Bovine anaplasmosis is an infectious vector-borne disease caused by bacteria of the genus Anaplasma, which have a wide global distribution and represent a high economic burden for agriculture. The use of molecular genetic techniques has increased our knowledge of the species diversity of Anaplasma spp. and naturally susceptible animals. Monitoring studies allow us to assess the level of infection in herds, as well as the involvement of natural vectors in the processes of maintaining and spreading infection. Despite the high prevalence of Theileria and Babesia in cattle in Kazakhstan, there is no information on the distribution and species diversity of Anaplasma spp in this country. As part of this work, 7027 DNA samples isolated from the whole blood of cattle from 175 settlements in all 17 Kazakhstan regions were PCR-tested for the presence of Anaplasma spp. Anaplasma carriers were found in 1.3% (90 out of 7027) of the tested animals in 9 of the 17 regions of Kazakhstan. The highest percentage of infected animals was recorded in Turkistan (South Kazakhstan) and North Kazakhstan with 4.46% and 2.48% positive samples, respectively. The partial sequencing of 16S rRNA and the groEL gene allowed us to identify five species of Anaplasma: A. centrale, A. marginale, Candidatus Anaplasma Mongolica, A. ovis, and Unknown Anaplasma with infection rates of 0.63%, 0.44%, 0.13%, 0.01%, and 0.01%, respectively.

Background and Aim: Anaplasmosis in small ruminants is a tick-borne infection caused mainly by the obligate intraerythrocytic bacterium Anaplasma ovis. It is usually subclinical, with persistent infection in affected animals, but acute disease can occur, particularly in young animals. The pathogen is widespread in Central Asia and neighboring regions. In Kazakhstan, the infection was first detected in 1929. However, until now, diagnosis in the country has been based on traditional microscopic examination of blood smears. There were no reliable data on the prevalence and genetic diversity of Anaplasma spp. in sheep in Kazakhstan. This study aimed to determine the occurrence of Anaplasma spp. infection in sheep in southern Kazakhstan, a high-risk region for tick-borne diseases, using PCR and to identify the species by sequencing. Materials and Methods: A cross-sectional study was conducted on apparently healthy adult ewes from 77 settlements in 34 districts of Kyzylorda, Turkistan, Zhambyl, Almaty, and Jetisu, southern Kazakhstan. A total of 2553 whole blood samples collected in midsummer 2022 and 2023 were analyzed for Anaplasma spp. using polymerase chain reaction targeting the 404 bp groEL gene fragment. The amplification products from the 441 positive samples were sequenced using the Sanger sequencing method. Phylogenetic analysis of the obtained sequences was performed using the maximum likelihood model. Results: Overall, 1017/2553 (39.8%; 95% confidence interval: 37.9%-41.7%) ewes tested were positive for Anaplasma spp. Positive animals were found in 68/77 (88%) of the settlements from which samples were taken. The percentage of Anaplasma spp.-positive ewes varied significantly from 21.3% to 50.1% in the provinces. Altitude 99.5% identical to isolates from Africa, Cyprus, and China. Conclusion: This first molecular study revealed a widespread of A. ovis infection in adult ewes in southern Kazakhstan. Altitude <500 m was identified as a risk factor. Therefore, clinical cases associated with A. ovis are expected in this region, especially in young animals. Future studies are needed to determine the clinical and economic impact of anaplasmosis on sheep production in the country, to investigate seasonal patterns of infection, and to identify tick species or other arthropods that act as local vectors. This information is useful for developing possible control measures and evaluating their effectiveness. Keywords: Anaplasma ovis, Kazakhstan, polymerase chain reaction, sequencing, sheep.

Background and Aim: Bovine leukemia virus (BLV) is a globally distributed retrovirus that causes enzootic bovine leu-kosis, a chronic infection associated with significant economic losses in cattle. Conventional serological diagnostic tools such as agar gel immunodiffusion and enzyme-linked immunosorbent assay detect anti-BLV antibodies but cannot identify proviral DNA, especially in early infections or in calves with maternal antibodies. This study aimed to develop a sensitive and specific duplex quantitative polymerase chain reaction (qPCR) assay targeting the env gene of BLV with β-actin as an internal control and apply it for molecular surveillance and genotyping of BLV in cattle from six regions of Kazakhstan. Materials and Methods: A total of 1,680 bovine DNA samples from cattle aged over 3 years were collected from six administrative regions of Kazakhstan. A duplex qPCR assay was developed using primers targeting a conserved region of the BLV env gene and bovine β-actin. Sensitivity was assessed using plasmid and genomic DNA dilutions, and specificity was tested against existing WOAH-recommended and commercial polymerase chain reaction (PCR) protocols. Positive samples with cycle threshold <28 were subjected to nested PCR and Sanger sequencing for genotyping. Phylogenetic analysis was conducted using the maximum likelihood method. Results: The developed qPCR assay demonstrated a sensitivity of 20 plasmid copies for the env gene and 6 genomic equivalents for β-actin per reaction, with high specificity comparable to international standards. The overall BLV infection rate was 38.9%, ranging from 13% in Pavlodar to 60.5% in East Kazakhstan. Among 149 sequenced positive samples, four genotypes (G1, G4, G7, and G8) were identified. Genotype G4 was predominant, comprising 79.2% of sequences and present in all six regions. Conclusion: The duplex qPCR assay is a robust, sensitive, and cost-effective diagnostic tool for detecting BLV provirus, including in animals with maternal antibodies or early-stage infections. The regional genotypic distribution underscores the need for tailored control strategies. This molecular surveillance provides essential baseline data for national BLV eradication programs and contributes to global BLV epidemiological mapping. Keywords: bovine leukemia virus epidemiology, bovine leukemia virus, cattle, env gene, genotyping, Kazakhstan, molecular diagnostics, proviral DNA, quantitative polymerase chain reaction, β-actin.

