Phenotypic Selection of Dairy Cattle Infected with Bovine Leukemia Virus Demonstrates Immunogenetic Resilience through NGS-Based Genotyping of BoLA MHC Class II Genes

Abstract

Characterization of the bovine leukocyte antigen (BoLA) DRB3 gene has shown that specific alleles associate with susceptibility or resilience to the progression of bovine leukemia virus (BLV), measured by proviral load (PVL). Through surveillance of multi-farm BLV eradication field trials, we observed differential phenotypes within seropositive cows that persist from months to years. We sought to develop a multiplex next-generation sequencing workflow (NGS-SBT) capable of genotyping 384 samples per run to assess the relationship between BLV phenotype and two BoLA genes. We utilized longitudinal results from milk ELISA screening and subsequent blood collections on seropositive cows for PVL determination using a novel BLV proviral load multiplex qPCR assay to phenotype the cows. Repeated diagnostic observations defined two distinct phenotypes in our study population, ELISA-positive cows that do not harbor detectable levels of provirus and those who do have persistent proviral loads. In total, 565 cows from nine Midwest dairy farms were selected for NGS-SBT, with 558 cows: 168 BLV susceptible (ELISA-positive/PVL-positive) and 390 BLV resilient (ELISA-positive/PVL-negative) successfully genotyped. Three BoLA-DRB3 alleles, including one novel allele, were shown to associate with disease resilience, *009:02, *044:01, and *048:02 were found at rates of 97.5%, 86.5%, and 90.3%, respectively, within the phenotypically resilient population. Alternatively, DRB3*015:01 and *027:03, both known to associate with disease progression, were found at rates of 81.1% and 92.3%, respectively, within the susceptible population. This study helps solidify the immunogenetic relationship between BoLA-DRB3 alleles and BLV infection status of these two phenotypic groupings of US dairy cattle.

Keywords: BoLA; ELISA; MHC Class II; bovine leukemia virus; dairy science; disease resilience; immunogenetics; molecular diagnostics; proviral load; qPCR; sequence-based typing.

Figure 1

Longitudinal DHI milk ELISA and blood PVL modeling of BLV susceptible and resilient cows selected for genotyping. All available DHI milk ELISA and blood derived PVL values were modeled over the age of each cow. 246 resilient DHI milk ELISA (blue), 384 resilient PVL (green), 116 susceptible ELISA (pink), and 169 susceptible (red) cow BLV diagnostic histories are represented here. Number of times individual cows were ELISA tested varies. Susceptible: 1× = 18, 2× = 35, 3× = 67, 4× = 2 and 5× = 2. Resilient: 1× = 96, 2× = 98, 3× = 83, 4× = 8 and 5× = 2. Line backdrop represents 95% confidence interval.

Figure 2

NGS-SBT experimental workflow. Cows were selected based on BLV phenotype, and stored gDNA samples were used for targeted amplification of the BoLA DQA1 and DRB3 genes.

Figure 3

Comparison of BoLA-DRB3 allele frequencies between BLV resilient (R) and susceptible cows (S). Allele frequency in 335 resilient (grey) and 153 susceptible (black) cows were calculated for each BoLA-DRB3 allele that showed at least 4% frequency in the total population.

Figure 4

Comparison of average PVL per BoLA-DRB3 allele. Error bars represent 95% confidence interval.

Figure 5

Molecular phylogeny of frequently found BoLA-DRB3 alleles. Neighbor-joining tree with maximum composite likelihood model. Green alleles indicate DRB3 alleles associated with resilience whereas red alleles show association with susceptibility.

Figure 6

Amino acid sequence MUSCLE alignment of highly associated BLV resilient and susceptible BoLA-DRB3 alleles.