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.
Materials and Methods
Sample
Brucella melitensis vaccine strain M5 was purchased from a commercial provider”; B melitensis wild-type strain M43 had been isolated from a sheep fetus collected from Xinjiang Uygur Autonomous Region13 and archived in frozen (−80°C) aliquots. To obtain a pure isolate of the vaccine strain for testing, selective Brucella agar medium was prepared in accordance with established protocols.14 The streaking technique was used to inoculate blood agar platesb with the obtained vaccine strain, and this medium was incubated for 82 hours at 37°C (the optimal incubation temperature for Brucella spp14) in 10% CO2. A single colony was collected from the medium, killed by heating to 100°C for 10 minutes, and fixed on a new plate for identification by means of the Ziehl-Neelsen staining method.14 Once the identity of the organism was confirmed, the vaccine strain isolate was stored in Eppendorf tubes at −80°C until used.
For testing, both B melitensis strains were prepared from frozen aliquots and inoculated onto trypticasesoy agar plates containing 5% serum. Inoculated plates were incubated in 10% CO2 at 37°C for 24 to 72 hours.15 Bacteria were harvested from plates by use of saline (0.85% NaCl) solution and preserved by the addition of 2 volumes of 100% methanol. The preserved cells were stored in Eppendorf tubes at 4°C until needed.
DNA extraction
Primers for extraction of DNA from B melitensis isolates were developed by use of the reported sequences of Brucella VNTR loci 1 through 87 and a biological analysis of flanking sequences for HOOF-Prints analysis of B melitensis by use of molecular biology software.c Eight pairs of upstream and downstream primers were designed and produceda accordingly (Appendix). Total genomic DNA was extracted and purified by use of a whole genome DNA extraction kit.d Purity and concentration of the extracted DNA were measured by use of a spectrophotometer.e
PCR assay
Extracted DNA was amplified by means of PCR assay. The reaction solution (25 μL) was composed of 2.5 μL of 10× Taq reaction buffer,b 0.8 μL of 2.5mM dNTP,b 0.3 μL of 20μM upstream primer,a 0.3 μL of 20μM downstream primer,a 1.0 μL of strain M5 as DNA template (50 ng/μL), 19.7 μL of deionized water, and 0.4 μL of Taq DNA polymeraseb (2.5 U/μL). Reaction conditions for the PCR assay included predenaturation for 3 minutes at 95°C; 35 cycles of denaturation for 80 seconds at 94°C, annealing for 50 seconds at 53°C, and extension for 60 seconds at 72°C; extension for 15 minutes at 72°C; and conservation at 4°C. The final step involved running the mixtures and a DNA markerf on a regular 1.2% agarose electrophoretic gel. Primer dimmera was used as a negative control substance for that analysis.
Bacterial identification by use of the HOOF-Prints technique
Eight PCR assays per strain were performed as previously described.5 Amplified DNA samples were cloned into pBluescript-T vectors by use of a commercial kit,g and transformation of competent cells was performed strictly in accordance with instructions from the manufacturers of the agarose gel DNA extraction kit. Bacterial isolates for which identities were confirmed by Ziehl-Neelsen staining and strain-specific PCR assays were sent for genetic sequencing.h Comparative sequence searches were accomplished by use of a bioinformatics search tool.i Phylogenic analysis was performed for each of the M5 and M43 strains by use of molecular genetics analysis software,j and incorporation of previously reported data for Brucella reference strains.7 Phylogenetic trees were visually compared.
Results
HOOF-Prints assay
The VNTR DNA segments of B melitensis vaccine strain M5 and wild-type strain M43 were successfully amplified by means of PCR assay. All target gene fragments ranged in size from 100 to 300 bp (Figures 1 and 2).
Photograph of an electrophoretic gel showing results of HOOF-Prints PCR amplification of Brucella melitensis vaccine strain M5 at 8 VNTR loci (lanes 1 through 8). M = DNA marker. N = Negative control sample (primer dimmer).
Citation: American Journal of Veterinary Research 78, 4; 10.2460/ajvr.78.4.495
Photograph of an electrophoretic gel showing results of HOOF-Prints PCR amplification of Brucella melitensis vaccine strain M5 at 8 VNTR loci (lanes 1 through 8). M = DNA marker. N = Negative control sample (primer dimmer).
