Copy number variations in BOLA-DQA2, BOLA-DQB, and BOLA-DQA5 show the genomic architecture and haplotype frequency of major histocompatibility complex class II genes in Holstein cows.

Bovine major histocompatibility complex (MHC) class II region contains many genes. The bovine leukocyte antigen (BoLA)-DRB3 was reportedly associated with susceptibility of various phenotypes of infections including bovine leukemia virus-induced lymphoma. However, the association of the remaining genes with various phenotypes has not been clarified due to the complicated genomic structure of the MHC class II region. Thus, in this study, we elucidated the MHC class II genomic structure, including the novel alleles and copy number variations (CNVs). We determined the copy numbers of BOLA-DQA2 (DQA2), BOLA-DQB (DQB2), BOLA-DQA5 (DQA5), BLA-DQB (DQB1), LOC100848815 (DQA1), and BOLA-DRB3 (DRB3) in 127 unrelated Holstein cows by TaqMan copy number assay. The genomes were sequenced using target next-generation sequencing (NGS) based on multiplex polymerase chain reaction. Combining the results of the copy numbers and alleles, we identified the BoLA alleles directly without haplotype estimation. Pairwise linkage disequilibrium (LD) analysis between alleles and genes were performed. The CNVs of DQA2, DQB2, and DQA5 in Holstein cows were detected. The frequency of the whole gene deletion in DQA2, DQB2, and DQA5 was 35.4%, 93.7%, and 93.7%, respectively. After target NGS, we identified 37 alleles in the six genes. Fifteen novel alleles (40.5%) were not registered in the IPD-MHC Database. LD analysis showed strong LD among the DQB2*deletion, DQA5*deletion, and DRB3*27:03 alleles. Our findings will provide important insights into the identification of the BoLA genes associated with various infection-related phenotypes.

Fukunaga K ，Yamashita Y ，Yagisawa T 《-》

Identification and diversity of bovine major histocompatibility complex class II haplotypes in Japanese Black and Holstein cattle in Japan.

Bovine leukocyte antigen (BoLA), the major histocompatibility complex of cattle, is one of the most polymorphic gene clusters. We genotyped a population of 109 Japanese Black and 39 Holstein cattle to analyze their BoLA class II haplotypes, BoLA-DRB3 locus, 5 BoLA-DQA loci, and 5 BoLA-DQB loci. We identified 26 previously reported DRB3 alleles, 22 previously reported and 3 new DQA alleles, and 24 previously reported and 6 new DQB alleles. A dendrogram was constructed based on the predicted amino acid sequences of the α1 or β1 domains encoded by BoLA-DQA or -DQB alleles, which revealed that DQA alleles were clustered into 5 loci, whereas DQB alleles could not be clearly assigned to specific DQB loci. The BoLA-DRB3-DQA-DQB haplotypes were sorted by sequential analytical processes, and 42 distinct haplotypes, including 11 previously published haplotypes and 31 novel haplotypes, were defined. Strong linkage disequilibrium was present in the BoLA genes. We also compared DRB3-DQA1 haplotype frequencies between 507 Japanese Black and 143 Holstein cattle. Thirty-nine DRB3-DQA1 haplotypes were identified, including 29 haplotypes from Japanese Black and 22 haplotypes from Holstein cattle. The majority of the haplotypes could be identified in both breeds, although several haplotypes were identified in only a single breed. This is the first report presenting a detailed study of the BoLA class II haplotype in Japanese Black and Holstein cattle in Japan.

Miyasaka T ，Takeshima SN ，Sentsui H ，Aida Y ... -  《-》

Bovine leukemia virus proviral load is more strongly associated with bovine major histocompatibility complex class II DRB3 polymorphism than with DQA1 polymorphism in Holstein cow in Japan.

