The present study was approved by the Institutional Committee of Post-Graduate Studies and Research at Shihezi University (Shihezi, China). All efforts were made to minimize animal suffering.

The GTPV strain G14-STV44-55, an attenuated live vaccine, was provided by Xinjiang Tecon Animal Husbandry Biotechnology Co., Ltd. (Urumqi, China). Sheep fetus fibroblast (SFF) cells and lamb testis (LT) cells (obtained from Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing, China) were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in 5% CO₂. The GTPV was propagated and titrated on SFF or LT cells in DMEM supplemented with 2% FBS. The plasmid pLSEG-GPT includes the GPT gene and 7.5K early/late promoter (P7.5). The plasmid pGM-TK-I1L-GPT1 was synthesized by the Key Laboratory of Xinjiang Endemic and Ethnic Disease, Shihezi University.

In total, 30 female six-week-old BALB/c mice (weight, 25 g) were obtained from the Experimental Animal Center of the Academy Military Medical Science (Beijing, China). Animals were maintained in barrier housing with filtered inflow air in a restricted-access room in pathogen-limited conditions. The light/dark cycle was 12/12 h, the temperature was 26°C and the humidity was 40–60%. They were acclimatized for a minimum of 1 week prior to experimentation. Water and commercial food were provided ad libitum. All experimental procedures and animal care were performed in compliance with institutional animal care regulations.

Total genomic DNA was extracted from infected LT cells using a commercial virus DNA extraction kit (Bioteke Corporation, Beijing, China). Polymerase chain reaction (PCR) amplification of the conserved TK gene was successfully performed on all isolates using Taq DNA polymerase (Takara Biotechnology, Co., Ltd., Dalian, China) for PCR with specific primers (Table I). The PCR reaction system contained the following: 1.5 µl 10X buffer, 0.2 µl dNTP (10 mmol/l), 1 µl DNA (20 ng/µl), 0.2 µl Taq enzyme, 0.2 µl primers (20 µmol/l), 0.6 µl MgCl₂ (25 mmol/l; Tiangen Biotech Co., Ltd., Beijing, China) and 11.1 µl H₂O. The total volume was 15 µl. The PCR reaction conditions were as follows: 5 min at 95°C; followed by 30 cycles at 60°C for 40 sec and 72°C for 1 min; and 10 min at 72°C. PCR products were analyzed on 1% agarose gels containing ethidium bromide under a UV transilluminator. Subsequently, PCR products were ligated to the plasmid pGEM-T (Tiangen Biotech, Co., Ltd.) for DNA sequencing.

Fick and Viljoen (12) previously identified the VVI1L promoter sequence; therefore, the present study used the reverse complementary sequence of the VVI1L promoter and the GPT gene promoter, overlapping synthesis of primers in the opposite direction (Table I).

The vaccinia virus p7.5 promoter that regulates xanthine-guanine GPT was obtained by XhoI/NotI digestion of plasmid pLSEG-GPT, and was used as a positive selection marker for recombinant virus screening. It was ligated to the XhoI and NotI restriction sites of pBS-T. This constructed element was inserted at the Acc65I restriction sites of the TK gene of strain G14-STV44-55, and was subsequently used for the construction of a transfer vector, which was termed pGM-TK-I1L-GPT.

The OMP25 DNA (642 bp) of Brucella was amplified with OMP25-F and OMP25-R, in which the Xho I sites products were ligated with pGM-TK-I1L-GPT vector to generate pGM-TK-I1L-GPT-OMP25.

Confluent LT cells were co-transfected with GTPV G14-STV44-55 and the expression vector pGM-TK-I1L-GPT-omp25, using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Cultures were subsequently harvested when 80% cytopathic effect (CPE) was observed. Cultured LT cells were digested with 0.25% trypsin and resuspended in screening solution (DMEM, 2.5% fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin/streptomycin, 25 µg MPA ml⁻¹, 250 µg xanthine ml⁻¹, 15 µg hypoxanthine ml⁻¹, 2 µg aminopterin ml⁻¹ and 2 µg thymidine ml⁻¹). Following centrifugation at 350 × g for 5 min at room temperature, cells were added to 12-well cell culture plates (1 ml well⁻¹; 1×10⁵ cells ml⁻¹). Cells were cultured at 37°C in 5% CO₂ for 16–24 h until 70–80% confluent. Subsequently, cultures were harvested once they reached 80% CPE, and were inoculated on LT cells following repeated freezing and thawing three times. Cells were cultured at 37°C in 5% CO₂ for 14 days and observed once daily. The cytopathic culture medium was collected, and centrifuged at 350 × g for 5 min at room temperature to remove cell debris, and used for further screening with this method. The recombinant virus was confirmed by PCR following the protocol mentioned above.

