Classification of Brucella strains isolated from marine mammals by infrequent restriction site-PCR and development of specific PCR identification tests

Cloeckaert, Axel; Grayon, Maggy; Grépinet, Olivier; Sidi-Boumedine, Karim

doi:10.1016/s1286-4579(03)00091-1

Cited by 84 publications

(74 citation statements)

References 27 publications

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“…However, PCR-based methods have been used increasingly due to their accuracy, sensitivity, and speed of identification and the ability to work with DNA as opposed to highly infectious live cultures. A wide array of genetic polymorphisms can be assayed for the differentiation of Brucella spp., including the insertion element IS711 (2,3,29,31) and genes of outer membrane proteins (7,10,20), and other assay techniques may be used, such as whole-gene differentiation (30), infrequent restriction site PCR (6), and amplified fragment length polymorphisms (37). PCR-based assays for identifying Brucella were recently reviewed (1).…”

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Real-Time PCR Assays of Single-Nucleotide Polymorphisms Defining the Major Brucella Clades

et al. 2008

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Members of the genus Brucella are known worldwide as pathogens of wildlife and livestock and are the most common organisms of zoonotic infection in humans. In general, brucellae exhibit a range of host specificity in animals that has led to the identification of at least seven Brucella species. The genomes of the various Brucella species are highly conserved, which makes the differentiation of species highly challenging. However, we found single-nucleotide polymorphisms (SNPs) in housekeeping and other genes that differentiated the seven main Brucella species or clades and thus enabled us to develop real-time PCR assays based around these SNPs. Screening of a diverse panel of 338 diverse isolates with these assays correctly identified each isolate with its previously determined Brucella clade. Six of the seven clade-specific assays detected DNA concentrations of less than 10 fg, indicating a high level of sensitivity. This SNP-based approach places samples into a phylogenetic framework, allowing reliable comparisons to be made among the lineages of clonal bacteria and providing a solid basis for genotyping. These PCR assays provide a rapid and highly sensitive method of differentiating the major Brucella groups that will be valuable for clinical and forensic applications.Brucella spp. are pathogenic bacteria that infect a wide variety of mammalian hosts worldwide, often causing reproductive failure. The genus Brucella has classically been divided into six species based on host specificity, including B. abortus (cattle and bison), B. melitensis (goats and sheep), B. suis (pigs), B. canis (dogs), B. neotomae (desert woodrat), and B. ovis (sheep) (12). Two new species have been discovered recently in marine mammals (B. cetaceae in dolphins and whales and B. pinnipediae in seals) (10). Taxonomic limits of the marine clade, however, are not fully defined, and this group may represent one to three species (8, 18). B. abortus, B. melitensis, B. suis, and B. canis are well-characterized zoonotic pathogens, annually infecting Ͼ500,000 people worldwide (26). In the United States, the first three of these species are defined as select agents due to their pathogenicity and potential use as biological weapons (11).Despite host-based segregation, Brucella spp. have proven challenging to differentiate using molecular techniques. Brucella genomes are highly conserved, with Ͼ90% homology among species based on DNA-DNA hybridization (35), identical 16S rRNA sequences among all species (15), and Ͼ90% of genes sharing Ͼ98% sequence identity (16,27). Serological methods and biochemical testing of isolates allow differentiation of species and biovars. However, PCR-based methods have been used increasingly due to their accuracy, sensitivity, and speed of identification and the ability to work with DNA as opposed to highly infectious live cultures. A wide array of genetic polymorphisms can be assayed for the differentiation of Brucella spp., including the insertion element IS711 (2, 3, 29, 31) and genes of outer membrane proteins (7,...

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Real-Time PCR Assays of Single-Nucleotide Polymorphisms Defining the Major Brucella Clades

et al. 2008

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“…However, B. canis, B. neotomae, some biovars of B. abortus and B. suis and the Brucella species isolated from marine mammals described later on, could not be detected by AMOS PCR. Novel IS711 chromosomal locations specific to marine mammal Brucella isolates have been identified allowing their identification and classification based on the same principle as AMOS PCR (Cloeckaert et al, 2003;Maquart et al, 2008;Zygmunt et al, 2010). Real-time PCR assays based on some of the genetic markers described above were later developed for Brucella species identification (Al Dahouk et al, 2007c) but these tests have the same limitations regarding B. suis and B. abortus detection.…”

Section: Molecular Detection and Identificationmentioning

confidence: 99%

Brucellosis at the animal/ecosystem/human interface at the beginning of the 21st century

Godfroid

Scholz²,

Barbier

et al. 2011

Preventive Veterinary Medicine

346

312

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“…However, several subgroups within each species have been identified by molecular typing methods, such as multilocus sequence analysis (MLSA), multilocus variable number of tandem repeats (VNTR) analysis (MLVA), or omp2a and omp2b porin gene diversity analysis (2,4,6,8,9,14,15,16,18,28,32). Among them, one subgroup within B. ceti, which is composed exclusively of strains isolated from various dolphin species, has been proposed to constitute a separate species with the name B. delphini (2,14,29).…”

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Novel IS 711 Chromosomal Location Useful for Identification of Marine Mammal Brucella Genotype ST27, Which Is Associated with Zoonotic Infection

et al. 2011

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We report a novel IS711 chromosomal location that is specific for the Brucella genotype ST27 previously associated with Pacific marine mammals and human zoonotic infection in New Zealand and Peru. Our data support the previous observation that this peculiar genotype is distinct from those commonly isolated from the Atlantic and currently classified within the species B. ceti and B. pinnipedialis.Brucellae are Gram-negative, facultative, intracellular bacteria that can infect humans and many species of animals. Six species were initially recognized within the genus Brucella: B. abortus, B. melitensis, B. suis, B. ovis, B. canis, and B. neotomae (13, 19, 29). This classification is based mainly on differences in pathogenicity, host preference, and phenotypic characteristics. Four additional species have been included since 2007 in the genus Brucella. These comprise the species B. ceti and B. pinnipedialis isolated from marine mammals, with cetaceans (dolphin, porpoise, and whale species) and pinnipeds (various seal species) as the preferred hosts, respectively (11,12). Brucella microti, first described in 2008, was isolated initially from the common vole but later also from the red fox and from soil (21-23). The latest species is B. inopinata, which was isolated from a human breast implant infection and represents the most distant Brucella species at the phenotypic and molecular levels relative to the others (10,

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Classification of Brucella strains isolated from marine mammals by infrequent restriction site-PCR and development of specific PCR identification tests

Cited by 84 publications

References 27 publications

Real-Time PCR Assays of Single-Nucleotide Polymorphisms Defining the Major Brucella Clades

Real-Time PCR Assays of Single-Nucleotide Polymorphisms Defining the Major Brucella Clades

Brucellosis at the animal/ecosystem/human interface at the beginning of the 21st century

Novel IS 711 Chromosomal Location Useful for Identification of Marine Mammal Brucella Genotype ST27, Which Is Associated with Zoonotic Infection

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