Babesiosis is a tick-borne infection caused by protozoan parasites of the genus Babesia, suborder Piroplasmida, family Babesiidae. Members of the genus Babesia have historically had evolutionary success, with more than 100 species of Babesia spp. now identified in mammals and birds. Bovine babesiosis is an acute and persistent disease characterized by high mortality. In large ruminants are detected: Babesia bovis (B.bovis), Babesia bigemina (B. bigemina), Babesia occultans, Babesia divergens, Babesia ovata, Babesia odocoilei and Babesia capreoli. Babesiosis of buffalo in China caused by Babesia orientalis has been described [6]. The most frequently reported species detected in cattle with babesiosis are B.bovis and B.bigemina. Animals that survive acute infection become long-term asymptomatic carriers of the parasites. Thus, persistently infected animals are a constant source of tick transmission and maintenance of collective immunity. One of the traditional methods for diagnosing babesiosis is microscopy of stained blood smears to detect merozoites of the pathogen. This is a fairly easy-to-perform test that allows detection of acute cases of infection, but is of limited use at low levels of parasitemia. There may also be difficulties in differentiating babesia morphologically from other hemosporidium species.
The main aim of the project is to study the species diversity of Babesia spp in Kazakhstan and to develop a PCR test system for differential diagnosis of babesiosis of cattle.
The idea is to study the species diversity of Babesia spp on the territory of Kazakhstan and to develop a PCR test system for diagnostics of cattle babesiosis. In order to obtain up-to-date data on Babesia species composition, DNA samples from whole blood of cattle and tick suspensions from different regions of Kazakhstan will be tested. The primers, fluorescent probes and optimized PCR reaction conditions will be selected for the established Babesia species. Developed conditions will be the basis for laboratory prototype of PCR test-system for detection and differentiation of cattle babesiosis by PCR with real-time detection. At the final stage, the sensitivity and specificity of the laboratory developed prototype PCR test system will be evaluated and laboratory regulations will be developed for further implementation.
Assel Akhmetova – project leader, 32 years old, PhD, senior researcher at the laboratory of applied genetics of NCB. Hirsch index = 4 (Scopus). Experience of scientific work for 9 years. Research interests: molecular epidemiology, development of diagnostic test systems, epizootology, microbiology, molecular biology, application of new generation sequencing methods. Provides overall leadership within the project. Participates in determining the design of experiments, development of PCR protocols, preparation of necessary reports and scientific publications on the results of research.
https://orcid.org/0000-0003-2206-7890
https://www.scopus.com/authid/detail.uri?authorId=56736565400
Dinara Kamalova – 36 years old, master, PhD student, researcher at the laboratory of applied genetics of NCB. Hirsch index=3 (Scopus). Experience of scientific research for 10 years. Research interests: development of diagnostic test systems for diagnostics of infectious diseases, genotyping by MLVA and MLST methods. Within the framework of the project she participates in the development of PCR test systems and gene identification, primer design, PCR and sequencing.
https://www.scopus.com/authid/detail.uri?authorId=56736413900
https://orcid.org/0000-0002-8444-3305
Alma Kairzhanova – 34 years old, PhD student, researcher at the laboratory of applied genetics of NCB. Hirsch index (Scopus) = 2. Experience of scientific research 12 years. Area of research: development of PCR for diagnostics of blood-parasitic diseases, sequencing. Within the framework of the project participates in the development of PCR protocols, evaluation of specificity and sensitivity of real-time PCR test systems.
https://orcid.org/0000-0002-9864-2700
https://www.scopus.com/authid/detail.uri?authorId=56737034300
Madina Kadyrova – 25 years old, master, laboratory assistant of the laboratory of applied genetics of NCB. Experience of scientific work for 2 years. Area of research: isolation of nucleic acids, PCR development, sequencing by Sanger method. Within the framework of the project participates in DNA isolation, study of samples taken from cattle and small ruminants by real-time PCR, horizontal electrophoresis, purification of PCR products.
https://orcid.org/0000-0002-9079-8743
Nailya Tursunbay – 23 years old, master’s student, laboratory assistant of applied genetics laboratory of NCB. Experience of scientific work 2 years. Area of research: isolation of nucleic acids, PCR development, sequencing by Sanger method. Participates in PCR staging, real-time PCR, purification of PCR products, sequencing.
https://orcid.org/0000-0002-6604-4247
Alexander Ostrovskii, 23 years old, master’s student, laboratory assistant at the Laboratory of Applied Genetics, NCB. Experience of scientific work 1 year. Area of research: development of PCR for diagnostics of infectious diseases, evaluation of test-systems efficiency. Within the framework of the project participates in PCR staging, real-time PCR, purification of PCR products, sequencing.
https://orcid.org/0009-0006-8139-8285
PCR testing of 7000 DNA samples isolated from whole blood of cattle was performed for the presence of DNA of parasites of the genus Babesia spp. PCR testing was performed using universal primers selected for the genus Babesia spp. The species affiliation was established by direct sequencing. It was found that the most common types of Babesia are: Babesia bigemina, Babesia major, Babesia occultans.
Primers and fluorescent TaqMan probes were selected to detect Babesia bigemina, Babesia major, Babesia occultans. Two pairs of primers and two fluorescent TaqMan probes were selected for each pathogen. The calculated annealing temperature of the primers ranged from 58 to 62ºС, the primers do not form refractory dimers when tested in PrimerSelect (Lasergene, DNASTAR). When tested in PrimerBLAST, the selected primers lead to the formation of the corresponding PCR products only in Babesia bigemina, Babesia major, Babesia occultans.
Optimization of PCR with real-time detection for detection and species identification of the prevailing pathogenic species of Babesia spp was carried out using the primer annealing temperature and the PCR cycling program, as well as the PCR reaction composition. The temperature gradient range was calculated based on the calculated primer annealing temperature in PrimerBlast. During optimization, it was found that the optimal primer annealing temperature for 3 primer pairs was 60 °C, 61 °C for 2 primer pairs and 59 °C for 1 primer pair.