Limits...
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.

Show MeSH

Related in: MedlinePlus

Multilocus allele analysis of B. abortus field strains. Graphical representation of the alleles generated by fluorescent tagged-PCR amplification of the genomic DNA of Brucella abortus biovar 1 field isolates. Each lane is the compilation of data from the independent amplification of each of the eight VNTR loci. Among cattle isolates, the samples are labeled with the state of origin; independent herds are indicated by an arbitrary number; and samples from the same herd are boxed. Each isolate was obtained from a single animal.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC183870&req=5

Figure 5: Multilocus allele analysis of B. abortus field strains. Graphical representation of the alleles generated by fluorescent tagged-PCR amplification of the genomic DNA of Brucella abortus biovar 1 field isolates. Each lane is the compilation of data from the independent amplification of each of the eight VNTR loci. Among cattle isolates, the samples are labeled with the state of origin; independent herds are indicated by an arbitrary number; and samples from the same herd are boxed. Each isolate was obtained from a single animal.

Mentions: Most bovine brucellosis in the USA is caused by B. abortus biovar 1. For a typing method to be of value for epidemiological trace-back, it is essential to be able to subtype biovar 1 isolates. A small panel of B. abortus biovar 1-field strains, consisting of 17 bovine isolates, 2 elk isolates and 3 bison isolates, was tested to look at strain diversity in the U.S. Cattle field isolates were acquired from 10 herds in 5 states. The compiled fingerprints, along with the fingerprint of B. abortus reference strain 544, are shown in Fig. 5. The allele patterns of Brucella isolates cultured from multiple animals in the same herd are boxed together. The first observation was that while the fingerprint pattern within a herd appeared to be stable, the profile for each herd was unique. It was also apparent that the individual loci were mutating at different rates. For example, among the 17 cattle field isolates, Allele-2 was the only allele found at VNTR Locus-5 and Locus-8. Allele-2 was also found at Locus-6 in 16 of the 17 cattle isolates. At the same time, VNTR Locus-1 (7 alleles), Locus-3 (6 alleles), and Locus-7 (5 alleles), showed the highest variation. The fingerprint profiles of the wildlife isolates were also unique and easily distinguished from the cattle isolates. Nonetheless, these isolates shared the same alleles found for VNTR Locus-5 (Allele-2) and Locus-8 (also Allele-2) in cattle. The bison isolates shared common alleles at VNTR Locus-3 (Allele-3) and Locus-4 (Allele-11). However, because of the very small sample size, it remains to be seen if this constitutes a true pattern.


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

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

Multilocus allele analysis of B. abortus field strains. Graphical representation of the alleles generated by fluorescent tagged-PCR amplification of the genomic DNA of Brucella abortus biovar 1 field isolates. Each lane is the compilation of data from the independent amplification of each of the eight VNTR loci. Among cattle isolates, the samples are labeled with the state of origin; independent herds are indicated by an arbitrary number; and samples from the same herd are boxed. Each isolate was obtained from a single animal.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC183870&req=5

Figure 5: Multilocus allele analysis of B. abortus field strains. Graphical representation of the alleles generated by fluorescent tagged-PCR amplification of the genomic DNA of Brucella abortus biovar 1 field isolates. Each lane is the compilation of data from the independent amplification of each of the eight VNTR loci. Among cattle isolates, the samples are labeled with the state of origin; independent herds are indicated by an arbitrary number; and samples from the same herd are boxed. Each isolate was obtained from a single animal.
Mentions: Most bovine brucellosis in the USA is caused by B. abortus biovar 1. For a typing method to be of value for epidemiological trace-back, it is essential to be able to subtype biovar 1 isolates. A small panel of B. abortus biovar 1-field strains, consisting of 17 bovine isolates, 2 elk isolates and 3 bison isolates, was tested to look at strain diversity in the U.S. Cattle field isolates were acquired from 10 herds in 5 states. The compiled fingerprints, along with the fingerprint of B. abortus reference strain 544, are shown in Fig. 5. The allele patterns of Brucella isolates cultured from multiple animals in the same herd are boxed together. The first observation was that while the fingerprint pattern within a herd appeared to be stable, the profile for each herd was unique. It was also apparent that the individual loci were mutating at different rates. For example, among the 17 cattle field isolates, Allele-2 was the only allele found at VNTR Locus-5 and Locus-8. Allele-2 was also found at Locus-6 in 16 of the 17 cattle isolates. At the same time, VNTR Locus-1 (7 alleles), Locus-3 (6 alleles), and Locus-7 (5 alleles), showed the highest variation. The fingerprint profiles of the wildlife isolates were also unique and easily distinguished from the cattle isolates. Nonetheless, these isolates shared the same alleles found for VNTR Locus-5 (Allele-2) and Locus-8 (also Allele-2) in cattle. The bison isolates shared common alleles at VNTR Locus-3 (Allele-3) and Locus-4 (Allele-11). However, because of the very small sample size, it remains to be seen if this constitutes a true pattern.

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