Citation: American Journal of Veterinary Research 78, 4; 10.2460/ajvr.78.4.495
Photograph of an electrophoretic gel showing results of HOOF-Prints PCR amplification of Brucella melitensis vaccine strain M5 at 8 VNTR loci (lanes 1 through 8). M = DNA marker. N = Negative control sample (primer dimmer).
Citation: American Journal of Veterinary Research 78, 4; 10.2460/ajvr.78.4.495
Photograph of an electrophoretic gel showing results of HOOF-Prints PCR amplification of B melitensis wild-type strain M43 at 8 VNTR loci (lanes 1 through 8). See Figure 1 for key.
Citation: American Journal of Veterinary Research 78, 4; 10.2460/ajvr.78.4.495
Photograph of an electrophoretic gel showing results of HOOF-Prints PCR amplification of B melitensis wild-type strain M43 at 8 VNTR loci (lanes 1 through 8). See Figure 1 for key.
Citation: American Journal of Veterinary Research 78, 4; 10.2460/ajvr.78.4.495
Photograph of an electrophoretic gel showing results of HOOF-Prints PCR amplification of B melitensis wild-type strain M43 at 8 VNTR loci (lanes 1 through 8). See Figure 1 for key.
Citation: American Journal of Veterinary Research 78, 4; 10.2460/ajvr.78.4.495
Sequence analysis
The 8-bp tandem repeat units involved in 8 Brucella VNTR loci could be used to differentiate B melitensis vaccine strain M5 from wild-type strain M43. Frequencies of repeated units were summarized (Table 1). A striking difference in frequencies was identified by the various loci within each strain, particularly differences in loci 1, 4, 5, and 7 of vaccine strain M5, loci 1 and 4 of wild-type strain M43, and reference strains.
Number of repetitions in VNTR sequences identified for Brucella melitensis vaccine strain M5, wild-type strain M43, and reference strain 16M and other Brucella strains.
| Forward primer | M5 | M43 | 16M | Brucella abortus 2308 | Brucella suis 1330 | Brucella ovis |
|---|---|---|---|---|---|---|
| Locus-1 fp1 | 0 | *1π+7Δ | 1π+4Δ | 2π | 4π | 11π |
| Locus-2 fp2 | *1ω+3π | *1ω+5π | 1ω+4π | 2π | 2π or 5π | |
| Locus-3 fp3 | *1π | *1π | 1π | 4π | 4π | 2π |
| Locus-4 fp4 | *2ω+4A | *3ω+5Δ | 1π+6Δ | 4π | 1π | 1π |
| Locus-5 fp5 | 0 | *12π | 7π | 2π | 7π | 0 |
| Locus-6 fp6 | 4π | *7π | 1π | 2π | 5π | 5π |
| Locus-7 fp7 | 7π | *7π | 8π | *14π | 11π | *14π |
| Locus-8 fp8 | *2π | *2π | 6π | 2π | 4π | 6π |
Sequence has been submitted to NCBI. Accession numbers are EF221600, EU232128, EU212130, EU255807, EU255808, EU265671, EU255809, EU265672, EU255811, EU255810, and EU255806, respectively.
Δ = Repeated unit GGGGCAGT. ω = Repeat unit AAGGCAGT. π = Repeated unit AGGGCAGT.
Phylogenic analysis
Comparison of the separate phylogenic trees revealed considerable differences between vaccine strain M5 (Figure 3) and wild-type strain M43 (Figure 4). In this indirect comparison, the M5 strain appeared more phylogenetically similar than the M43 strain to the B melitensis reference strain 16M.16
Phylogenetic tree of B melitensis vaccine strain M5 and reference strains B melitensis strain 16M (meli), Brucella ovis (ovis), Brucella suis strain 1330 (suis), and Brucella abortus strain 2308 (abor). The separate bar indicates genetic distance (0.05 cM).
Citation: American Journal of Veterinary Research 78, 4; 10.2460/ajvr.78.4.495
Phylogenetic tree of B melitensis vaccine strain M5 and reference strains B melitensis strain 16M (meli), Brucella ovis (ovis), Brucella suis strain 1330 (suis), and Brucella abortus strain 2308 (abor). The separate bar indicates genetic distance (0.05 cM).
Citation: American Journal of Veterinary Research 78, 4; 10.2460/ajvr.78.4.495
Phylogenetic tree of B melitensis vaccine strain M5 and reference strains B melitensis strain 16M (meli), Brucella ovis (ovis), Brucella suis strain 1330 (suis), and Brucella abortus strain 2308 (abor). The separate bar indicates genetic distance (0.05 cM).
