Production of foetally stimulated lymphocytotoxic antibodies by primiparous cows

Serum samples from groups of heifers at different pregnancy-related stages (preconception, month of pregnancy, and postpartum) were examined for the presence of lymphocytotoxic antibodies. Only a few weak and transitory antibodies were observed before or during the first pregnancy. These did not appear to be foetaliy stimulated. Strong or medium strength antibodies were detected in first or second month postpartum samples from 8 of 27 heifers (30%). The reactive sera were in every case reactive with the offspring of the cows from which the respective sera were collected.     

Production of foetally stimulated lymphocytotoxic antibodies by primiparous cows

Serum samples from groups of heifers at different pregnancy-related stages (preconception, month of pregnancy, and postpartum) were examined for the presence of lymphocytotoxic antibodies. Only a few weak and transitory antibodies were observed before or during the first pregnancy. These did not appear to be foetally stimulated. Strong or medium strength antibodies were detected in first or second month postpartum samples from 8 of 27 heifers (30%). The reactive sera were in every case reactive with the offspring of the cows from which the respective sera were collected.     

Breed differences in the distribution of BoLA-A locus antigens in American cattle

A total of 627 cattle representing seven breeds from south central Nebraska, USA were tested for 37 BoLA antigens which behave as products of 37 distinct alleles of the class I BoLA-A locus. Four antigens were absent from all breeds tested. The other antigens showed marked and statistically significant differences in breed distribution. There was no evidence for blank (null) alleles. The number of alleles in each breed ranged from 10 to 20. The Hereford and Simmental populations tested were less polymorphic than the Angus, Brown Swiss, Charolais, Gelbvieh and Limousin populations.     

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.     

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).     

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.     

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.     

Studies on the bovine lymphocyte antigens and the production of lymphocytotoxic antibodies by parous cattle

Studies were carried out to determine the time of appearance, frequency, titre and specificity of lymphocytotoxic antibodies in the plasma of parous Hereford cattle. Cytotoxic antibody was first detected in a small proportion (3162 = 4.8 % ) of primigravid cattle during the last third of pregnancy. Titres were low (neat or 1 in 2) at this time and decreased in one animal so that antibodies were not detectable in samples obtained on the day of calving or 9 days beforehand.
Following parturition, the proportion of primiparous cattle producing lymphocytotoxic antibodies increased markedly and reached a maximum value (8/19 = 42.1 %) during the third month post partum. Antibody levels also rose over the same period. An increase in the parity of the dam also resulted in an increase in the proportion of cattle with lymphocytotoxic plasma. These antibodies appeared earlier in pregnancy, were at a higher titre and had a wider specificity than those found in primigravida.
Non-foetally stimulated antibody was detected in 4 cattle. In one plasma sample, lymphocytotoxic activity was present prior to mating, and in the 3 others it was not directed against cells from either the bull to which the dam was mated or the calf produced by the sire and dam.     

A bovine 'Ia-like' antigen detected by a xenogeneic monoclonal antibody

A monoclonal antibody termed B2 Val 7C7, was produced by the fusion of xenoimmune mouse spleen cells with Sp2/0.Ag 14 myeloma cells. This antibody is specific for a polymorphic lymphocyte antigen; it was detected on cells from 138 out of 177 cattle by both ¹²⁵I-labelled protein A (solid-phase radioimmunoassay, SPRIA) and gold-labelled protein A (immunogold). Its binding was tested on various cell types (peripheral blood lymphocytes, monocytes, polymorphonuclear cells (PMN), thymocytes) from a variety of normal bovine donors. On the one hand, B2 Val 7C7 detects a determinant present on all IgG-bearing lymphocytes, on 20 % of the non-IgG-bearing lymphocytes and on the majority of the monocytes. On the other hand, no binding occurs on any PMN or thymocytes. The detected membrane antigen was isolated by immunoprecipitation from an NP 40 extract of ³H-leucine-labelled cells. On SDS-PAGE, it appears to be composed of two sub-units: a 32 000-dalton chain and a 27 000-dalton chain. These results show that B2 Val 7C7 recognizes an alloantigenic specificity present on an Ia-like antigen     

Interspecies cross-reactivity of bovine lymphocytotoxic sera with pig lymphocytes

Forty-three bovine BoLA antisera were tested on pig lymphocytes by a microlymphocytotoxicity test. Twenty-five were found to be cytolytic. Fifteen sera detected the A blood group antigen on porcine lymphocytes but showed no reaction with the J antigen on bovine lymphocytes. Six BoLA reagents reacted with all pig cells tested. Cross-reactions with SLA antigens were observed in only four sera, the highest correlation being recorded with SLA-W7 (r = 0.87). Bovine alloantisera are not of value for SLA typing.     

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.     

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.     

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.     

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.     

A study on associaton between mastitis and serologically defined class I bovine lymphocyte antigens (BOLA-A) in Norwegian cows

A total of 102 cows was tested for class I antigens of the bovine major histocompatibility complex. Half of the animals (51) had completed at least four lactations without any veterinary treatment for mastitis. The distribution of BoLA-A antigens among these relative mastitis-resistant cows was compared to that in the other half of the material (51), which comprised animals with at least one recorded treatment for mastitis. There were no statistically significant differences in BoLA-A antigen frequency between cows with mastitis and cows without mastitis. The two most common antigens were A2 and w16. The frequency of these two antigens deviated from earlier estimates within the Norwegian cattle (NRF) population, the difference for w16 being statistically significant.     

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.     

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.     

High levels of linkage disequilibria between serologically defined class I bovine lymphocyte antigens (BOLA-A) and class II DQB restriction fragment length polymorphism (RFLP) in Norwegian cows

Serum defined BoLA-A antigens, together with BoLA-DQB RFLP patterns, were determined in 87 almost unrelated Norwegian cattle. Statistical analysis revealed strong linkage disequilibria between these loci at the population level. A total of 13 haplotypes were found to be present at frequencies significantly greater than those predicted on the basis of their component gene frequencies. Among these, the subgroups 1A and 1B of the DQ1 haplotype were found to be closely associated with the class I antigens A11 and w16, respectively. The association between A11 and DQ1A is of particular interest, as two independent studies, one employing class I serology, and the other RFLP analysis of the class II locus DQ, have previously indicated that A11 and DQ1A confer relative susceptibility to mastitis.     

Studies on the bovine major histocompatibility class I and class II antigens using homozygous typing cells and antigen-specific BoT4+ blast cells

Animals were identified from two sire lines as being homozygous for the class I bovine lymphocyte antigen (BoLA-A) w23. These animals were also shown to be homozygous for class II antigens (BoLA-D) which, however, differed between the two sire lines. Lymphocytes from these animals were then used either as stimulator cells in one-way mixed lymphocyte reactions (MLR) with all animals in the herd carrying the w23 antigen or as antigen presenting cells to bovine T4+ cell blasts. It was shown that, within each sire line, the genes encoding the MHC class I and class II antigens were closely linked. There were no detected recombinations between the MHC class I and class II regions nor within the BoLA-D region responsible for mixed lymphocyte reactivity. MLR typing of MHC class II antigens correlated with the results from T-lymphocyte proliferation studies. Cells from these cattle, which are homozygous at the class I and II MHC loci but differ in the class II antigen expressed, could be used to type the BoLA-D of other cattle.