Bovine leukemia virus (BLV) is a retrovirus that causes enzootic bovine leukosis (EBL) and has worldwide distribution. Infections with BLV have been reported in cattle from Kazakhstan but the virus has not yet been thoroughly characterized. In this study, we detect and estimate the level of BLV proviral DNA by qPCR in DNA samples from 119 cattle naturally infected with BLV, from 18 farms located in four different geographical regions of Kazakhstan. Furthermore, we conducted the phylogenetic and molecular analysis of 41 BLV env-gp51 gene sequences from BLV infected cattle. Phylogenetic analysis showed the affiliation of sequences to two already known genotypes G4 and G7 and also to a new genotype, classified as genotype G12. In addition, a multivariate method was employed for analysis of the association between proviral load and different variables such as the geographical location of the herd, cattle breeds, age of animals, and the presence of particular BLV genotypes. In summary, the results of this study provide the first evidence on molecular characterization of BLV circulating in cattle from Kazakhstan.

Viral diarrhoea is one of the most common and economically important diseases in cattle, causing significant losses in productivity and increased mortality in young livestock. Effective diagnosis of the disease requires the use of standardised sera that ensure high accuracy and reproducibility of laboratory results. Selection of sera to obtain standard sera for the diagnosis of bovine viral diarrhea represents a key challenge in the development of a standard sera panel. During monitoring in some regions of the Republic of Kazakhstan (Almaty, Mangistau, Karaganda, Turkistan oblasts) 399 samples of biomaterial from cattle were collected in private subsidiary farms and serological method of research was applied. In a serological test, specific diarrhea virus antibodies were detected in 310 serum samples. Comparative studies of sensitivity and stability of the positive sera with positive standard serum from the kit (ID Vet) were performed. During selection of sera, several factors were determined: specificity and sensitivity of the sera used, with consideration of the peculiarities of the epizootic situation in the region. This research work culminates in the production of a positive serum for the candidate from field-derived natural BVDV disease infections, and shortens the production cycle and saves costs.

Bovine leukemia virus (BLV) remains a major concern for cattle industries worldwide due to its persistent nature, economic impact, and challenges in control. In this study, we conducted a comprehensive nationwide survey of BLV in Kazakhstan between 2014 and 2024, utilizing serological diagnostics to assess prevalence and characterize viral genotypes (2024). A total of 433,537 serum samples were screened by agar gel immunodiffusion (AGID), revealing an overall seroprevalence of 5.87%, with the highest rates observed in the North Kazakhstan, Kostanay, and East Kazakhstan regions. In 2024, a targeted analysis of 3736 serum and 536 whole blood samples across 17 regions was performed using AGID, ELISA, real-time PCR, and nested PCR. ELISA demonstrated higher sensitivity than AGID (10.4% vs. 8.2%), confirmed by statistical correlation (r = 0.97, p < 0.001) and a Wilcoxon signed-rank test (p = 0.026). Real-time PCR detected BLV DNA in 4.7% of samples, with the highest positivity in the East Kazakhstan and Abai regions, confirming active viral circulation. Validation of a domestically developed AGID diagnostic kit showed full concordance with commercial assays (IDEXX, IDvet), supporting its use in national surveillance programs. These findings highlight the endemic status of BLV in Kazakhstan. Molecular analysis of sequenced isolates revealed the presence of genotype G-7, consistent with strains circulating in neighboring countries. Together, these results underscore the importance of integrated serological and molecular approaches for effective monitoring and control.

Background. Enzootic bovine leukosis (EBL) remains a significant problem in cattle production. Agar gel immunodiffusion (AGID) is widely used for screening; however, diagnostic performance depends on test-kit manufacturing parameters. Aim. To develop and optimize manufacturing conditions for an AGID test system for serological diagnosis of EBL, ensuring reproducibility and compliance with World Organisation for Animal Health (WOAH) recommendations. Methods. BLV antigen was produced on a persistently infected FLK cell line and standardized to gp51 using WOAH reference serum E05. We optimized agarose concentration (0.8–2.0% in 0.2 M Tris, pH 7.2, with 8.5% NaCl), antigen dilutions (1:2–1:64), and control sera dilutions. Plates were incubated in a humid chamber at 20–27 °C with readings at 24–72 h. Validation was performed on bovine sera (positive/weak-positive/negative) and compared with commercial kits. Results. An agarose concentration of 1.0–1.2% provided clear, stable precipitin lines within 24–72 h, balancing diffusion rate and line quality. Optimal antigen dilutions were 1:4 (eliminating nonspecific lines with negative serum while maintaining high sensitivity) and 1:8. Positive control serum was optimal at 1:4–1:8; weak-positive control at 1:32 yielded a reproducible faint line suitable for sensitivity assessment. Negative serum produced no lines. Stable performance was achieved under specified storage and operating conditions; diagnostic characteristics were comparable to those of commercial tests. Conclusions. The optimized AGID test system demonstrates high reproducibility, specificity, and adequate sensitivity, aligns with international requirements, and is suitable for broad implementation in veterinary laboratories in Kazakhstan. Accounting for regional features further enhances its practical value.

