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

Bricker BJ, Ewalt DR, Halling SM - BMC Microbiol. (2003)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: United States Department of Agriculture, Agricultural Research Service, National Animal Disease Center, 2300 Dayton Rd, Ames, IA 50010, USA. bbricker@nadc.ars.usda.gov

ABSTRACT

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.

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Alignment of DNA sequences downstream from the octameric repeat units. The flanking downstream sequences for each of the 9 VNTR loci from the three sequenced genomes were aligned by the Sequencher 4.0 software program. A consensus sequence, derived by alignment of VNTR Loci 1 through 8, is shown in the top line. The individual sequences are labeled "ab" for B. abortus strain 9–941; "mel" for B. melitensis strain 16M; and "suis" for B. suis strain 1330. Bold letters designate the last octameric repeat unit; lower case indicates divergence from the consensus sequence; colons represent missing nucleotides. An asterisk, "*", below the consensus sequence signifies identity for all Brucella species in all VNTR Loci 1 through 8. The two arrows above the consensus sequence mark the positions of the two reverse primers used for allele amplification. Y = C or T; R = A or G; S = G or C.
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Figure 2: Alignment of DNA sequences downstream from the octameric repeat units. The flanking downstream sequences for each of the 9 VNTR loci from the three sequenced genomes were aligned by the Sequencher 4.0 software program. A consensus sequence, derived by alignment of VNTR Loci 1 through 8, is shown in the top line. The individual sequences are labeled "ab" for B. abortus strain 9–941; "mel" for B. melitensis strain 16M; and "suis" for B. suis strain 1330. Bold letters designate the last octameric repeat unit; lower case indicates divergence from the consensus sequence; colons represent missing nucleotides. An asterisk, "*", below the consensus sequence signifies identity for all Brucella species in all VNTR Loci 1 through 8. The two arrows above the consensus sequence mark the positions of the two reverse primers used for allele amplification. Y = C or T; R = A or G; S = G or C.

Mentions: The homologous sequences were aligned revealing variable numbers of repeats at eight of the nine loci (Fig. 1). Among the three sequenced Brucella genomes, the number of complete repeat units ranged from one copy (e.g. VNTR Locus-4 of B. suis) to fourteen copies (VNTR Locus-7 of B. abortus). A notable feature of the repeat loci was the significant conservation of the sequence immediately downstream of the repeat units in eight of the nine loci. Alignment of the downstream sequences of B. abortus, B. melitensis and B. suis (Fig. 2), identified a conserved sequence stretch of about 100-bp. These sequences had a higher than normal level of sequence divergence than is typically found among Brucella species, but a consensus sequence was evident (top line of Fig. 2). VNTR Locus-9 was the only locus that contained the same number of repeat units (3 complete units) in all three genomes. It was also the only locus lacking the conserved downstream sequence (data not shown).


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

Bricker BJ, Ewalt DR, Halling SM - BMC Microbiol. (2003)

Alignment of DNA sequences downstream from the octameric repeat units. The flanking downstream sequences for each of the 9 VNTR loci from the three sequenced genomes were aligned by the Sequencher 4.0 software program. A consensus sequence, derived by alignment of VNTR Loci 1 through 8, is shown in the top line. The individual sequences are labeled "ab" for B. abortus strain 9–941; "mel" for B. melitensis strain 16M; and "suis" for B. suis strain 1330. Bold letters designate the last octameric repeat unit; lower case indicates divergence from the consensus sequence; colons represent missing nucleotides. An asterisk, "*", below the consensus sequence signifies identity for all Brucella species in all VNTR Loci 1 through 8. The two arrows above the consensus sequence mark the positions of the two reverse primers used for allele amplification. Y = C or T; R = A or G; S = G or C.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC183870&req=5

Figure 2: Alignment of DNA sequences downstream from the octameric repeat units. The flanking downstream sequences for each of the 9 VNTR loci from the three sequenced genomes were aligned by the Sequencher 4.0 software program. A consensus sequence, derived by alignment of VNTR Loci 1 through 8, is shown in the top line. The individual sequences are labeled "ab" for B. abortus strain 9–941; "mel" for B. melitensis strain 16M; and "suis" for B. suis strain 1330. Bold letters designate the last octameric repeat unit; lower case indicates divergence from the consensus sequence; colons represent missing nucleotides. An asterisk, "*", below the consensus sequence signifies identity for all Brucella species in all VNTR Loci 1 through 8. The two arrows above the consensus sequence mark the positions of the two reverse primers used for allele amplification. Y = C or T; R = A or G; S = G or C.
Mentions: The homologous sequences were aligned revealing variable numbers of repeats at eight of the nine loci (Fig. 1). Among the three sequenced Brucella genomes, the number of complete repeat units ranged from one copy (e.g. VNTR Locus-4 of B. suis) to fourteen copies (VNTR Locus-7 of B. abortus). A notable feature of the repeat loci was the significant conservation of the sequence immediately downstream of the repeat units in eight of the nine loci. Alignment of the downstream sequences of B. abortus, B. melitensis and B. suis (Fig. 2), identified a conserved sequence stretch of about 100-bp. These sequences had a higher than normal level of sequence divergence than is typically found among Brucella species, but a consensus sequence was evident (top line of Fig. 2). VNTR Locus-9 was the only locus that contained the same number of repeat units (3 complete units) in all three genomes. It was also the only locus lacking the conserved downstream sequence (data not shown).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: United States Department of Agriculture, Agricultural Research Service, National Animal Disease Center, 2300 Dayton Rd, Ames, IA 50010, USA. bbricker@nadc.ars.usda.gov

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus