An IS 711 Element Downstream of the bp26 Gene Is a Specific Marker of Brucella spp. Isolated from Marine Mammals

Abstract

DNA polymorphism of the bp26 gene, coding for a diagnostic protein antigen for brucellosis, was assessed by PCR and restriction fragment length polymorphism analysis using primers to amplify the bp26 gene with its flanking regions. Surprisingly, whereas PCR performed on DNA of the reference strains of the six recognized Brucella species produced a product of the expected size (1,029 bp), PCR performed on DNA of three representative strains from marine mammals (from a seal, a dolphin, and a porpoise) produced a larger product, of about 1,900 bp. Nucleotide sequencing of the 1,900-bp PCR products revealed the presence of an insertion sequence, IS711, downstream of the bp26 gene and adjacent to a Bru-RS1 element previously described as being a hot spot for IS711 insertion. PCR performed on a large number of field strains from different geographic origins and from marine mammal isolates indicated that the occurrence of an IS711 element downstream of the bp26 gene was a feature specific to the marine mammal Brucella strains. Thus, this PCR assay is able to differentiate Brucella terrestrial isolates from marine mammal isolates and could be applied for diagnostic purposes.Brucellae are gram-negative, facultative, intracellular bacteria that can infect many species of animals, as well as humans. Six species are recognized within the genus Brucella: B. abortus, B. melitensis, B. suis, B. ovis, B. canis, and B. neotomae (8). This classification is mainly based on differences in pathogenicity and host preference (8). The main pathogenic species worldwide are B. abortus, which is responsible for bovine brucellosis, B. melitensis, the main etiologic agent of ovine and caprine brucellosis; and B. suis, which is responsible for swine brucellosis. These three Brucella species may cause abortion in their hosts, which results in huge economic losses. B. ovis and B. canis are responsible for ram epididymitis and canine brucellosis, respectively. For B. neotomae, only strains isolated from desert rats have been reported. Distinction between species and biovars is currently performed by differential tests based on phenotypic characterization of lipopolysaccharide antigens, phage typing, dye sensitivity, CO 2 requirement, H 2 S production, and metabolic properties (2).Brucella strains have also been isolated from a great variety of wildlife species, such as bison, elk, feral swine, wild boars, foxes, hares, African buffalo, reindeer, and caribou (9).The broad spectrum of Brucella hosts has recently been enlarged to include marine mammals. A number of recent reports have described the isolation and characterization of Brucella strains from a wide variety of marine mammals, such as bottlenose dolphins (Tursiops truncatus), common seals (Phoca vitulina), harbor porpoises (Phocoena phocoena), common dolphins (Delphinus delphis), Atlantic white-sided dolphins (Lagenorhynchus acutus), striped dolphins (Stenella caeruleoalba), hooded seals (Cystophora cristata), grey seals (Halichoerus grypus), a minke whale (Balaenoptera acutorostrata), ...

Epizootiology of Brucella Infection in Australian Fur Seals

“…DNA template was added to a PCR reaction mix containing HotStar TaqMaster Mix (Qiagen) primers targeting the Brucella-specific bp26 gene and downstream IS711 element (Cloeckaert et al, 2000). In addition to the sample templates, positive and negative controls were included.…”

Isolation and Characterization of Brucella From the Lungworms of a Harbor Porpoise (Phocoena Phocoena)

“…PCR amplification of an IS711 element downstream of the bp 26 gene (Cloeckaert et al, 2000) confirmed isolates originating from the lungworms and kidney of the harbor porpoise possess the specific marker of Brucella spp. unique to isolations from marine mammals.…”

“…Brucella has been reported to cause abortion in bottlenose dolphins (Tursiops truncatus) in the USA (Ewalt et al, 1994;Miller et al, 1999). Studies using molecular techniques have shown isolates differ from the six recognized species originating from terrestrial mammals (Cloeckaert et al, 2001). Phenotypically, these strains can be differentiated with respect to their requirement for increased carbon dioxide (CO 2 ) for primary growth; generally isolates originating from pinnipeds require increased CO 2 whereas isolates from cetaceans do not.…”

“…unique to isolations from marine mammals. PCR amplification of the outer membrane proteins (omp) 2 locus and subsequent restriction digestion with the use of a selection of enzymes as described by Cloeckaert et al (2001) was carried out on the lungworm and kidney isolates in order to characterize the strain. The IS711 fingerprint analysis was generated by the digestion of genomic DNA with Eco R1, the products separated on a 0.8% agarose gel and southern blotted with the use of a vacuum method.…”

“…PCR amplification of an IS711 element downstream of the base-pair (bp) 26 gene was carried out as described by Cloeckaert et al (2000) to assess whether the isolates possessed the specific marker of Brucella spp. unique to isolations from marine mammals.…”