Bovine leukemia virus (BLV) causes enzootic bovine leukosis and is closely related to the human T-lymphotropic virus. Bovine major histocompatibility complex (BoLAs) are used extensively as markers of disease and immunological traits in cattle. For BLV diagnosis, proviral load is a major diagnosis index for the determination of disease progression and transmission risk. Therefore, we investigated the frequency of BoLA-DRB3 alleles, BoLA-DQA1 alleles, and haplotypes of BoLA class II isolated from the heads of 910 BLV-infected cows out of 1290 cows assessed from BLV-positive farms, in a nationwide survey from 2011 to 2014 in Japan. Our aim was to identify BoLA class II polymorphisms associated with the BLV proviral load in the Holstein cow. The study examined 569 cows with a high proviral load and 341 cows with a low proviral load. Using the highest odds ratio (OR) as a comparison index, we confirmed that BoLA-DRB3 was the best marker for determining which cow spread the BLV (OR 13.9 for BoLA-DRB3, OR 11.5 for BoLA-DQA1, and OR 6.2 for BoLA class II haplotype). In addition, DRB3*002:01, *009:02, *012:01, *014:01, and *015:01 were determined as BLV provirus associated alleles. BoLA-DRB3*002:01, *009:02, and *014:01 were determined as resistant alleles (OR > 1), and BoLA-DRB3*012:01 and *015:01 were determined as susceptible alleles (OR < 1). In this study, we showed that BoLA-DRB3 was a good marker for determining which cow spread BLV, and we found not only one resistant allele (BoLA-DRB3*009:02), but also two other disease-resistant alleles and two disease-susceptible alleles. This designation of major alleles as markers of susceptibility or resistance can allow the determination of the susceptibility or resistance of most cows to disease. Overall, the results of this study may be useful in eliminating BLV from farms without having to separate cows into several cowsheds.

Takeshima SN ，Ohno A ，Aida Y 《Retrovirology》

Identification of bovine leukocyte antigen class II haplotypes associated with variations in bovine leukemia virus proviral load in Japanese Black cattle.

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis, which is the most common neoplastic disease of cattle. Bovine leukocyte antigen (BoLA) is strongly involved in the subclinical progression of BLV infections. Recent studies show that the BoLA-DRB3 gene might play a direct role in controlling the number of BLV-infected peripheral B lymphocytes in vivo in Holstein cattle. However, the specific BoLA class II allele and DRB3-DQA1 haplotypes determining the BLV proviral load in Japanese Black cattle are yet to be identified. In this study, we focused on the association of BLV proviral load and polymorphism of BoLA class II in Japanese Black cattle. We genotyped 186 BLV-infected, clinically normal cattle for BoLA-DRB3 and BoLA-DQA1 using a polymerase chain reaction-sequence-based typing method. BoLA-DRB3*0902 and BoLA-DRB3*1101 were associated with a low proviral load (LPVL), and BoLA-DRB3*1601 was associated with a high proviral load (HPVL). Furthermore, BoLA-DQA1*0204 and BoLA-DQA1*10012 were related to LPVL and HPVL, respectively. Furthermore, we confirmed the correlation between the DRB3-DQA1 haplotype and BLV proviral load. Two haplotypes, namely 0902B or C (DRB3*0902-DQA1*0204) and 1101A (DRB3*1101-DQA1*10011), were associated with a low BLV proviral load, whereas one haplotype 1601B (DRB3*1601-DQA1*10012) was associated with a high BLV proviral load. We conclude that resistance is a dominant trait and susceptibility is a recessive trait. Additionally, resistant alleles were common between Japanese Black and Holstein cattle, and susceptible alleles differed. This is the first report to identify an association between the DRB3-DQA1 haplotype and variations in BLV proviral load.

Miyasaka T ，Takeshima SN ，Jimba M ，Matsumoto Y ，Kobayashi N ，Matsuhashi T ，Sentsui H ，Aida Y ... -  《-》

Evidence for an additional cattle DQA locus, BoLA-DQA5.

Studying the genetic polymorphism of the major histocompatibility complex class II genes in cattle, we identified an allele (BNI13) which encodes a typical class II alpha chain. Its transcription was confirmed by RNA analysis. Sequence comparisons, Southern blot, and phylogenetic analyses indicate that (1) BNI13 represents a distinct DQA locus which we propose to designate BoLA-DQA5, (2) BoLA-DQA1 and BoLA-DQA5 separated after the divergence of BoLA-DQA1 and BoLA-DQA2, but prior to the separation of sheep DQA1 and cattle DQA1, and (3) DQA5 is distributed among various cattle breeds but is confined to certain haplotypes.

Gelhaus A ，Förster B ，Wippern C ，Horstmann RD ... -  《IMMUNOGENETICS》