Background and Aim: One of the reasons for the decline in the number of wild species of artiodactyls is poaching and the illegal trading of animal products. Molecular genetic identification of animals from a biological sample effectively proves poaching cases and illegal trade of animal products. This study aimed to develop a polymerase chain reaction (PCR) test that allows for species identification of artiodactyl animals that are most often subject to poaching. Materials and Methods: Genomic DNA was extracted from meat and blood samples of animals killed by poachers using commercial kits. Three pairs of primers were designed and used to amplify the cytochrome b gene fragment of Roe deer, Saiga antelope, and Siberian stag. Results: The proposed protocol allows amplification of specific PCR products of 542 bp with Roe deer DNA, 587 bp with Saiga DNA, and 525 bp with Siberian stag DNA. Specificity analysis showed no cross activity with DNA from other animal species. The detection limit of PCR ranged from 15.6 pg to 1.9 pg of DNA in 25 μL of the reaction mixture. Conclusion: Sequencing the amplified products and subsequent comparison with the corresponding reference sequence showed a similarity ranging from 99.99% to 100%. The PCR based on the developed primers demonstrated high sensitivity and specificity when using DNA from homogeneous and heterogeneous animals.

Background and Aim: Salmonella abortion in mares is caused by Salmonella enterica subspecies enterica serovar abortus equi infection and is characterized by premature (abortion) or non-viable fetus birth. Although all horses are susceptible to infection, the condition is more often clinically manifested in pregnant mares, with most abortions recorded in young females. In addition, nonspecific clinical disease signs and poorly sensitive and effective bacteriological diagnostic methods hinder rapid and reliable infection diagnoses. Immunochemical methods such as enzyme-linked immunosorbent assay (ELISA) and immunochromatography assays can facilitate effective and rapid diagnoses. However, they require highly specific and active antigens and antibodies. This study aimed to generate a recombinant S. enterica outer membrane protein X (OmpX) and evaluate its suitability for serological diagnosis of Salmonella abortion in mares. Materials and Methods: Outer membrane protein X from the S. enterica antigen was synthesized de novo and expressed in Escherichia coli using the pET28 vector. Transformed E. coli cells were cultured under different conditions to detect recombinant OmpX (rOmpX) expression, and rOmpX purification and refolding were both conducted using metal affinity chromatography. Refolded and purified rOmpX was characterized by western blotting, liquid chromatography with tandem mass spectrometry, and ELISA. Results: After optimized rOmpX expression, a 23 kDa molecular weight protein was identified. Amino acid sequence analysis using Mascot program suggested that these peptides were the OmpX protein from S. enterica. High specificity and diagnostic efficiency were recorded when rOmpX was used in ELISA against 89 serum samples from aborted and contact mares. Conclusion: Recombinant outer membrane protein, in comparison to the O antigen, demonstrated better diagnostic characteristics against sera from mares who aborted and contact horses. Keywords: diagnostics, outer membrane proteins, recombinant antigens, Salmonella abortion in mares, Salmonella enterica, specific antibodies.

Background and Aim: The rapid spread of lumpy skin disease (LSD) globally poses a serious threat to the agricultural sector. The timely and accurate diagnosis of the disease is crucial to control LSD. This study aimed to determine the effect of thioredoxin on the immunogenicity of the recombinant P32 (rP32) protein of LSD virus (LSDV). Since the P32 protein is poorly soluble, it is often expressed by adding an auxiliary sequence of a highly soluble partner protein such as thioredoxin. Materials and Methods: The P32 gene fragment was amplified using a polymerase chain reaction from genomic DNA used as a template. The resulting DNA fragments were cloned into the pET32a vector, and transformed into Escherichia coli BL21 (DE3) cells through electroporation. Purification of the rP32 protein was performed using a HisTrap column. Purified rP32 protein fused with thioredoxin (rP32Trx) was characterized by western blotting, liquid chromatography with tandem mass spectrometry and indirect enzyme-linked immunosorbent assay (ELISA). Results: Indirect ELISA revealed that, despite the lower molecular weight, the main part of the antibodies in the serum of immunized mice was directed against thioredoxin and not the target P32 protein. Thus, the antibody titers against rP32Trx were 1:102400, whereas antibody titers against heterologous recombinant 3BTrx and PD1Trx proteins were 1:25600 and 1:51200, respectively. Concurrently, the antibodies did not bind to the heterologous recombinant PD1 protein, which did not contain thioredoxin. Conclusion: The results showed that the rP32 protein fused with the partner protein thioredoxin could not be used to obtain polyclonal and monoclonal antibodies. However, the recombinant fusion protein rP32Trx can be used to develop a serological test to detect antibodies, since antibodies against thioredoxin were not detected in the animal sera.

