Development of TaqMan PCR assay for genotyping SNP rs211250281 of the bovine agpat6 gene.

Milk fat percentage is an important production trait of dairy cattle and is one of the goals of breeding programs. Over 95% of the milk fat accounts for triacylglycerols. AGPAT6 (1-acylglycerol-3-phosphate O-acyltransferase 6) catalyzes an intermediary step of triglyceride synthesis in the mammary cells. Genome-wide association studies identified SNP rs211250281 (g27: 36520069 G/T) in the agpat6 gene associated with milk fat content and fat-to-protein ratio in dairy cattle. The article presents data on the development of TaqMan PCR assay for genotyping SNP rs211250281 of the bovine agpat6 gene. In this method, a primer pair, initiating amplification of 75-bp fragments of the agpat6 gene, and two allele-specific TaqMan probes are used. Identification of the G and T alleles is based on a comparison of the final fluorescence intensity of FAM and VIC dyes, respectively. The developed TaqMan PCR assay was validated by Sanger sequencing method. The results of both methods fully coincided, that confirmed high accuracy of the developed TaqMan PCR assay. This reliable, simple, rapid, and high-throughput method could be a suitable tool for studying the distribution of the SNP rs211250281 among different cattle breeds and its association with milk fat content.

Identification of novel integration sites for bovine leukemia virus proviral DNA in cancer driver genes in cattle with persistent lymphocytosis.

Enzootic bovine leukosis is one of the unsolved problems of cattle breeding in many countries. The etiological agent of the disease is the bovine leukemia virus (BLV) - an oncogenic retrovirus, that infects B-lymphocytes in cattle. The number and genetic content of BLV provirus integration sites in the bovine genome were reported to can be used as an early diagnostic sign of leukemogenesis in the infected cattle, but patterns of BLV provirus integration into the bovine genome and associations between genomic features of the integration sites and development of lymphocytosis and B-cell lymphomas remain poorly elucidated. Here we present data on five novel BLV provirus integration sites in the genome of cattle with persistent lymphocytosis. Two of these sites were located in introns of scfd2 and pgpep1 genes, which have been recognized as cancer driver genes. Three of the rest integration sites were found in the intergenic spaces between ctps1 and cited4, nampt and ccdc71, skp2 and lmbrd2 genes, from which cited4 and skp2 also possess oncogenic properties. These data support previous findings of the association between localization of BLV proviral DNA near cancer driver genes and leukemogenesis in the BLV-infected cattle.

Development of TaqMan PCR assay for detection of A and B variants of the bovine ß-lactoglobulin.

ß-Lactoglobulin (BLG) is one of the prevalent whey protein in cattle. To date, several variants of bovine BLG have been found, but the most common are A and B, which differ from each other by SNPs rs109625649 and rs110066229. Numerous studies showed effects of A and B variants of BLG on milk yield, fat and protein content and cheese-making properties. To date, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), allele-specific polymerase chain reaction (ASPCR), PCR single-strand conformation polymorphism (PCR-SSCP) and high resolution melting (HRM) methods have been proposed for detection of A and B variants of bovine BLG. These methods involve multistep sample processing, which is an essential disadvantage in conducting large-scale cattle genotyping projects. This article describes a development of TaqMan PCR assay for detection of A and B variants (rs109625649) of bovine BLG. In this method a primer pair, initiating amplification of 101-bp fragment of BLG gene, and two allele-specific TaqMan probes are used. Identification of B and A variants of BLG is based on comparison of final fluorescence intensity of FAM and VIC dyes, respectively. The developed one-step method requires less time and is more suitable for large-scale genotyping of cattle compared to the commonly used PCR-RFLP.

Real-time PCR assay with an endogenous internal amplification control for detection and quantification of Anaplasma marginale in bovine blood.

Bovine anaplasmosis is a tick-borne rickettsial disease, causing significant economic losses in many countries. The main causative agent of bovine anaplasmosis is Anaplasma marginale (Rickettsiales, Anaplasmataceae). To date, several PCR assays for A. marginale DNA detection were proposed, but most of them do not provide an internal amplification control, which allows to prevent false-negative results and is required for reliability of the results of pathogen DNA detection by PCR assay. In the present study, a real-time PCR assay based on the species-specific and highly conserved fragment of msp1α gene was developed for detection and quantification of A. marginale in bovine blood. The real-time PCR assay is able to detect as few as one copу of msp1α gene per reaction. To prevent false-negative results, simultaneous amplification and detection of the bovine genomic DNA fragment as an endogenous internal amplification control (IAC) was provided. The assay can be used as a highly specific and sensitive method for detection and quantification of A. marginale in infected cattle, and for the evaluation of the efficacy of anti-rickettsial drugs and anaplasmosis vaccines.

Molecular survey and genetic characterization of Anaplasma marginale isolates in cattle from two regions of Russia.

Anaplasma marginale is an intraerythrocytic tick-borne rickettsial pathogen that causes bovine anaplasmosis, an economically important disease of cattle worldwide. Major surface protein MSP1α has been used as a stable marker in identifying geographical strains of A. marginale. The genetic diversity of A. marginale based on MSP1α has been reported in several countries all over the world. Only a few molecular surveys of A. marginale strains have been conducted in Russia. The aim of this study was molecular detection and characterization of A. marginale isolates in cattle from two regions of Russia. Blood samples from 62 cattle were collected and screened for the presence of A. marginale by real-time PCR targeting the msp4 gene. Anaplasma marginale DNA was detected in 26 cattle (42%). The partial msp1α gene containing tandem repeat sequences and msp4 gene were amplified from msp4-positive samples, cloned and sequenced. Sequence analysis revealed that two msp4 genotypes were found. The genetic diversity of A. marginale strains was analyzed based on the MSP1α tandem repeats structure and 5'-UTR microsatellite. Sixteen new genotypes of A. marginale were found in 17 animals. Seven animals (41%) were infected by more than one genotype. Eight new tandem repeats are described for the first time. The number of repeats differed between 1 and 6 across the isolates. The msp1α microsatellite analysis revealed that six genotypes were identified; one of them was not previously described. Phylogenetic analysis revealed that Russian isolates formed four separate clades. The tandem repeat and microsatellite analyses of the msp1α gene showed a high genetic diversity among the isolates. The present study provided the first evidence of genetic diversity of A. marginale in cattle in Russia.