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: Bovine babesiosis, caused by Babesia bovis, poses significant economic challenges to Kazakhstan’s cattle industry. Early and accurate detection is crucial for interrupting transmission cycles, particularly in regions lacking advanced diagnostic infrastructure. This study aimed to develop a rapid lateral flow immunoassay (LFIA) using a recombinant C-terminal fragment of the recombinant rhoptry-associated protein 1 (rRap1) antigen for the serodiagnosis of bovine babesiosis. Materials and Methods: A C-terminal fragment (amino acids 345–480) of the B. bovis Rap1 gene was codon optimized and expressed in Escherichia coli. The recombinant protein was purified using metal-affinity chromatography and validated through sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, and nanoflow liquid chromatography-tandem mass spectrometry. A diagnostic evaluation was performed using enzyme-linked immunosorbent assay (ELISA) and LFIA on sera from 102 uninfected and 15 infected cattle, all of which had been pre-tested using polymerase chain reaction. Colloidal gold-protein G conjugates were prepared for LFIA, and test conditions were optimized for antigen concentration and serum dilution. Assay performance was compared with previously published LFIAs. Results: A 21-kDa rRap1 protein was successfully expressed and demonstrated high specificity to positive control sera. ELISA and LFIA both detected antibodies in 13 of 15 infected samples (sensitivity 86.6%). Specificity was 90.1% for ELISA and 88.2% for LFIA. Receiver operating characteristic analysis showed an area under the curve of 0.83, and Cohen’s Kappa indicated fair-to-moderate agreement between ELISA and LFIA. The LFIA exhibited comparable performance to assays based on merozoite surface antigen 1 or spherical body protein antigens, marking the first successful use of a B. bovis Rap1 C-terminal fragment for LFIA-based field diagnostics in Kazakhstan. Conclusion: The developed rRap1-based LFIA is a promising, field-deployable diagnostic tool for bovine babesiosis, offering rapid results without the need for laboratory equipment. Despite slightly lower sensitivity than ELISA, its simplicity, cost-effectiveness, and specificity support its use in large-scale epidemiological surveillance. Further validation in diverse field conditions and cattle populations is recommended to refine sensitivity and broaden applicability. Keywords: Babesia bovis, bovine babesiosis, enzyme-linked immunosorbent assay, lateral flow immunoassay, rapid diagnostics, recombinant rhoptry-associated protein 1, serodiagnosis.

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.

Background and Aim: Toxoplasma gondii is an intracellular protozoan and a major foodborne pathogen worldwide. Nearly, all warm-blooded animals are susceptible to toxoplasmosis, with raw and undercooked meat and animal products serving as the primary transmission routes. To date, the distribution of T. gondii among farm animals in Kazakhstan has been inadequately studied. This study aimed to determine the occurrence of antibodies against T. gondii in cattle and sheep in the Akmola and Kostanay regions. Materials and Methods: Blood samples were randomly collected from 437 cattle and 397 sheep from two regions of Kazakhstan: Akmola and Kostanay. A commercial enzyme-linked immunosorbent assay based on the native protein Toxoplasma surface antigen 1 was used for serological analysis. Results: The occurrences of T. gondii were 8.0% and 3.8% among cattle and 42.1% and 19.0% among sheep in the Akmola and Kostanay regions, respectively. Antibodies against T. gondii were detected in all study areas. The greatest frequency of seropositive reactions in cattle was observed in the Arshaly region (9.0%), whereas the least frequent was observed in Arkalyk (3.3%). The highest occurrence of seropositive reactions among sheep was found in the Zerenda region (54.5%), whereas the lowest was found in the Auliekol region (15.6%). Conclusion: The obtained results confirmed the circulation of the T. gondii pathogen among cattle and sheep in the investigated regions. These findings provide insight into the current distribution of this zoonotic parasite among farm animals in Kazakhstan. Keywords: antibodies, cattle, enzyme-linked immunosorbent assay, sheep, surface antigen 1, Toxoplasma gondii.

Bovine babesiosis is a disease caused by parasites of the genus Babesia. One of the main Babesia genera causing disease in cattle, according to the International Office of Epizootics (OIE), is Babesia bovis. Recovered cattle become permanent latent carriers and can be a source of tick infestation and spread of infection. Despite susceptibility and specificity, diagnostic methods such as enzyme-linked immunosorbent assays (ELISA) and indirect fluorescent antibody assays (IFA) have some disadvantages. These methods require a long analysis time, the use of specialized equipment and trained specialists. At the same time, immunochromatographic tests (ICA) are widely used in veterinary practice today. ICA is characterized by a short analysis time, ease of performance and interpretation of results. The main components of the ICA test system are monoclonal antibodies. As a result of the studies, three mAbs against the recombinant Rap1 antigen of B. bovis were obtained. The resulting mAbs have high diagnostic characteristics. A conjugate of colloidal gold with mAbs is capable of detecting the Rap1 antigen of the causative agent of babesiosis in cattle.