Brucellosis is a major bacterial zoonosis caused by bacteria of the genus Brucella.1 This infectious disease occurs worldwide and has resulted in substantial economic losses in the food animal industry. It is also a public health concern, particularly to people in developing countries.2 In China, Brucella melitensis has been the predominant species responsible for both human and livestock infections, and a clinical diagnosis of brucellosis in sheep is typically made on the basis of results of serologic tests.
Vaccination is considered the most effective strategy for preventing brucellosis in veterinary species. The B melitensis vaccine strain M5 has been used widely as a live attenuated Brucella vaccine for sheep and goats. Use of this vaccine is considered an important contributor to the rapid decrease in the incidence of brucellosis in nonhuman animals in China from the 1970s to the 1990s.3 Although vaccination with this strain confers protection against brucellosis,4 serologic tests are unable to distinguish antibody against the vaccine strain from antibody against wild-type strains because of the high similarity between the two.5
As a result of the widespread use of live attenuated Brucella vaccine and disinfectants and the overuse of antimicrobials, numerous atypical Brucella strains and variants have evolved, which account for 10% to 30% of all isolated strains of the organism. Furthermore, the genetic sequences of the same bacterial species have mutated, presumably as the result of mismatches and recombination during bacterial reproduction.6 Consequently, it is now impossible to identify the taxonomic status of those variants on Brucella classification lists when traditional methods are used. Several methods must therefore be used to improve the ability to classify and identify these strains.
Use of pulsed-field gel electrophoresis has revealed that each species of Brucella yields its own unique fingerprint of conservative bands on the electrophoreticgel. Variationsin repeated units (ie, VNTRs) of the Brucella genomic sequence can be used for the identification and discrimination of Brucella spp, biotypes, and even strains.7–11 The HOOF-Prints technique, which has been used in molecular epidemiological analyses (ie, strain typing by multilocus analysis of VNTRs), was first reported in 2003.7 Brucella vaccines are produced by inoculating non-susceptible animals in an unfavorable environment, where gene mutation of the vaccines can easily occur.12 The purpose of the study reported here was to determine whether the vaccine and wild-type strains of B melitensis could be differentiated by use of the HOOF-Prints technique. We believed that such an ability would enable discrimination between animals that are seropositive because of vaccination against B melitensis and those that are seropositive because of B melitensis infection and would decrease the likelihood of importing Brucella-infected animals.
Supported by grants from the International Science and Technology Cooperation Project of China (Nos. 2015DFR31110 and 2013DFA32380), Project of Sichuan Provincial Department of Education (No. 15ZB0210), Talents Introduction Project of Sichuan University of Science and Engineering (No. 2015RC12), Project of Sichuan Key Laboratory of Winemaking Biotechnology and Application (No. NJ2015-06), and Technology Foundation for Selected Overseas Chinese Scholar, Ministry of Human Resources and Social Security of the People's Republic of China.
This manuscript has been reviewed by all authors. All authors have contributed sufficiently to the scientific work and therefore share collective responsibility and accountability for the results.
The authors declare that there were no conflicts of interest.
The authors thank Dr. Thomas A. Gavin for assistance with the writing of this report.
Hypervariable octameric oligonucleotide fingerprints
Variable-number tandem repeat
Sangon Biotech (Shanghai) Co Ltd, Shanghai, China.
TIANGEN Biotech (Beijing) Co Ltd, Beijing, China.
DNAman, version 7.0, Lynnon LLC, San Ramon, Calif.
GENEray bacteria whole genome DNA extraction kit GK1072, GENEray Biotechnology, Shanghai, China.
NanoDrop spectrophotometer, Thermo Fisher Scientific Inc, Shanghai, China.
DNA marker II, TIANGEN Biotech (Beijing) Co Ltd, Beijing, China.
pBS-T Kit, TIANGEN Biotech (Beijing) Co Ltd, Beijing, China.
BGI, Shenzhen, China.
BLAST, National Center for Biotechnology Information, National Institutes of Health, Bethesda, Md. Available at: blast.ncbi.nlm.nih.gov/. Accessed Apr 18, 2008.
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Forward and reverse primersa used for HOOF-Prints loci amplification of Brucella VNTR loci 1 through 8.
|Forward primer||Sequence of forward primer (5’ to 3′)||Reverse primer|
|Locus-1 fp1||TAT CGA CTG GTC TTC GGG TCG CA||rp1|
|Locus-2 fp2||AAC AGC TGG ATG CGG CGG CGT GAA TA||rp2|
|Locus-3 fp3||AGG CGC TTG AGG ATG AGG CGG CAG T||rp1|
|Locus-4 fp4||AGA ATT TTC GAG GCA TTC GGC G||rp2|
|Locus-5 fp5||ACG GCT ACA AGA TCG AAG TGC TCC||rp1|
|Locus-6 fp6||AGG CGA TCT GGA GAT TAT CGG GAA G||rp1|
|Locus-7 fp7||AGA GCC GTC GGT GGT TAC TTG AGT||rp2|
rp1 = 5′-GTT AAG GGA ATA GGG GAA TAA GGG-3′. rp2 = 5′-GTA TGT TTT GGT TGC GCA TG-3′.