Piroplasmosis of cattle has a significant negative economic impact due to loss of productivity, animal mortality, costs of tick control, disease detection, prevention and treatment. In addition, piroplasmosis is a deterrent to the intensification of livestock farming with highly productive cattle, since highly productive cattle brought to an unfavorable region, when infected, suffer from a severe acute form of the disease with a high mortality rate and loss of productivity, while native cattle are more resistant to the disease and suffer from it in a milder form. Babesiosis (pyraplasmosis) is a tick-borne infection caused by protozoan parasites of the genus Babesia, order Piroplasmida, family Babesiidae. Representatives of the genus Babesia have historically had evolutionary success, due to which more than 100 species of Babesia spp have now been identified in mammals and birds. In large ruminants, Babesia bovis, Babesia bigemina, Babesia occultans, Babesia divergens, Babesia ovata, Babesia odocoilei and Babesia capreoli are detected. Bovine babesiosis caused by B.bovis and B. bigemina is an acute and persistent disease characterized by high mortality. Animals that survive acute infection become long-term asymptomatic carriers of the parasite. Thus, persistently infected animals are a constant source of tick transmission and maintenance of herd immunity. Compared with B. bovis, B. bigemina is considered to be lower in virulence; however, infection caused by B. bigemina can also lead to serious economic losses in livestock production.
The goal of the project is to identify and synthesize immunodominant antigens of Babesia bigemina and develop an ELISA test system for the serological diagnosis of bovine babesiosis.
The idea is to study the immunodominant proteins of Babesia bigemina and determine their diagnostic value in ELISA. For this purpose, recombinant antigens described in the literature will be obtained, as well as new proteins that potentially have diagnostic value, determined by “reverse vaccinology” methods. Recombinant analogs will be obtained for candidate proteins, the specificity of which will be determined by ELISA and Western blotting. Based on the selected specific antigens, an ELISA test system will be developed. Widespread use of the development in the diagnosis of babesiosis will contribute to the timely and prompt detection of infected livestock, will have a positive effect on increasing productivity, and will prevent the spread of the disease to new regions. The proposed research design corresponds to an interdisciplinary approach that ensures collaboration between broad scientific areas: veterinary medicine, bioinformatics, molecular biology, and immunology.
Marat Kuibagarov, 49 years old, Candidate of Veterinary Sciences, Senior Researcher of the Applied Genetics Laboratories of the National Center for Biotechnology. Trained in research facilities in Germany and the United States Research interests: molecular biology, genomic technologies, development of PCR and ELISA test systems, genetic engineering, 20 years of scientific experience. Hirsch index by Scopus/WoS =3. As part of the submitted project, he carries out general project management, takes part in optimizing ELISA parameters, interpreting and analyzing the results obtained, and preparing scientific and technical documentation.
https://www.scopus.com/authid/detail.uri?authorId=57220278412
https://orcid.org/0000-0001-7428-7620
Kassym Mukanov – 69 years old, Doctor of Veterinary Sciences, Professor, Chief Researcher of the Laboratory of Applied Genetics of the National Center of Biotechnology, 43 years of scientific experience. H-index according to Scopus/WoS=7. Has experience in developing diagnostic test systems based on ELISA, PCR, obtaining monoclonal antibodies, and molecular genetic characterization of infectious disease agents. Area of scientific interests: immunology, development of diagnostic systems, epidemiology, virology and microbiology. As part of the project, work will be carried out on identifying genes, performing PCR and ELISA, obtaining recombinant antigens, and developing laboratory regulations.
https://www.scopus.com/authid/detail.uri?authorId=56340590800
https://orcid.org/0000-0002-0502-9238
Alexandr Shevtsov, 42 years old, candidate of biological sciences, Head of the Laboratory of Applied Genetics of the National Center for Biotechnology, 16 years of scientific work experience. He has experience in the development of diagnostic test systems based on PCR, the genetic characteristics of pathogens that cause infectious diseases. Research interests: PCR, Sanger sequencing, and using next-generation sequencers (NGS) such as Ion Torrent, MiSeq, MLVA typing, cloning, primer design, and the development of PCR test systems. Hirsch index by Scopus/WoS = 11. As part of the project, it will carry out work on gene identification, sequencing, primer design, PCR and RT-PCR, ELISA, and obtaining recombinant proteins.
http://orcid.org/0000-0002-0307-1053
https://www.scopus.com/authid/detail.uri?authorId=57201604158
Asylulan Amirgazin, 31 years old, researcher at the Laboratory of Applied Genetics of the National Central Library, 6 years of scientific experience, H-index (Scopus) = 42. Area of research: Sanger sequencing, NGS, real-time PCR, bioinformatics analysis. As part of the project, she takes part in bioinformation analysis using reverse vaccinology methods, sequencing, cultivation of recombinant strains, isolation and purification of target proteins, and ELISA.
https://orcid.org/0000-0001-9418-7758
https://www.scopus.com/authid/detail.uri?authorId=57218628470
Anara Ryskeldina, 27 years old, is a master, junior researcher at the Laboratory of Applied Genetics of the National Center for Biotechnology. Research experience of 4 years, Hirsch index (Scopus) = 2. Field of research: Sanger sequencing, real-time PCR. Within the framework of the project, she takes part in cloning (pGEM-T), sequencing, cultivation of recombinant strains, isolation and purification of target proteins, and ELISA.
https://www.scopus.com/authid/detail.uri?authorId=57218871763
https://orcid.org/0000-0002-7100-2711
Ayan Dauletov, 22 years old, laboratory assistant at the Laboratory of Applied Genetics of the National Central Library. Research experience 2 years. Area of research: PCR, ELISA, Western blot. As part of the project, she takes part in the isolation and purification of target proteins, ELISA, Western blot, SDS-PAGE, and preparation of reagent solutions.
https://orcid.org/0000-0003-1906-6889
DNA samples from whole blood collected from cattle were tested using universal primers targeting the ribosomal operon of Babesia spp. and Theileria spp.. To determine species diversity, sequencing of the 18S rRNA gene fragment was performed on PCR-positive samples. The results showed that the highest percentage of cattle infected with Theileria spp. and Babesia spp. was found in two southern regions of Kazakhstan (Zhambyl and Turkestan). Two species of Theileria (T. annulata and T. orientalis) and three species of Babesia (B. bigemina, B. occultans, and B. major) were identified.
To identify diagnostically relevant antigens, the proteome of B. bigemina was analyzed. Bioinformatic analysis of predicted proteins was conducted using reverse vaccinology (in silico) methods. The genome of B. bigemina was analyzed to identify open reading frames of proteins, and proteins predicted to be accessible to the immune system were determined. Conserved genes of the selected proteins were analyzed across pathogenic Babesia species. Strategies and methods for cloning the selected candidates were defined, including expression systems, vectors, cultivation conditions, and purification processes. Amplification and de novo synthesis of nucleotide sequences for two proteins identified in silico were performed.
De novo amplification/synthesis of nucleotide sequences of diagnostically significant B. bigemina protein genes described in the literature was carried out. To produce the AMA-1 protein, de novo synthesis of regions of ectodomains I and II was performed. For the production of RAP-1 antigens, amplification of the C-terminal fragment was carried out.