Citation: American Journal of Veterinary Research 78, 4; 10.2460/ajvr.78.4.495
Phylogenetic tree of B melitensis strain M43 and reference strains B melitensis strain 16M, B ovis, B suis strain 1330, and B abortus strain 2308. See Figure 3 for key.
Citation: American Journal of Veterinary Research 78, 4; 10.2460/ajvr.78.4.495
Phylogenetic tree of B melitensis strain M43 and reference strains B melitensis strain 16M, B ovis, B suis strain 1330, and B abortus strain 2308. See Figure 3 for key.
Citation: American Journal of Veterinary Research 78, 4; 10.2460/ajvr.78.4.495
Phylogenetic tree of B melitensis strain M43 and reference strains B melitensis strain 16M, B ovis, B suis strain 1330, and B abortus strain 2308. See Figure 3 for key.
Citation: American Journal of Veterinary Research 78, 4; 10.2460/ajvr.78.4.495
Discussion
In conventional bacterial identification involving culture of organisms, smooth Brucella spp will sometimes lose antigens A and M, thereby exposing antigen R, which can then be agglutinated with R antiserum. This process can occasionally result in unavoidable confusion as to the identities of Brucella S-R variants. This confusion, along with the difficulty distinguishing vaccine-induced antibody against B melitensis from infection-induced antibody through serologic testing, supports the potential usefulness of the HOOF-Prints technique for detection of animals infected with (vs immunized against) B melitensis. The technique also has some advantages with respect to safety, efficiency, and convenience.
Results of this preliminary study suggested that the described HOOF-Prints technique would be helpful for accurate identification and discrimination of Brucella variants. This technique may be useful not only during performance of conventional bacteriologic tests, but also for distinguishing between clinical Brucella isolates (such as the M43 isolate from a sheep fetus) from currently used vaccine strains (such as the live, attenuated M5 strain). More interestingly, the technique could be used to develop evolutionary models of pathogens or to trace the source of infections. In another study,17 we compared 8 VNTR loci of B melitensis strain M43 (isolated in Shihezi, China, in 2005) with those of B melitensis strain 80/23 (isolated from ram semen in Shihezi in 1980) and found that the HOOF-Prints data were identical between these 2 strains. This result suggested that the M43 isolate was a local pathogen and that infection with the M43 strain had been endemic in sheep for at least 25 years.
The B melitensis strains M5 and 16M had a close genetic distance in the phylogenetic analysis, even though some differences were evident at certain HOOF-Prints loci between the M5 strain and reference strains Brucella abortus 2330,18 Brucella ovis,19 and Brucella suis 1330.20 Variation in vaccine strain M5 at the nucleic acid level is typically quite small, and clinical immunologic testing has shown that the immunity induced by the vaccine M5 is still effective. Obviously, wild-type M43 had depth variation derived from the phylogenetic tree, in that almost every HOOF-Prints locus had a certain genetic distance (Figure 4). The genetic distance between strains M43 and 16M indicated these strains were still the most similar. However, the genetic distance appeared greater between strains M5 and M43 on indirect examination. The most likely reasons for this difference were mutation of B melitensis due to the harsh culture environment or existing defects in current taxonomic methods, by which the rightful phylogenetic position for atypical strains or depth-mutant strains cannot be determined.
Comparison between results of the HOOF-Prints assay (Table 1) and phylogenetic analysis (Figures 3 and 4) yielded good evidence of the reliability of the HOOF-Prints technique. Results were consistent between these 2 methods. Furthermore, results suggested that Brucella vaccine strain M5 still has a resistance to wild-type strains. The genetic findings also indicated that wild-type strain M43 (which originated from B melitensis biotype 3) that was used in the present study may provide more protection than vaccine strain M5 (which originated from B melitensis biotype 1) if used in a vaccine. However, immunologic testing is needed to test this supposition.
Acknowledgments
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.
ABBREVIATIONS
| HOOF-Prints | Hypervariable octameric oligonucleotide fingerprints |
| VNTR | Variable-number tandem repeat |
Footnotes
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.
MEGA, version 5.0. Available at: external.informer.com/megasoftware.net/. Accessed May 5, 2008.
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Appendix
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′.
