobial susceptibility of 20 Brachyspira intermedia field isolates from laying hen flocks to components of essential oils (EO). Minimal inhibitory concentration (MIC) distributions, obtained for eight EO components, were all monomodal. Cinnamaldehyde had the lowest MIC values (40-80 mg/l), followed by nerolidol, capsaicin, carvacrol and thymol (80-320 mg/l), eugenol (160-640 mg/l) and linalool (320-1280 mg/l). The MIC ranges of piperine were mostly above the test-range of 1280 mg/l. In an in vivo experiment, coated cinnamaldehyde was supplemented to the feed of rearing pullets. A completely randomized experimental design with 4 treatments and 3 replicates each (replicate = group of 7 one-day-old birds) was applied. The negative and positive control birds received a conventional feed during the whole trial. The positive control group was orally inoculated on 3 consecutive days (day 22, 23 and 24) with 1 ml 1.0 x 108 cfu/ml of a B. intermedia field isolate. Two treatment groups (preventive and curative), identically inoculated, were fed the coated cinnamaldehyde supplemented feed (final cinnamaldehyde concentration in the feed of 500 ppm), the preventive group from day 1 and the curative from day 25. On day 32, ceca were collected for bacteriologic B. intermedia enumeration. The number of Brachyspirapositive birds and the mean enumeration of Brachyspira cells was decreased (P < 0.05) in the curative treated group versus the positive control group. The in vitro results of the present study demonstrate the potential of EO components as antimicrobials against poultry Brachyspira isolates, including isolates with acquired resistance for classic antimicrobial drugs. Reduction of Brachyspira colonization in young pullets was obtained, on a curative way, in an in vivo study using feed supplemented with coated cinnamaldehyde. Further studies are necessary to investigate the mode of action of the coated cinnamaldehyde in reducing Brachyspira colonization of the ceca

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

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

Importance of microbial culture to identify (novel) highly beta-hemolytic Brachyspira species

Brachyspira hyodysenteriae is considered the primary etiological agent of swine dysentery, causing severe mucohemorrhagic diarrhea. Up till now, the presence of strong beta-hemolysis and ring-phenomenon differentiates the highly pathogenic B. hyodysenteriae from weak hemolytic - and less pathogenic - species (B. innocens, B.murdochii and B. intermedia). However, recent data suggest that also other strongly beta-hemolytic Brachyspira species, identified as B. intermedia, can induce a dysentery-like disease in pigs (1). In a routine monitoring protocol, two gilts were presented for necropsy. Soft, watery colonic content was found. Microbial culture of fecal content revealed strongly beta-hemolytic, ring phenomenon-positive spirochetes, indicative for B. hyodysenteriae. In additional samples taken at the farm, strongly beta-hemolytic Brachyspira isolates, with ring phenomenon were again found on microbial culture, whereas PCR analysis (ADIAVET BRACHY) did not confirm the presence of B. hyodysenteriae. In order to elucidate this discrepancy, additional PCR’s were performed targeting the 16S rRNA, 23S rRNA, nox, hlyA and tlyA genes of different swine-related Brachyspira spp (2,4). PCR’s targeting nox and 23s rRNA specific for B. intermedia were positive. However, all isolates were also positive for tlyA and hlyA gene, hemolysins considered being virulence factors of B. hyodysenteriae. The hlyA gene was properly located between fabG en fabF genes encoding an ACP-reductase and –synthetase(4). A sequencing protocol, targeting a highly variable region of the Brachyspira nox gene(3), was performed, identifying the isolates as B. intermedia. Additionally, the obtained sequence showed high (>99%) similarity with sequences of previously published strongly hemolytic B. intermedia isolates(1). This case report highlights the fact that strongly hemolytic isolates, belonging to the “less pathogenic” Brachyspira species can be isolated from field cases in Europe. While the presence of strong beta-hemolysis and ring phenomenon might indicate the pathogenicity (1), intense genotypic testing is required to correctly identify these isolates to the species level. References 1. Burrough et al. (2012) Comparative virulence of clinical Brachyspira spp. isolates in inoculated pigs. Journal of Veterinary Diagnostic Investigation 24(6) 1025-1034. 2. Backhäns et al. (2010) Phenotypic and molecular characterization of Brachyspira spp isolated from wild rodents. Environmental Microbiology Reports 2(6) 720–727. 3. Weissenböck et al. (2005) Amplification and sequencing of Brachyspira spp. specific portions of nox using paraffin-embedded tissue samples from clinical colitis in Austrian pigs shows frequent solitary presence of Brachyspira murdochii. Veterinary Microbiology 111(1-2) 67-75. 4. Barth et al. (2012) Demonstration of genes encoding virulence and virulence life-style factors in Brachyspira spp. isolates from pigs. Veterinary Microbiology 155(2-4) 438-434