Background and Aim: Bovine interleukin 15 (bIL15) is a potential immunotherapy that can block the spread of bovine leukemia virus (BLV). However, immune checkpoints that maintain body homeostasis may reduce their effectiveness. Thus, an analysis of the effectiveness of bIL15 while blocking negative immune regulators is necessary. We aimed to obtain recombinant bIL15 (rbIL15) and determine its percentage using monoclonal antibodies against bovine cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death ligand 1 (PD-L1). To achieve this goal, peripheral blood mononuclear cells (PBMCs) from healthy and BLV+ cattle were treated with bIL15 using a CTLA-4– and PD-L1–blocking algorithm. Materials and Methods: The codon-optimized bIL15 gene was synthesized under de novo conditions using polymerase chain reaction (PCR). The synthesized gene was cloned into pET28 and transformed into electrocompetent Escherichia coli BL21 cells; rbIL15 was purified using metal affinity chromatography and analyzed using sodium dodecyl-sulfate– polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. The expression of the Bcl2, STAT3, and STAT5 genes was studied using qualitative PCR. An enzyme-linked immunosorbent assay (ELISA) was used to analyze interferon (IFN)-γ production by rbIL15-treated mononuclear cells. Results: Analysis of rbIL15 using SDS-PAGE and western blotting revealed a specific product weighing 24 kDa. The optimal conditions for rbIL15 induction were 0.2 mM isopropyl-β-D-1-galactopyranoside and 37°C. When rbIL15 was added to PBMCs from healthy cattle, the Bcl2, STAT3, and STAT5 genes were expressed. ELISA of the culture medium of rbIL15-treated PBMCs revealed IFN-γ production. When PBMCs from healthy cows were treated with rbIL15, CTLA-4, and PD-L1 blockade together, they did not produce more IFN-γ than the rbIL15 group. Using PBMCs from BLV+ cattle, combination treatment increased IFN-γ production. Conclusion: The biological activity of rbIL15 is characterized by the induction of transcription factors and the production of IFN-γ. Using rbIL15 with CTLA-4 and PD-L1 blockade in PBMCs from healthy and BLV+ cows led to the production of a transcription factor and cytokine. The results demonstrate the possibility of using this method to improve immunity and immunological memory in patients with chronic viral infections. Keywords: bovine interleukin-15, bovine leukemia virus, cytotoxic T-lymphocyte antigen 4, monoclonal antibody, programmed cell death ligand 1.

Background and Aim: Campylobacteriosis causes gastrointestinal tract lesions in adults and children and may result in severe complications. The primary sources of infection are infected animals and animal products. Immunochemical methods effectively diagnose intestinal infections but require highly specific antigens to detect their antibodies. This study aimed to obtain two recombinant immunogenic antigens of Campylobacter jejuni, an outer membrane protein with a molecular weight of 18 kDa (Omp18) and the major outer membrane protein (MOMP) with a molecular weight of 45 kDa, and evaluate their suitability for the serological diagnosis of campylobacteriosis using immunochromatographic assay (ICA). Materials and Methods: The C. jejuni Omp18 and MOMP gene sequences were synthesized de novo (Macrogen, Korea) and cloned into the pET32 expression plasmid. Using these genetic constructs, electrocompetent cells of the Escherichia coli BL21 strain were transformed and cultured under various conditions. Antigens were purified and refolded using metal affinity chromatography. The properties of the purified proteins were studied by western blotting, liquid chromatography with tandem mass spectrometry, and enzyme-linked immunosorbent assay (ELISA). Results: We developed two recombinant E. coli BL21 cells producing rOmp18 and Recombinant MOMP (rMOMP) antigens with molecular weights of 36 and 64 kDa, respectively. Amino acid sequence analysis of the obtained antigens showed complete homology with the reference sequences in the PubMed NCBI database. Western blotting using positive-control sera demonstrated the specificity of the recombinant antigens. The results of ELISA with 94 bovine sera showed the interaction of recombinant antigens with specific antibodies. Conclusion: The obtained rOmp18 and rMOMP antigens can detect antibodies in the serum of infected or recovered animals and can be used to develop ICA.

Background and Aim: In chronic viral infections, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed death ligand 1 (PD-L1) significantly suppress immune responses. The CTLA-4 receptor abundance in regulatory T cells showed a positive association with viral load and a negative association with interferon-gamma (IFN-γ) production in bovine leukemia virus (BLV)-infected cattle. Blocking this receptor boosted IFN-γ production, recovering immune response against this illness. In human cancer patients, not everyone responded positively to non-immunotherapy using CTLA-4 receptor antibodies. The present study analyzed the synergistic effects of CTLA-4 and PD-L1 receptor blockade on IFN-γ production in BLV+ cattle in vitro. Materials and Methods: The genes for bovine CTLA-4 and PD-L1 were artificially produced. The amino acid sequences of the extracellular receptor domains were sourced from the National Center for Biotechnology Information PubMed database. The western blotting and liquid chromatography with tandem mass spectrometry (LC-MS/MS) techniques were employed for the characterization of recombinant CTLA-4 (rCTLA-4) and recombinant PD-L1 (rPD-L1) proteins. The immunoinhibitory effects of recombinant proteins in Staphylococcus enterotoxin B (SEB)-stimulated cattle peripheral blood mononuclear cells (PBMCs) were investigated. Enzyme-linked immunosorbent assay (ELISA) was used to analyze monoclonal antibodies against rCTLA-4 and rPD-L1. Antibodies generated from peripheral blood mononuclear cells of healthy and BLV-seropositive cows were employed to evaluate their blocking capabilities. Results: The resulting recombinant proteins specifically reacted with commercial homogeneous monoclonal antibodies (mAbs) using ELISA and anti-His-tag mAbs using western blotting. Analysis of the proteins using LC-MS/MS revealed correspondence with the sequences of rCTLA-4 and rPD-L1 located in the Mascot database. rCTLA-4 and rPD-L1 proteins inhibited IFN-γ production in bovine PBMCs of activated SEB. When PBMCs from cows were cultured with activated SEB containing rCTLA-4 and rPD-L1, the mAbs increased IFN-γ production in PBMCs. The combined cultivation of mAbs and PBMCs from BLV+ cattle enhanced IFN-γ production in the cells. Conclusion: These findings suggest that the combined blockade of bovine CTLA-4 and PD-L1 receptors can be used as a therapy for bovine leukemia. However, it was shown that a single PBMC sample from a BLV-positive donor did not amplify the synergistic effect. Therefore, it is necessary to perform further studies on a larger population and assessing a wider range of cytokines. Keywords: bovine, bovine leukemia virus, cytotoxic T-lymphocyte-associated antigen 4, interferon-gamma, programmed death ligand 1.