The definition of five B lymphocyte alloantigens closely linked to BoLA class I antigens

B lymphocyte alloantigens in cattle were identified by serological analysis. Alloantisera were raised by skin implant immunization or leucocyte immunization and were absorbed with platelets to reduce class I-specific antibody activity. Leucocyte absorptions were done to reduce the complexity of some antisera. A panning technique was used to prepare B-enriched and B-depleted lymphocytes. Antisera which displayed anti-B cell activity over a number of dilutions were tested against 115 Charolais cattle, and 13 antisera were used to define five B lymphocyte alloantigens. These antigens were present on B lymphocytes but did not appear to be present, at least at the same density, on the majority of T lymphocytes or platelets. Family studies suggested that these antigens are coded by one or two loci which are closely linked to the bovine class I loci. These results suggest the five antigens are class II antigens of the major histocompatibility complex (MHC) of cattle.     

Joint Report of the Third International Bovine Lymphocyte Antigen (BoLA) Workshop, Helsinki, Finland, 27 July 1986

Summary. Two hundred and eighty-two alloantisera were submitted by 20 participating laboratories from 13 countries and tested against lymphocytes of 1298 cattle. The cell panel consisted of samples from 38 Bos taurus breeds, 11 Bos taurus crossbreeds, 4 Bos indicus breeds, 6 Bos taurus X Bos indicus , and a variety of other crossbred populations. Using a standardized lymphocytotoxicity test, all 17 previously identified BoLA specificities were confirmed. The workshop produced agreement on 16 new lymphocyte alloantigenic specificities. Three of the new specificities behaved as splits of previously identified BoLA specificities. Four of the new specificities behaved as alleles at the agreed BoLA-A locus. Seven new specificities are tentatively assigned to the BoLA-A locus but require further definition. Two new specificities may represent products of a second closely-linked BoLA locus.     

Genetic variation in BoLA microsatellite loci in Portuguese cattle breeds

Major histocompatibility complex (MHC) typing based on microsatellites can be a valuable approach to understanding the selective processes occurring at linked or physically close MHC genes and can provide important information on variability and relationships of populations. Using microsatellites within or in close proximity with bovine lymphocyte antigen (BoLA) genes, we investigated the polymorphisms in the bovine MHC, known as the BoLA, in eight Portuguese cattle breeds. Additional data from non-BoLA microsatellite loci were also used to compare the variability between these regions. Diversity was higher in BoLA than in non-BoLA microsatellites, as could be observed by the number of alleles, allelic richness and observed heterozygosity. Brava de Lide, a breed selected for aggressiveness and nobility, presented the lowest values of observed heterozygosity and allelic richness in both markers. Results from neutrality tests showed few statistically significant differences between the observed Hardy–Weinberg homozygosity ( F ) and the expected homozygosity ( F _E), indicating the apparent neutrality of the BoLA microsatellites within the analysed breeds. Nevertheless, we detected a trend of lower values of observed homozygosity compared with the expected one. We also detected some differences in the levels of allelic variability among the four BoLA microsatellites. Our data showed a higher number of alleles at the BoLA-DRB3 locus than at the BoLA-DRBP1 locus. These differences could be related to their physical position in the chromosome and may reflect functional requirements for diversity.     

Breed differences in frequency of BoLA specificities*

A total of 675 cattle of five purebred and one crossbred group were tested for lymphocyte antigens. The purebred animals represented progeny of 107 sires. Lymphocytotoxicity sera obtained from parous cows were used to detect six antigens which are controlled by codominant alleles at the BoLA-TxA locus. Gene frequencies for the six alleles varied within breeds and large differences were observed between breeds for a given allele.     

Production of foetally stimulated lymphocytotoxic antibodies by multiparous cows

Serum samples were collected monthly from second gestation cows and examined for the presence of lymphocytotoxic antibodies. Of 25 cows studied 16 (64 %) raised antibodies during or immediately after second gestation. Ten of these cows (40 %) raised antibodies during gestation, some as early as 5 months before parturition. Reactors which had had first-gestation reactivity responded earlier, but had peak antibody titers no higher than cows without observable first gestation reactions. Cows with antibodies of similar specificity in the first two gestations responded earlier than those with different antibody specificities. Regardless of the time of first antibody detrection, peak titers were usually achieved during the first month postpartum. Antibody persistence increased with parity. Among cows of all ages sampled at random times, there was a linear relationship of serum reactivity to cow age.     

