Genotyping Brucella canis isolates using a highly discriminatory multilocus variable-number tandem-repeat analysis (MLVA) assay

Baoyan An,Xuming Liu,Jianfa Bai,Chengming Wang,Russell Ransburgh,Elizabeth Poulsen,Gary Anderson,Yin Wang,Nanyan Lu,Yi Yang

doi:10.1038/s41598-017-01114-7

Abstract

Differentiation of Brucella canis from other Brucella species are mainly performed through PCR-based methods and multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) procedures. Both PCR-based and MLVA methods are limited in discriminating B. canis strains. A new MLVA-13Bc method for B. canis genotyping was established by combining eight newly-developed VNTRs with five published ones. During 2010 and 2016, 377 B. canis PCR-positives were identified from 6,844 canine blood samples from 22 U.S. states, resulting in 229 B. canis isolates. The MLVA-13Bc method was able to differentiate each of these 229 isolates. The Hunter-Gaston Discriminatory Index of the individual VNTR loci ranged from 0.516 to 0.934 and the combined discriminatory index reached 1.000. Three major clusters (A, B and C) and 10 genotype groups were identified from the 229 B. canis isolates. Cluster A mainly contains genotype groups 1 and 2, and a few group 3 isolates; nearly all Cluster B isolates were from group 6; other genotype groups were classified into Cluster C. Our newly developed MLVA-13Bc assay is a highly discriminatory assay for B. canis genotyping, and can serve as a useful molecular epidemiological tool, especially for tracing the source of contamination in an event of a B. canis outbreak.

Highlights

Brucella canis is a gram-negative, facultative intracellular pathogen mainly responsible for causing canine brucellosis
The approach based on 21 Variable number of tandem repeat (VNTR) loci provided better strain genotyping information for B. abortus, B. melitensis and B. suis, but was less informative in differentiating strains of B. canis, B. ovis and B. neotomae
With the combination of the 13 VNTRs, each of the 229 isolates were differentiated. This multilocus VNTR analysis (MLVA)-13Bc assay was designed for genotyping of B. canis strains with high discriminatory power

Summary

Introduction

Brucella canis is a gram-negative, facultative intracellular pathogen mainly responsible for causing canine brucellosis. Bricker et al.[21] used a MLVA method named HOOF-Prints with eight tandem repeat (TR) loci (MLVA-8) and was able to differentiate Brucella isolates at both the species and biovar levels. A rather comprehensive screening of 107 TRs identified 15 TRs (MLVA-15) that were more informative and were able to differentiate most Brucella species and for some strains even at the biovar level[22]. The study identified 110 genotypes that differentiated most of the 128 strains, yet the MLVA-16 method provided much lower discriminatory power against the eight B. canis and 18 B. ovis strains in the study. The approach based on 21 VNTR loci provided better strain genotyping information for B. abortus, B. melitensis and B. suis, but was less informative in differentiating strains of B. canis, B. ovis and B. neotomae. The goal of this study were: 1) to develop and validate a MLVA genotyping method for the differentiation of B. canis strains; and 2) to study genetic diversity of a collection of 229 B. canis isolates collected from the US in recent years using the newly developed MLVA-13Bc method

Objectives

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Results

Conclusion