Bovine Viral Diarrhea Virus (BVDV) is a globally important cattle pathogen causing substantial economic losses. In Kazakhstan, BVDV’s epidemiological status remains poorly characterized due to the absence of systematic surveillance. We carried out a cross-sectional study of cattle herds across Kazakhstan, using ELISA to detect anti-BVDV antibodies and RT-PCR to identify active infections. Positive samples underwent sequencing for phylogenetic analysis of circulating strains. Additionally, a standard reference serum panel was developed to measure virus neutralization titers (ND₅₀) and to evaluate cross-neutralization with Border Disease virus (BDV). Antibodies against BVDV were prevalent, with seropositivity ranging from 28.89% to 96.13% across surveyed regions. Active BVDV infection was confirmed by RT-PCR in 17 animals. Phylogenetic analysis with 2 samples from Mangystau region classified the virus as BVDV2 genotype. The reference serum panel exhibited high neutralizing titers ND₅₀ up to 1:286 against the local BVDV-1 isolate. Notably, these sera also neutralized BDV, albeit at lower titers ND₅₀ 1:45. These findings provide crucial baseline epidemiological data and enhanced diagnostic tools for BVDV in Kazakhstan. They highlight the need for improved surveillance and will inform strategic control measures against this economically significant cattle disease.

За последние десять лет стремительно развивается использование стандартных сывороток для стандартизации диагностических наборов при лабораторном обследовании различных инфекционных заболеваний, в том числе при выявлении вирусных инфекций. С диагностической целью используются серологические методы выявления лейкоза крупного рогатого скота (КРС), рекомендованные Всемирной организацией здравоохранения животных (ВОЗЖ, МЭБ), иммуноферментный анализ (ИФА) и реакция иммунодиффузии в агаровом геле (РИД). При проведении исследований обоими указанными методами диагностические наборы для ИФА и РИД проверяются сывороткой международного стандарта Е05, который одобрен контрольными лабораториями Европейского экономического союза (European Economic Community, EC, теперь European Union). Большое значение имеет применение стандартных сывороток крови для оценки качества диагностических тест-систем, выпускаемых в стране и зарубежных производителей, в целях повышения качества выполнения анализов в диагностических лабораториях. Сыворотки, которые вырабатывают в странах-членах ЕС для определения антител против вируса лейкоза КРС должны быть непременно стандартизированы с использованием сыворотки Е05. С этой целью в лаборатории вирусологии ТОО «Казахский научно - исследовательский ветеринарный институт» разработан национальный стандарт сыворотки крови (Q-1/19-положительные, К-1/19-отрицательные) для контроля качества тест-систем для выявления лейкоза КРС. Разработанные сыворотки крови национального стандарта получили положительную оценку в референтной лаборатории Международного эпизоотического бюро по изучению энзоотического лейкоза КРС (PiWet, Польша), получен сертификат о соответствии разработанной национальной стандартной сыворотки международному стандарту Е05. В настоящее время национальная стандартная сыворотка используется для проверки качества диагностических тест-систем. Были проведены сравнительные исследования чувствительности и стабильности разработанной стандартной сыворотки Q-1/19 в течение трех лет хранения с международными стандартными сыворотками крови Е05 и Р9 (Польша). В статье представлены сравнительные результаты проверки разработанных стандартных сывороток в методиках ИФА для серологического определения IDEXX Leukosis Blocking, IDEXX Leuskosis Serum X2, IDEXX leukosis Serum Ѕсгеепіпд известных зарубежных производителей тест-систем и инструкция по срокам хранения стандартной сыворотки.