Polymorphism of bovine major histocompatibility complex (MHC) class I genes revealed by polymerase chain reaction (PCR) and restriction enzyme analysis

The polymorphic exon 2-exon 3 region of bovine major histocompatibility complex (MHC) class I genes was amplified by polymerase chain reaction (PCR) from genomic DNA samples with characterized class I polymorphism. The primers for amplification were designed in conserved regions at the borders of exons 2 and 3, based on all available cDNA sequences. The primers should, therefore, amplify most expressed class I genes, but may also amplify non-expressed class I genes. The PCR amplified class I gene fragments of 700 bp were characterized on the basis of restriction fragment length polymorphism (RFLP). The PCR-RFLP analysis of class I genes showed that the bands in each digestion could be classified as non-polymorphic, as shared between several bovine lymphocyte antigen (BoLA)-A types, or as specific to a single BoLA-A type. The same primers were then used for amplification of class I gene fragments from eight Sahiwal animals, a breed which originated in the Indian subcontinent. These studies showed that BoLA class I PCR-RFLP could be used to study class I polymorphism in family groups.     

Cloning of the bovine major histocompatibility complex class II genes

Summary. Class II genes of the bovine major histocompatibility complex (MHC) have been cloned from a genomic library. The library was constructed in the bacteriophage Λ vector EMBL3 and comprises approximately 10 times the equivalent of the haploid genome. Half the library was screened with the human DQA, DQB, DRA and DRB cDNA probes. Of the 100 positively hybridizing phage clones, 37 were eventually fully characterized and mapped by means of Southern blot analysis. The exons encoding the first, second and transmembrane domain of all different A and B genes were subcloned and mapped in more detail. These analyses showed that these 37 clones were derived from five different A and 10 different B genes. The hybridization studies indicate that we have cloned and mapped two DQA genes, one DRA gene, two other A genes, four DQB genes, three DRB genes and three other B genes. Since the library was made from a heterozygous animal, this would suggest that there are at least one DQA, one DRA one other undefined A, two DQB, two DRB and one or two other undefined B genes in the haploid genome of Holstein Friesian cattle.     

Analysis of alloantisera against bovine lymphocytes. Joint report of the 1st International Bovine Lymphocyte Antigen (BoLA) Workshop

The results and agreements of the 1st international BoLA workshop, held in Edinburgh, Scotland in August 1978, are reported. Most of these concern the results from a comparison test of 249 alloantisera to bovine lymphocytes, the antisera being contributed by 9 laboratories. These sera were compared directly in Edinburgh on a panel of lymphocytes from 130 cattle of 21 breeds. In the micro-lymphocytotoxicity test used 75% of the sera reacted. Sixty eight of these sera were grouped into clusters according to their reaction patterns against the lymphocyte panel. Eleven of these clusters were clearly defined and were given workshop BoLA designations. In addition 22 sera were assigned to subgroups of the agreed clusters. There was no evidence that the method of production of the sera had any effect on their specificity.
Although genetic data was not available, the phenotypes of the test panel of lymphocytes are consistent with the clusters detecting antigens controlled by multiple alleles at a single autosomal locus. It was agreed to name the genetic region where this putative locus is located BoLA (bovine lymphocyte antigen).     

Failure to find an association between ocular squamous cell carcinoma and class I antigens of the bovine major histocompatibility system*

Summary. We tested 53 cattle with ocular squamous cell carcinoma (cancer-eye) and 53 paired, matched controls for 25 class I antigens of the bovine major histocompatibility system. The most common antigen was W5 which was present in 40% of the animals with cancer-eye and 36% of the controls. There were no statistically significant differences in BoLA antigen frequency between cattle with and cattle without cancer-eye.     

Restriction fragment length polymorphism of DQ and DR class II genes of the bovine major histocompatibility complex

DQ alpha, DQ beta, DR alpha and DR beta class II genes of the bovine major histocompatibility complex (MHC) were investigated by Southern blot hybridizations using human probes. Hybridizations of these probes to genomic DNA, digested with PvuII or TaqI, revealed extensive restriction fragment length polymorphisms (RFLPs). The polymorphisms were interpreted genetically by analysing a family material, comprising five sires, 48 dams and 50 offspring, and a population sample comprising 197 breeding bulls. The analysis resolved 20 DQ alpha, 17 DQ beta, 5 DR alpha and 25 DR beta RFLP types. The segregation data were consistent with simple Mendelian inheritance of the RFLPs. The analysis of the bull sample showed that it is possible to apply the RFLP method for routine typing of class II polymorphism in population samples. The linkage disequilibrium in the DQ-DR region was found to be extremely strong as only about 20 DQ and about 30 DQ-DR haplotypes were observed despite the large number of possible haplotypes. Close linkage to the blood group locus M was also found; the M' allele occurred in strong linkage disequilibrium with the class II haplotype DQ1BDR alpha 4DR beta 1B. A population genetic analysis of the DQ data in the sample of breeding bulls revealed that the frequency of homozygotes was significantly lower than Hardy-Weinberg expectation and that the allele frequency distribution deviated significantly from the one expected for selectively neutral alleles.     

