Multiplex Assay Based on Single-Nucleotide Polymorphisms for Rapid Identification of Brucella Isolates at the Species Level

Abstract

The genus Brucella includes a number of species that are major animal pathogens worldwide and significant causes of zoonotic infections of humans. Traditional methods of identifying Brucella to the species level can be time-consuming, can be subjective, and can pose a hazard to laboratory personnel in the absence of suitable biocontainment facilities. Using a robust phylogenetic framework, a number of single-nucleotide polymorphisms (SNPs) that define particular species within the genus were identified. These SNPs were used to develop a multiplex SNP detection assay, based on primer extension technology, that can rapidly and unambiguously identify an isolate as a member of one of the six classical Brucella species or as a member of the recently identified marine mammal group.Brucellosis is a major cause of disease in livestock worldwide, with substantial implications for animal welfare and economic output. Furthermore, as the most common zoonotic disease, it remains a significant public health concern (22). Although a few parts of the developed world have eradicated the disease by a combination of strict veterinary hygiene measures, monitoring programs, and improved food safety measures, it remains endemic in large areas. Human disease usually reflects occupational exposure or the consumption of unpasteurized dairy products and can lead to a chronic debilitating infection.The causative organisms of brucellosis are members of the genus Brucella. Traditionally, the group is divided into six species with distinct host specificities (1), although at the genetic level, members of the genus are highly conserved (14, 32). Reflecting this, DNA hybridization experiments led to the traditional view of Brucella taxonomy being challenged and the group being described as monospecific (28). Practical considerations meant that this classification system never found widespread support, and recently, moves were made to return to the classical species designations (20). Of the currently recognized species, B. abortus is predominantly associated with bovine brucellosis, B. melitensis is predominantly associated with small ruminant brucellosis, B. canis is predominantly associated with canine brucellosis, B. ovis is predominantly associated with ram epididymitis, and B. neotomae has been reported only in the desert wood rat. The remaining species, B. suis, has a broader host range, being predominantly associated with porcine brucellosis (biovars 1, 2, and 3) but also being found in hares (biovar 2), rangifers (biovar 4), and rodents (biovar 5). More recently, novel Brucella isolates were detected in a variety of marine mammal species (7,10,12,17,23). These isolates clearly form new Brucella groups, although they have yet to receive formal taxonomic recognition (13a, 20).Classically, Brucella isolates are divided into species (and biovars in the case of B. abortus, B. melitensis, and B. suis) by a biotyping procedure that assesses a range of cultural, phenotypic, and antigenic properties (1). While biotyping has long been the mainst...

Brucella canis Is an Intracellular Pathogen That Induces a Lower Proinflammatory Response than Smooth Zoonotic Counterparts

“…One representative B. canis strain named bcanCR12 (here B. canis) isolated from a vaginal swab of a Pomeranian bitch after abortion was chosen for biological studies. The results of bacteriological analysis (33), Bruce-ladder multiplex PCR assay (31), multiplex SNP detection (32), and multilocus variable-number tandem-repeat analysis based on 16 loci (MLVA16) (34) were consistent with the B. canis genotype.…”

“…Seventeen isolates of Gram-negative bacteria compatible with B. canis were isolated from seminal fluid of stud males, from vaginal swabs of bitches, or from aborted fetuses between 2009 and 2013. The bacterial strains were characterized as B. canis by bacteriological analysis (3), molecular Bruce-ladder multiplex PCR assay, and multiplex single nucleotide polymorphism (SNP) detection as reported previously (31,32). One representative B. canis strain named bcanCR12 (here B. canis) isolated from a vaginal swab of a Pomeranian bitch after abortion was chosen for biological studies.…”

Whole-Genome-Based Phylogeny and Divergence of the Genus Brucella

“…Multilocus sequence typing, which assesses single nucleotide polymorphisms (SNPs) and other mutations in housekeeping genes, has revealed considerable variation among Brucella isolates that is taxonomically informative (48). Single SNPs can then be used to identify Brucella species because they are evolutionarily stable and can be incorporated into genotyping methods (13,14,41). Multilocus sequencing, however, does not capture enough variation in many species because conserved genomes often have too few polymorphic loci.…”

Development of a Multiplex Primer Extension Assay for Rapid Detection of Salmonella Isolates of Diverse Serotypes

“…We have adopted an approach used previously for phylotyping Brucella species (28) and Escherichia coli (16). We have studied 152 isolates, representing 50 STs and 33 KauffmannWhite Salmonella serovars, and have selected key alleles from the Salmonella MLST database.…”