Vet World   Vol.18   August-2025  Article - 15 

Development and application of a quantitative polymerase chain reaction assay for the detection and genotyping of bovine leukemia virus in cattle from Kazakhstan

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.

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How to cite this article: Ostrovskii A, Shevtsov A, Kuibagarov M, Kamalova D, Dauletov A, Rsaliyev A, Abduraimov Y, and Mukanov K (2025) Development and application of a quantitative polymerase chain reaction assay for the detection and genotyping of bovine leukemia virus in cattle from Kazakhstan, Veterinary World, 18(8): 2320-2331.

Received: 08-05-2025   Accepted: 15-07-2025   Published online: 14-08-2025

Corresponding author: Kassym Mukanov    E-mail: mukanov@biocenter.kz

DOI: 10.14202/vetworld.2025.2320-2331

Copyright: Ostrovskii, et al. This article is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.