Identification of Brachyspira species by cpn60 universal target sequencing is superior to NADH oxidase gene sequencing.

The pig colon is the habitat of diverse Brachyspira species, of which only a few are of clinical importance. Methods for identification have shifted from phenotypic to molecular testing over the last two decades. Following the emergence of B. hampsonii it became evident that relying on species-specific PCRs carries the risk of overlooking important new species. Consequently, sequencing was proposed as an unbiased alternative for identification of isolates. So far, the main target for identification across species has been the NADH oxidase gene (nox). However, multiple copies of this gene in the genome and potential lateral gene transfer reduce confidence when using this gene. This study compared identification and phylogentic relationship inferred from nox sequencing to that inferred from sequencing of the cpn60 universal target using a collection of 168 isolates from different Brachyspira species. The majority of isolates had an identical identification with both methods. There were a few outliers in the trees with uncertain assignment to a species by BLAST analysis. A few major discrepancies pertained to the pathogenic species B. hampsonii (2), B. pilosicoli (1) and B. suanatina (1). Weakly haemolytic variants of B. hyodysenteriae were assigned to the correct species by both methods. Some of the isolates identified as B. hampsonii also had a weakly haemolytic phenotype.

"Brachyspira hampsonii" clade I isolated from Belgian pigs imported to Germany.

This report describes the detection of "Brachyspira (B.) hampsonii" clade I in Belgian pigs imported to Germany. Two of seventeen pigs from one herd were reported positive for Brachyspira hyodysenteriae by culture in a Belgian diagnostic laboratory, but negative for this Brachyspira species by specific PCR. In this study, from 22 fecal samples and 2 colon contents of these animals various Brachyspira species were cultured and identified by nox-RFLP as Brachyspira murdochii, Brachyspira innocens and Brachyspira intermedia. Albeit the six B. intermedia isolates proved to be negative in a species specific PCR. Sequencing of the nox-gene of three of these isolates revealed that the sequences were 99% identical to published sequences of "B. hampsonii" clade I. From one pig which was positive for "B. hampsonii" clade I histopathology was done and showed moderate lesions consistent with brachyspiral disease.

An up-date on the differentiation of Brachyspira species from pigs with nox-PCR-based restriction fragment length polymorphism.

Different Brachyspira (B.) species colonize the porcine intestinal tract, some of which are pathogens of significant clinical and economic importance. In 2002 we published a novel molecular method for differentiation of Brachyspira species from pigs based on the amplification of the nox-gene and the generation of species-specific restriction patterns (nox-RFLP) using the enzymes BfmI and DpnII (Rohde et al., 2002). We applied this method for identification in addition to biochemical testing in doubtful cases until 2008. Since 2009 we have used it as the first line method of identification. The current study documents the results of examining 2050 Brachyspira isolates collected from January 2009 to December 2011. In addition to identifying isolates with previously described patterns, four novel restriction fragment length patterns were observed, and isolates with these patterns could be assigned to the species B. intermedia and the B. innocens/murdochii complex on the basis of their phenotypic properties and by nox-sequence analysis. In 2007 a potentially new Brachyspira species, "B. suanatina", was described in Swedish pigs (Råsbäck et al., 2007). From the published nox-gene sequence it could be expected that this Brachypira species should show a new restriction pattern making nox-RFLP a suitable technique for identification of "B. suanatina". In this study the new restriction fragment length pattern could be demonstrated in one of the strains described by Råsbäck et al. (AN4859/03). Nevertheless, no isolates with this new pattern corresponding to "B. suanatina" were identified amongst the 2050 Brachyspira isolates examined from northern Germany.