Brucella 'HOOF-Prints': strain typing by multi-locus analysis of variable number tandem repeats (VNTRs)

BACKGROUND: Currently, there are very few tools available for subtyping Brucella isolates for epidemiological trace-back. Subtyping is difficult because of the genetic homogeneity within the genus. Sequencing of the genomes from three Brucella species has facilitated the search for DNA sequence variability. Recently, hypervariability among short tandem repeat sequences has been exploited for strain-typing of several bacterial pathogens. RESULTS: An eight-base pair tandem repeat sequence was discovered in nine genomic loci of the B. abortus genome. Eight loci were hypervariable among the three Brucella species. A PCR-based method was developed to identify the number of repeat units (alleles) at each locus, generating strain-specific fingerprints. None of the loci exhibited species- or biovar-specific alleles. Sometimes, a species or biovar contained a specific allele at one or more loci, but the allele also occurred in other species or biovars. The technique successfully differentiated the type strains for all Brucella species and biovars, among unrelated B. abortus biovar 1 field isolates in cattle, and among B. abortus strains isolated from bison and elk. Isolates from the same herd or from short-term in vitro passage exhibited little or no variability in fingerprint pattern. Sometimes, isolates from an animal would have multiple alleles at a locus, possibly from mixed infections in enzootic areas, residual disease from incomplete depopulation of an infected herd or molecular evolution within the strain. Therefore, a mixed population or a pool of colonies from each animal and/or tissue was tested. CONCLUSION: This paper describes a new method for fingerprinting Brucella isolates based on multi-locus characterization of a variable number, eight-base pair, tandem repeat. We have named this technique "HOOF-Prints" for Hypervariable Octameric Oligonucleotide Finger-Prints. The technique is highly discriminatory among Brucella species, among previously characterized Brucella strains, and among unrelated field isolates that could not be differentiated by classical methods. The method is rapid and the results are reproducible. HOOF-Printing will be most useful as a follow-up test after identification by established methods since we did not find species-specific or biovar-specific alleles. Nonetheless, this technology provides a significant advancement in brucellosis epidemiology, and consequently, will help to eliminate this disease worldwide

Evaluation of the HOOF-Print assay for typing <it>Brucella abortus </it>strains isolated from cattle in the United States: results with four performance criteria

<p>Abstract</p> <p>Background</p> <p>A fundamental question that arises during epidemiological investigations of bacterial disease outbreaks is whether the outbreak strain is genetically related to a proposed index strain. Highly discriminating genetic markers for characterizing bacterial strains can help in clarifying the genetic relationships among strains. Under the auspices of the European Society of Clinical Microbiology and Infectious Diseases, the European Study Group for Epidemiological Markers (ESGEM) established guidelines for evaluating the performance of typing systems based of a number of criteria. Recently, HOOF-Print genotype analysis, a new method for typing <it>Brucella abortus </it>strains based on hypervariability at eight tandem repeat loci, was described. This paper evaluates the HOOF-Print assay by four of the criteria set out by the ESGEM: typeability, reproducibility, power of discrimination, and concordance with other typing methods.</p> <p>Results</p> <p>The HOOF-Print Assay was evaluated with a test population composed of 97 unrelated field isolates and 6 common laboratory strains of <it>B</it>. <it>abortus</it>. Both typeability and reproducibility of the assay were excellent. Allele diversity and frequency varied widely among the eight loci, ranging from 1 to 13 alleles. The power of discrimination, measured by the Hunter-Gaston discrimination index (HGDI), varied by locus ranging from 0 to 0.89, where a maximal value of 1.0 indicates discrimination of all strains. The HGDI values calculated for subgroups sorted by biovar were similar to the values determined for the whole population. None of the individual loci achieved the recommended HGDI threshold of 0.95, but the HGDI of the composite profiles was 0.99 (93 unique genotypes from 97 field strains evaluated), well above the recommended threshold. By comparison, the HGDI value for biovar typing was 0.61 in a test population biased with disproportionate numbers of the less common biovars. Cluster analysis based on HOOF-Print genotypes assembled the strains into hierarchical groups with no apparent association with the time or location of strain isolation. Likewise, these hierarchical groups were not homogeneous with regard to biotype. In one extreme case, two field isolates with identical fingerprints were identified as different biovars by conventional methods.</p> <p>Conclusion</p> <p>The main purpose of this study was to assess the ability of HOOF-Print genotyping to discriminate unrelated field strains of <it>B</it>. <it>abortus</it>, and whether the assay met established requirements for bacterial strain typing methods. The discriminatory power of the assay was remarkable, considering the genetic homogeneity found among species within the genus. The assay met or exceeded all of the recommended levels for the performance criteria of typeability, reproducibility, and power of discrimination, however some inconsistencies with conventional biovar typing were observed. Nevertheless, the results indicate that with cautious interpretation, multilocus genotyping of polymorphic tandem repeats by HOOF-Print analysis could be a valuable complement to routine epidemiological investigations into localized <it>B</it>. <it>abortus </it>outbreaks.</p