Anaplasma marginale is a gram-negative, obligate intracellular bacterium that infects cattle, buffalo, deer, and antelope, causing significant economic losses worldwide. In recent years, due to an increase in the area of uncultivated land and uncontrolled use of pastures, there has been a tendency for a sharp increase in the population of blood sucking ticks - carriers of blood-parasitic animal diseases. In this regard, the development of molecular genetics methods for the diagnosis of blood parasitic diseases is relevant for taking effective measures to prevent the spread of tick-borne infections. The lack of sensitivity and specificity of the microscopic and immunological diagnostic methods used, as well as the tendency for a sharp increase in the population of blood-sucking ticks, aggravates the epizootological situation. As part of this research, a real time PCR test system for the identification of Anaplasma marginale was developed. Species-specific primers and a fluorescence-labelled TaqMan probe were developed for the highly conserved gene of the heat shock family protein groEL. The developed realtime PCR test system showed high specificity and sensitivity, allowing the detection of the groEL gene of A. marginale at the level of 8 copies in the reaction. The developed real-time PCR test system can be used for early diagnosis of anaplasmosis in cattle and conducting monitoring studies.

Bovine babesiosis is a tick-borne disease of major veterinary and economic importance, yet molecular epidemiological data from Kazakhstan remain limited. This study investigated the prevalence, distribution, and species composition of Babesia spp. in cattle across all 17 administrative regions of Kazakhstan. A total of 6455 cattle blood samples from 171 villages were screened by nested PCR targeting the cytochrome b (cytb) gene, and positive samples were characterized by sequencing and phylogenetic analysis. The overall positive rate for Babesia spp. were 3.87% (95% CI: 3.41–4.37%). Of 250 PCR-positive samples, 215 yielded high-quality sequences and were assigned to four species: Babesia major (31.2%), B. occultans (29.6%), B. bigemina (18.0%), and B. bovis (7.2%). Marked regional heterogeneity was observed, with B. major and B. occultans predominating across most regions, whereas B. bigemina was dominant in Atyrau. Turkistan was the only region where all four species co-circulated. First molecular evidence of B. bovis and B. bigemina was obtained for Atyrau, Akmola, and West Kazakhstan regions. These findings expand current knowledge of bovine babesiosis in Kazakhstan and reveal a species composition dominated by B. major and B. occultans, contrasting with the B. bovis/B. bigemina-dominated patterns commonly reported elsewhere. The detection of pathogenic Babesia species in previously unsurveyed regions highlights the need for continued molecular surveillance.

Tularaemia is caused by the Gram-negative bacterium Francisella tularensis, which has three subspecies: holarctica, mediacia and tularensis. Due to the high virulence of the pathogen, the wide range of susceptible animals and the presence of numerous vectors and natural reservoirs, the development of sensitive diagnostic methods for the epidemiological surveillance of tularemia is crucial. New opportunities for understanding the epidemiological situation of tularemia are opening up with the use of new molecular analysis technologies for typing F. tularensis. The development and use of molecular methods for the diagnosis of tularemia is important in this context. The present study developed a protocol for detection of F. tularensis by real-time polymerase chain reaction (qPCR). The selection of primers and a fluorescent probe was based on the aligned sequences of ISFtu1 (multi-copy insertion element). qPCR conditions were optimised, including the determination of the optimal annealing and extension temperatures for the primers. Sensitivity was tested using successive 4-fold dilutions of DNA derived from F. tularensis subsp. mediasiatica, indicating a minimum sensitivity threshold of 15 genomic equivalents per reaction. Two subspecies of F. tularensis (subsp. mediasiatica and subsp. holarctica), 27 non-target bacterial species and 3 eukaryotic organisms were used to assess specificity.