Evidence of positive selection towards Zebuine haplotypes in the BoLA region of Brangus cattle

The Brangus breed was developed to combine the superior characteristics of both of its founder breeds, Angus and Brahman. It combines the high adaptability to tropical and subtropical environments, disease resistance, and overall hardiness of Zebu cattle with the reproductive potential and carcass quality of Angus. It is known that the major histocompatibility complex (MHC, also known as bovine leucocyte antigen: BoLA), located on chromosome 23, encodes several genes involved in the adaptive immune response and may be responsible for adaptation to harsh environments. The objective of this work was to evaluate whether the local breed ancestry percentages in the BoLA locus of a Brangus population diverged from the estimated genome-wide proportions and to identify signatures of positive selection in this genomic region. For this, 167 animals (100 Brangus, 45 Angus and 22 Brahman) were genotyped using a high-density single nucleotide polymorphism array. The local ancestry analysis showed that more than half of the haplotypes (55.0%) shared a Brahman origin. This value was significantly different from the global genome-wide proportion estimated by cluster analysis (34.7% Brahman), and the proportion expected by pedigree (37.5% Brahman). The analysis of selection signatures by genetic differentiation (F_st) and extended haplotype homozygosity-based methods (iHS and Rsb) revealed 10 and seven candidate regions, respectively. The analysis of the genes located within these candidate regions showed mainly genes involved in immune response-related pathway, while other genes and pathways were also observed (cell surface signalling pathways, membrane proteins and ion-binding proteins). Our results suggest that the BoLA region of Brangus cattle may have been enriched with Brahman haplotypes as a consequence of selection processes to promote adaptation to subtropical environments.     

Organization and polymorphism of bovine major histocompatibility complex class II genes as revealed by genomic hybridizations with bovine probes

Previous studies on restriction fragment length polymorphism of bovine major histocompatibility complex class II genes have primarily been based on the use of human probes. In the present study bovine probes for DQA, DQB, DRB and DYA were used for RFLP analysis of cattle genomic DNA digested with PvuII and TaqI. There was an excellent agreement between the RFLP results obtained with homologous and heterologous probes. Although a few 'new' restriction fragments were revealed with the bovine probes there was no discrepancy with regard to the classification of allelic types with the two types of probes. The major advantages of using bovine probes were a better hybridization signal and reduced cross-hybridization between loci. Hybridization experiments with DQA probes for the first domain exon from two different genomic clones revealed the presence of two distinct types of bovine DQA genes. Surprisingly, these probes did not cross-hybridize at high stringency, indicating that the two genes are quite divergent. Hybridization with a recently described genomic clone for a novel bovine alpha-chain gene confirmed that it corresponds to the DYA gene which had previously been identified by cross-hybridization to a human DQA probe.     

Production of alloantibodies against caprine lymphocyte antigens

In all, 217 primiparous goats were each injected with blood cells from their own newborn kid. Eighty-five goats were given mononuclear cells, 61 were given leucocytes and 71 received whole blood. The goats were injected one, two or three times before collection of sera. Sera were also collected from 42 non-injected, primiparous goats. The sera were compared with regard to their potential value in class I histocompatibility typing. The percentage of potentially valuable sera was highest in the group of animals injected twice with whole blood (66 X 7%). However, this percentage was not significantly higher than the percentage in the group of animals injected once with whole blood (54 X 7%). It is concluded that injecting primiparous goats once with whole blood from their own newborn kid, is a rapid and easy method, which gives a high yield of alloantisera with potential value in class I histocompatibility typing.     

Lymphocyte antigens in Norwegian goats: serological and genetic studies

Altogether, 292 goat alloantisera were screened for antilymphocyte reactivity in a two-step dye exclusion microcytotoxicity test. Fifteen different lymphocyte antigen specificities were characterized by cluster analysis and absorption studies. The specificities were designated N1-N15 (N for Norwegian). Lymphocytes from 247 Norwegian dairy goats were tested. Each animal displayed from none to four of the characterized specificities. Lysostrip testing and family studies indicated that the specificities N1-N14 were coded for by multiple alleles belonging to at least two closely linked loci. It is suggested that these loci are part of the caprine major histocompatibility complex. Family studies gave strong evidence that the specificity N15 was not coded for by genes located in the same region as the other 14 specificities. Absorption studies showed that this specificity was located on both lymphocytes and erythrocytes.     