LT cells were infected with the recombinant virus. For transmission electron microscopy, at 14 days post-infection, cells were fixed with 4% glutaraldehyde for 1 h at room temperature overnight at 4°C, dehydrated in an ascending ethanol series and embedded for 1 h at room temperature in Epoxi resin (Epoxi Embedding Medium kit; Sigma-Aldrich; Merck KGaA). Ultrathin sections (45 nm) were stained with 2% uranyl acetate and Reynold solution for 1 h at room temperature. For electron microscopy, cells were fixed with 4% paraformaldehyde and 0.25% glutaraldehyde for 24 h at room temperature. Ultrathin sections were collected in nickel grids and observed using a Zeiss EM109T transmission electron microscope (Zeiss GmbH, Jena, Germany; magnification, ×400).

The recombinant virus was screened to confirm its genetic stability. The recombinant virus was passaged for 15 generations, and viral genomes were extracted every 2–3 generations. PCR was performed to amplify the attachment gene of rGTPV-OMP25.

The recombinant virus-infected LT/SFF cells were harvested when 90% CPE was observed, cells were subsequently freezed/thawed three times and the cellular debris was pelleted. Lysis buffer [60 mM Tris-HCl (pH 7.1), 1 mM MgCl₂, 0.05% NP40, and 20 µg DNase ml⁻¹] was added to lyse the cells. Samples were centrifuged at 350 × g for 5 min at room temperature to remove the supernatant. Supernatants were mixed with an equal volume of 2X sample buffer [0.1 M Tris-HCl (pH 6.8), 4% sodium dodecylsulfate, 20% glycerol, 12% 2-mercaptoethanol and bromophenol blue] and boiled for 10 min. The protein concentrations were determined by a bicinchoninic acid assay. Samples (25 µg) were separated by 12.5% SDS-PAGE and subsequently transferred to nitrocellulose membranes. Membranes were blocked in blocking solution [5% nonfat milk in Tris-buffered saline Tween-20 (TBST)] for 1 h at room temperature, and incubated with Brucella-vaccinated sheep serum (Center of Chinese Disease Prevention and Control, Beijing, China; 1:300) diluted in blocking solution at 37°C for 1 h. Following three 10-min washes with TBS with Tween-20 (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% Tween-20), membranes were incubated with horseradish peroxidase (HRP)-conjugated rabbit anti-sheep immunoglobulin G (IgG) secondary antibodies (1:5,000; cat. no. SE134; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 1 h in 5% milk/TBST at room temperature. Membranes were washed again and bands were visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer's protocol.

The levels of OMP25 and GTPV were determined in the sera of mice by ELISA Quantikine Mouse kit (Elabscience Biotechnology, Inc., Houston, TX, USA; cat. no. E-EL-M0692c). The OMP25 protein served as the coating antigen and was diluted with carbonate buffer to 1:500, 1:600 and 1:400. Following this, 100 µl was added to the micro-ELISA strip plates for 1 h at 37°C and coated overnight at 4°C. Strips were washed by filling each well with wash solution (400 µl) three times, adding 150 µl blocking solution (Sangon Biotech Co. Ltd., Shanghai, China) to each well, washing three times, adding 100 ml test serum (test serum from mice that immunized with OMP25 protein; 1:40) to each well for 1 h at 37°C, adding 100 ml sheep anti-mouse HRP-conjugated IgG secondary antibody (1:5,000; cat. no. ab6808; Abcam, Cambridge, UK) for 1 h in 5% milk/TBST to each well for 1 h at 37°C, adding 100 µl fresh chromogen solution (TransGen Biotech Co., Ltd.) to each well, and finally incubating for 15 min at 37°C. Similarly, purified GTPV vaccine strains were used as a coating antigen, and test serum (1:40) was used as a primary antibody. PBS-treated cells served as a control.