PATHOLOGY OF BRUCELLOSIS IN BISON FROM YELLOWSTONE NATIONAL PARK

Between February 1995 and June 1999, specimens from seven aborted bison (Bison bison) fetuses or stillborn calves and their placentas, two additional placentas, three dead neonates, one 2-wk-old calf, and 35 juvenile and adult female bison from Yellowstone National Park (USA) were submitted for bacteriologic and histopathologic examination. One adult animal with a retained placenta had recently aborted. Serum samples from the 35 juvenile and adult bison were tested for Brucella spp. antibodies. Twenty-six bison, including the cow with the retained placenta, were seropositive, one was suspect, and eight were seronegative. Brucella abortus biovar 1 was isolated from three aborted fetuses and associated placentas, an additional placenta, the 2- wk-old calf, and 11 of the seropositive female bison including the animal that had recently aborted. Brucella abortus biovar 2 was isolated from one additional seropositive adult female bison. Brucella abortus was recovered from numerous tissue sites from the aborted fetuses, placentas and 2-wk-old calf. In the juvenile and adult bison, the organism was more frequently isolated from supramammary (83%), retropharyngeal (67%), and iliac (58%) lymph nodes than from other tissues cultured. Cultures from the seronegative and suspect bison were negative for B. abortus. Lesions in the B. abortus-infected, aborted placentas and fetuses consisted of necropurulent placentitis and mild bronchointerstitial pneumonia. The infected 2-wk-old calf had bronchointerstitial pneumonia, focal splenic infarction, and purulent nephritis. The recently-aborting bison cow had purulent endometritis and necropurulent placentitis. Immunohistochemical staining of tissues from the culture-positive aborted fetuses, placentas, 2-wk-old calf, and recently-aborting cow disclosed large numbers of B. abortus in placental trophoblasts and exudate, and fetal and calf lung. A similar study with the same tissue collection and culture protocol was done using six seropositive cattle from a B. abortus-infected herd in July and August, 1997. Results of the bison and cattle studies were similar

Molecular Characterization of Brucella Strains Isolated from Marine Mammals

Recently, gram-negative bacteria isolated from a variety of marine mammals have been identified as Brucella species by conventional phenotypic analysis. This study found the 16S rRNA gene from one representative isolate was identical to the homologous sequences of Brucella abortus, B. melitensis, B. canis, and B. suis. IS711-based DNA fingerprinting of 23 isolates from marine mammals showed all the isolates differed from the classical Brucella species. In general, fingerprint patterns grouped by host species. The data suggest that the marine mammal isolates are distinct types of Brucella and not one of the classical species or biovars invading new host species. In keeping with historical precedent, the designation of several new Brucella species may be appropriate

Characteristics of a Brucella species from a bottlenose dolphin (Tursiops truncatus

Abstract. A culture isolated from an aborted fetus of a bottlenose dolphin (Tursiops truncatus) was characterized. The isolate was a gram-negative coccobacillus, and the colonial morphology was typical of a smooth Brucella. The isolate was positive for catalase, oxidase, nitrate reduction, and urease. Hydrogen sulfide was not produced. It grew in air at 37 C but required 72 hours for good growth. There was growth on media containing basic fuchsin, thionin, thionin blue, penicillin, and erythritol. The M antigen was dominant, and the isolate was lysed by 4 of 10 brucellaphages tested. The oxidative metabolic profile of the isolate was similar to that for B. abortus but differed in utilization of L-asparagine, L-glutamic acid, and DL-citrulline. Whole-cell lysates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein profiles were markedly different from the protein profiles of reference strains of Brucella species. Biochemical and oxidative metabolism profiles indicated that the isolate belongs in the genus Brucella but did not match the profiles of any established species or biovars. This isolate may be an atypical strain of a recognized Brucella species or a new biovar or species of Brucella. Brucella species have been isolated from numerous animal species, including cattle, swine, goats, sheep, dogs, bison, and elk. 7,22 These bacteria cause abortion in the majority of infected hosts. There are no reports of Brucella infection in marine mammals. A bacterium was isolated from an aborted fetus of a bottlenose dolphin (Tursiops truncatus) at the Balboa Hospital, San Diego, California. The isolate was tentatively identified as a Brucella species and submitted for identification to the National Veterinary Services Laboratories (NVSL), Ames, Iowa. This report describes the characterization of this isolate using tests recommended for identification of species and biovars of the genus Brucella