Equine lymphocyte antigens and reproduction in the Standardbred mare

Summary. Equine lymphocyte antigen (ELA) gene frequencies were estimated for pacing and trotting Standardbred mares residing on a breeding farm in central Ohio. The ELA gene frequencies for Ohio Standardbreds did not differ significantly from the ELA gene frequencies of Kentucky Standardbreds, determined by Bailey (1983). No significant differences were found in the distribution of ELA class I antigens in horses with lower overall fertility or a history of abortion on the investigated breeding farm. Likewise, no significant association was observed when the ELA types of both the mare and the stallion to which she was mated were compared with the reproductive efficiency of the mare.     

Lymphocyte antigens in sheep

This paper describes the detection of 13 lymphocyte antigens in sheep. The results obtained from family studies are consistent with the hypothesis that at least 12 antigens are under the control of a single genetic system. The distribution of antigens in the population suggests that the system contains two loci. The 13 antigens were compared with those previously reported. Only one additional specificity was found.     

Selective forces shaping diversity in the class I region of the major histocompatibility complex in dairy cattle

The major histocompatibility complex (MHC) is one of the most diverse regions of the mammalian genome. Diversity in MHC genes is integral to their function in the immune system, and while pathogens play a key role in shaping this diversity, the contribution of other selective forces remains unclear. The controlled breeding of cattle offers an excellent model for the identification and exploration of these forces. We characterized the MHC class I genes present in a sample of Canadian Holstein A.I. bulls and compared the results with those obtained in an earlier study. No evidence for a reduction in MHC diversity over 20 years was observed, but the relative frequency of some haplotypes had changed: the formerly rare A12 (w12B) haplotype had become the most common, together with A15, while A19, which dominated the earlier sample, had significantly reduced in frequency. Only 7% of bulls in the current study were MHC homozygous compared with the 14% expected under Hardy-Weinberg. To identify the selective forces at work, a gene substitution model was used to calculate the effects of MHC on selection traits using estimated breeding values for each bull. Significant associations between MHC and production, disease and fertility traits were identified, suggesting that MHC diversity is not merely shaped by disease in this controlled breeding system. The decrease in a common haplotype, the reduced number of homozygous bulls and the associations with disease and production traits together indicate that MHC diversity in dairy cattle is maintained by heterozygote advantage.     

BoLA antigens are associated with increased frequency of persistent lymphocytosis in bovine leukaemia virus infected cattle and with increased incidence of antibodies to bovine leukaemia virus

The association between bovine major histocompatibility system (BoLA) type and persistent lymphocytosis in cattle with antibodies to bovine leukaemia virus was examined by comparing antigen frequencies in cattle with persistent lymphocytosis to controls matched for age, sex, breed and presence of antibodies to BLV. The cattle came from nine dairy herds in south-east Queensland, Australia; six herds were Australian Illawarra Shorthorn (AIS), two herds were Jersey and one herd was Friesian. Antigen W6 and Eu28R were more common in cattle with persistent lymphocytosis than in controls. Antigen W8 was less common in AIS cattle with persistent lymphocytosis. A study of 24 offspring from one sire, heterozygous for W10 and Eu28R, showed that offspring inheriting Eu28R from the sire were significantly more likely to have antibodies to BLV than offspring inheriting the opposing W10 haplotype.     

Proceedings of the Second International Bovine Lymphocyte Antigen (BoLA) Workshop

The results of the second International BoLA Workshop, held in Wageningen, Netherlands in July 1980, are reported. These results arise from a comparison of 362 alloantisera to bovine lymphocytes, originating from 9 laboratories. The an-tisera were tested against a selected panel of 144 lymphocyte samples, originating from 7 laboratories. Some of the antisera and lymphocytes had also been tested during the First International BoLA Workshop in 1978.
Ten of the eleven specificities defined at the first workshop were confirmed, and an additional six new specificities were designated. Two of the additional specificities are subgroups of the w6 specificity. The data from this workshop are consistent with the hypothesis that BoLA antigens are controlled by a series of codominant alleles at a single autosomal locus.     

Serological and genetic identification of a bovine B lymphocyte alloantigen system

A two-colour fluorescence micro cytotoxicity test was used to screen antisera for antibodies specific for bovine B lymphocytes. A total of 114 cattle alloantisera were screened against peripheral blood lymphocytes from 100 unrelated individuals. Anti-B lymphocyte activity was detected in 47 antisera. Cytotoxic antibodies to antigens other than B lymphocyte specific antigens were removed by absorbing the antisera with buffy coat cells or platelets isolated from whole blood. Selected antisera were used to type paternal half-sib families. The presence of a polymorphic, MHS-linked antigen system on B lymphocytes was demonstrated. The tissue distribution and MHS linkage of these antigens suggests this system is analogous to the class II or Ia antigens of other species.