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Joint Report of the Fourth International Bovine Lymphocyte Antigen (BoLA) Workshop, East Lansing, Michigan, USA, 25 August 1990.

Blood samples from 54 animals were exchanged between 15 laboratories in nine countries to improve and expand BoLA class I and class II typing. A total of 27 out of 33 (82%) of previously accepted BoLA-w specificities were represented within the cell panel. Seventeen new serum-defined BoLA specificities were accepted by the workshop participants, thus expanding the number of internationally recognized BoLA specificities to 50. The large number of new specificities detected resulted from the number of serological reagents used (n = 1139) and the genetic diversity of the cell panel. Confidence derived from the high percentage of agreement between the laboratories on antigen detection (97.3%; r = 0.84) permitted the removal of the workshop (w) notation from 23 BoLA-w specificities and their acceptance as full status BoLA-A antigens. Two new non-BoLA antigens were also detected, one completely included within the red blood cell factor S' (BoLy-S'), whereas a second (BoLy-w1) did not show any association with tested red blood cell factors. A comparison between serological, isoelectric focusing (IEF) and DNA typing for BoLA class II polymorphism was conducted with a subset of workshop cells. Correlation between the three methods was significant for three combinations of alleles. Three other serologically defined class II specificities were correlated with DR and/or DQ restriction fragment length polymorphism (RFLP) types, whereas six additional IEF types were correlated with DR and/or DQ RFLP types (r greater than or equal to 0.50). Several new IEF, DRB, DQA and DQB RFLP patterns were identified. In 46 animals that were typed for BoLA-DR and DQ genes by RFLP analysis, 46 different BoLA haplotypes were tentatively defined. These 46 haplotypes were distinguished by 31 serologically-defined BoLA-A alleles (and 2 'blanks'), 15 DRB RFLP types (plus up to 10 new DRB RFLP patterns) and 23 DQA-DQB haplotypes.

Animals

Association between BoLA and subclinical bovine leukemia virus infection in a herd of Holstein-Friesian cows.

The role of the bovine major histocompatibility system (BoLA) in subclinical bovine leukemia virus (BLV) infection was investigated in a herd of Holstein-Friesian cows (n = 240). The BoLA W8.1 allele was negatively associated with the presence of antibodies to the major BLV envelope glycoprotein, BLV-gp51 (corrected P less than 0.001, relative risk = 0.31). These results suggest that a BoLA-linked gene(s) may influence the early spread of BLV infection. Since B cells are the primary target of BLV infection, we then determined the relationship between BoLA-A locus phenotypes and B-cell numbers in peripheral blood of seropositive and seronegative cows. There were no significant differences between BoLA-A alleles for any hematological parameter in seronegative cows. Seropositive cows with the W12.1 allele had significantly greater absolute numbers of lymphocytes per microliter and B cells per microliter than did seropositive cows with other BoLA-A phenotypes (P less than 0.01, respectively). The average effect associated with the W12.1 allele in BLV-infected cows was an increase of 2010 B cells per microliter of whole blood relative to BLV-infected cows with other BoLA-A phenotypes. These results demonstrate that susceptibility to the polyclonal expansion of BLV-infected B lymphocytes is associated with the W12.1 allele in Holstein-Friesian cattle. Compared with results of a previous study in a herd of Shorthorn cattle, it appears that resistance and susceptibility to subclinical progression of BLV infection are associated with different BoLA-A locus alleles in different cattle breeds.

Animals

Evidence for BoLA-linked resistance and susceptibility to subclinical progression of bovine leukaemia virus infection.

The role of the bovine major histocompatibility complex in bovine leukaemia virus (BLV) infection and disease progression was investigated in a herd of Shorthorn cattle (n = 117). The frequency of cows that were seropositive to BLV-glycoprotein antigen was 51%. Twenty-three per cent of the seropositive cows were lymphocytotic. At the herd level, relative resistance to BLV-dependent B-cell proliferation and lymphocytosis among seropositive cows was associated with bovine lymphocyte antigen (BoLA)-DA7, whereas susceptibility was associated with BoLA-DA12.3. These associations were also confirmed at the family level, where BoLA phenotypes were used as haplotypic markers. Among the offspring of one BoLA-heterozygous sire (n = 33), resistance segregated with the DA7 haplotype and susceptibility with the DA12.3 haplotype. In this sire group, maternal transmission of the BoLA-w8 allele was associated with increased susceptibility to B-cell proliferation and lymphocytosis in w8/DA12.3 heterozygotes. These data provide the first evidence that subclinical progression of BLV infection is under the control of the BoLA complex, and suggest that the BoLA system can be used to select for resistance to B-cell proliferation and the development of lymphocytosis in BLV-infected herds.

Alleles

The influence of the BoLA-A locus on reproductive traits in cattle.