Histopathological studies were performed as previously described (13). Samples of the liver and kidney and from mice immunized with rGTPV were fixed in 10% neutral buffered formalin solution at necropsy for 3 days at room temperature. They were dehydrated, embedded in paraffin, and cut into 5–7-µm sections, which were subsequently stained with hematoxylin and eosin for 2 h at room temperature. Pathological sections were observed using a Motic ordinary light microscope (Motic BA310 DIGITAL; Motic Incorporation, Ltd., Causeway Bay, Hong Kong, China; magnification, ×400).

Antibody response and cytokine production are expressed as the mean ± standard deviation of the mean of three independent experiments. Two-tailed one-way analysis of variance followed by Bonferroni's post hoc pairwise comparison was used to assess the differences between groups. Statistical comparisons between different groups were conducted using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The GTPV-specific primers amplified fragments of 2400 bp, as expected, that were visualized on a 1% agarose gel using ethidium bromide staining. Sequence analysis demonstrated 99.54% homology between the TK gene and GTPV G20-LKV strain (14).

The pLSEG plasmid was digested with XhoI and NotI restriction enzymes, and the reporter gene GPT of 1,200 bp was obtained, as expected, including E. coli. xgpt (459 bp) and the upstream VVP7.5 promoter gene, indicating that a complete construct had been generated. After insertion into the PBS-T vector, transformation and confirmation were performed.

The Brucella OMP25 gene was amplified by PCR; yielding fragments had sizes of ~642 bp. PCR fragments derived from the genomic DNA of Brucella were digested and ligated to the XhoI restriction sites of the transfer plasmid pGM-TK-I1L-GPT, to construct the recombinant vector pGM-TK-I1L-GPT-OMP25 (Fig. 1).

Recombinant viruses were screened in LT and SFF cells until a CPE was observed. Viruses were tested for genetic stability at defined intervals after repeated passages. In the present study, in a total of 10 generations, viral genomes were extracted every 2–3 generations, and PCR was performed using customized primers to yield amplified fragments with sizes of 642 bp.

The transfection product rGTPV-OMP25 was inoculated into confluent SFF and LT cells that were cultured in screening solution for 24 h, and then transfected cells were observed daily for 14 days by microscopy. With the extension of the infection time by rGTPV, the cytopathic phenomenon gradually increased (Fig. 2). The electron microscope examination demonstrated that the viral particles had an oval or round appearance, and brick-shaped virion were identified in cytoplasm (Fig. 2).

The recombinant virus was passaged for 15 generations, and the viral genome was extracted every 2–3 generations; the genetic stability of rGTPV was confirmed by PCR (Fig. 3).

To confirm that the OMP25 was expressed in the recombinant vector, whole cell extracts of LT/SFF cells transfected with the GTPV or rGTPV strains were separated by electrophoresis. A band with a molecular mass of 25 kDa was present in rGTPV G14-STV44-55-OMP25 and GTPV G14-STV44-55; however, was missing in untransfected SFF cells. Furthermore, when western blot analysis of the expressed protein was performed using an anti-native Brucella serum, the absence of OMP25 in rGTPV G14-STV44-55-OMP25 was confirmed (Fig. 4).

The levels of OMP25 and GTPV in the sera of mice was measured at 450 nm using a microtiter plate reader. Compared with the control group, OMP25 levels were significantly upregulated in the OMP25 group. However, the rGTPV-OMP25 group exhibited reduced OMP25 levels compared with the OMP25 group (Fig. 5A). GTPV levels were significantly upregulated in the GTPV group compared with the control group; however, there were no significant differences between the GTPV and rGTPV-OMP25 groups (Fig. 5B).

The liver and kidney are important organs that harbor rGTPV during infection. To assess whether the increased bacterial burden in mice altered liver and kidney pathology, the number and area of granulomas in these organs were determined. As presented in Fig. 6, the mice did not exhibit alterations in liver granulomas after rGTPV infection, indicating that the recombinant vaccine did not cause visible damage to the liver or kidney.