Multilocus sequence typing as a tool for studying the molecular epidemiology and population structure of Brachyspira hyodysenteriae

Abstract

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The purpose of this study was to develop and apply a multilocus sequence typing 27 (MLST) scheme to study the molecular epidemiology of Brachyspira hyodysenteriae, 28 the aetiological agent of swine dysentery. Sequences of seven conserved genomic loci 29 were examined in 111 B. hyodysenteriae strains. Fifty-eight of these previously had 30 been analysed by multilocus enzyme electrophoresis (MLEE), and for some the 31 results of pulsed field gel electrophoresis (PFGE), restriction endonuclease analysis 32 (REA) and/or serotyping also were available. The discriminatory power of these 33 methods was compared. The strains were divided into 67 sequence types (STs) and 46 34 amino acid types (AATs) by MLST. The Index of Association value was significantly 35 different from zero, indication that the population was clonal. Eleven clonal 36 complexes (Cc) comprising between 2 and 10 STs were recognised. A population 37 snapshot based on AATs placed 77.5% of the isolates from 30 of the AATs into one 38 major cluster. The founder type AAT9 included 13 strains from nine STs that were 39 isolated in Australia, Sweden, Germany and Belgium, including one from a mallard. 40The MLST results were generally comparable to those produced by MLEE. The 41 MLST system had a similar discriminatory power to PFGE, but was more 42 discriminatory than REA, MLEE or serotyping. MLST data provided evidence for 43 likely transmission of strains between farms, but also for the occurrence of temporal 44 "micro-evolution" of strains on individual farms. Overall, the MLST system proved to 45 be a useful new tool for investigating the molecular epidemiology and diversity of B. 46 hyodysenteriae. 47 48

Multiple-Locus Variable-Number Tandem-Repeat Analysis of the Swine Dysentery Pathogen, Brachyspira hyodysenteriae

“…There are few published HunterGaston diversity index results for other typing techniques using large nonlocal collections of B. hyodysenteriae strains. However, a recent study by MLST reported a diversity index of 0.974 for 111 strains (24), and a previous MLEE study performed on 231 isolates gave a haplotypic diversity of 0.94 (34). Although no confidence limits were defined in the previous studies to enable an accurate evaluation of the techniques (14), MLST seems to be slightly more discriminatory than MLVA.…”

“…The field isolates were recovered from different herds, except for 26 Spanish isolates that were additionally isolated from 11 herds on different sampling occasions. B. hyodysenteriae isolates from the University of León and Murdoch University bacterial collections were identified and cultured, and DNA was extracted in each supplying laboratory by previously reported methods (19,24). Working dilutions of extracted DNA were prepared by adjusting them to 1 to 20 ng/l using a NanoDrop 1000 UV-Vis spectrophotometer (Thermo Scientific, Wilmington, DE).…”

“…The methods used have included serotyping (3), DNA restriction endonuclease analysis (REA) (6), random amplification of polymorphic DNA (RAPD) (8), restriction fragment length polymorphism of DNA (21), pulsed-field gel electrophoresis (PFGE) (2), multilocus enzyme electrophoresis (MLEE) (25), and multilocus sequence typing (MLST) (24). These techniques provide different levels of discrimination between isolates, and those that are highly discriminating present associated drawbacks such as difficulties in comparing results between laboratories (for example, RAPD and PFGE).…”

Identification of weakly haemolytic Brachyspira isolates recovered from pigs with diarrhoea in Spain and Portugal and comparison with results from other countries

“…Analysis of nox sequences has emerged as a robust method for identification of Brachyspiraspecies as the gene is relatively conserved but also shows speciesspecific variation (Atyeo et al, 1999;Burrough et al, 2012;Chander et al, 2012;Rubin et al, 2013). A more discriminatory multilocus sequence typing (MLST) scheme analyzing the sequence of seven genes encoding "housekeeping" enzymes has been described for identification and typing of Brachyspira isolates (Råsbäck et al, 2007b), but it is laborious and has not been widely used apart from analysis of B. hyodysenteriae (La et al, 2009;Osorio et al, 2012) and other indole-positive species (Phillips et al, 2010).…”

A preliminary study of the molecular epidemiology of Brachyspira hyodysenteriae isolates in Australia

“…including primers described by Elder et al (1997) was compared with the PCR targeting NADH oxidase (nox) as described by La et al (2006). Multi-locus sequence typing (MLST) was used to determine the relatedness of the B. hyodysenteriae isolates (La et al 2009). The hypothesis was that isolates that were test-negative using the multiplex PCR but test-positive using the simple PCR were related but different to those positive on both PCR tests.…”