Associations between the major histocompatibility complex (MHC) and reproductive performance have been reported in humans, mice, rats, pigs and chickens. Only the A locus of the bovine major histocompatibility complex (BoLA-A) has been well characterized, and 42 alleles of this locus have been identified in American cattle. Four studies were conducted to examine the association between alleles of the BoLA-A locus and reproductive performance. Testis size, which is an indicator of early puberty and increased fertility in young bulls, was examined in 440 yearling bulls from nine breeds with a gene substitution model that included the effects of breed, sire, age of dam and age or weight of the bull. Estimated breeding value for twinning was examined with a gene substitution model with 204 cattle from a herd with a high frequency of twinning. Fertility of potential partners having BoLA-A locus alleles in common was examined in a prospective study involving 101 pure-bred Hereford cows mated by artificial insemination to four pure-bred Hereford bulls. The effect of homozygosity on birth weight, preweaning weight gain and post-weaning weight gain was estimated in a sample of 683 calves from nine breeds; 22% of the calves were apparently homozygous and 78% were heterozygous at the BoLA-A locus. There were significant and large effects of some BoLA-A locus alleles on paired testicular volume, but the analyses on the other traits did not show significant associations. Substitution of the W6.1 allele for the W9A allele reduced paired testicular volume by 150 +/- 44 cm3. The W6.1 allele has now been shown to influence a reproductive trait, a production trait and susceptibility to an economically important disease. Selection for these traits may influence the frequency of the large number of alleles at the BoLA-A locus.

Alleles

Induction of a growth-phase-dependent promoter triggers transcription of bolA, an Escherichia coli morphogene.

The bolA gene, which is involved in the morphogenetic pathways of Escherichia coli, was sequenced and two potential promoters were identified. Expression from promoter P1, proximal to the bolA structural gene is specifically induced during the transition to the stationary phase of growth. This promoter contains an unusual--10 region (CGGCTAGTA), which defines a new class of E. coli promoters necessary for the dramatic increase in the rate of synthesis of a large set of proteins during the cessation of logarithmic growth. This conclusion was confirmed by identifying two additional E. coli promoters and one plasmid promoter, which also were induced during the transition to the stationary phase of growth. Analysis of proteins produced during the exponential and stationary phases of growth in a bolA null mutant suggest a possible role for the BolA protein in the induction of the expression of penicillin-binding protein 6 (PBP6) in the transition to the stationary phase. Supporting this hypothesis is the presence of a putative DNA-binding domain within the bolA coding sequence.

Amino Acid Sequence

Mapping of bovine markers CYP21, PRL, and BOLA DRBP1 by genetic linkage analysis in reference pedigrees.

We have analyzed DNA from 13 bovine reference pedigrees using primers specific for microsatellite markers derived from the 21-steroid hydroxylase (CYP21) and prolactin (PRL) genes and the leukocyte antigen (BOLA DRBP1) pseudogene. Linkage was demonstrated between PRL and BOLA DRBP1 (theta = 0.05; Z = 19.6), cyp21 and PRL (theta = 0.13; Z = 6.8), and BOLA DRBP1 and CYP21 (theta = 0.17; Z = 10.4). These results suggest an order BOLA DRBP1-PRL-CYP21, although in a multilocus analysis the alternative order PRL-BOLA DRBP1-CYP21 was also possible. The data confirm and extend the previously established syntenic relationship between these markers on bovine chromosome 23 and provide points of anchorage for further linkage studies in the reference pedigrees described.

Animals

Expression and genetic segregation of parental BoLA serotypes in bovine embryos.

The detection of parentally derived BoLA serotypes was attempted in 68 bovine embryos. 23 bovine embryos were tested for genetic segregation of maternally derived BoLA serotypes. 45 bovine embryos were tested for genetic segregation of paternally derived BoLA serotypes. The expected parentally derived BoLA gene products were detectable on approximately 50% of the embryos tested. A 1:1 segregation ratio of expression or non-expression of parental BoLA serotypes in 7-day-old preimplantation bovine embryos, which is expected for codominant alleles, could not be rejected.

Animals

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

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.

Animals

Extensive polymorphism of the BoLA-DRB3 gene distinguished by PCR-RFLP.

A polymerase chain reaction (PCR)-based method is described for typing of alleles of the bovine lymphocyte antigen (BoLA)-DRB3 gene. A total of 30 DRB3 alleles were distinguished by digestion of PCR amplification products of BoLA-DRB3 exon 2 with RsaI, BstYI and HaeIII (PCR-RFLP). All restriction fragment patterns, with the exception of one HaeIII pattern, were consistent with restriction sites that were found among 14 previously sequenced DRB3 alleles. The PCR-RFLP typing method was evaluated on 168 genomic DNA samples collected from animals of 10 cattle breeds, 48 of which were typed in the Fourth International BoLA Workshop for BoLA-DRB and -DQ by conventional restriction fragment length polymorphism (RFLP) analysis using heterologous and homologous DNA probes. Thirty-one DRB/DQ haplotypes containing 23 DRB3 alleles were identified among the 48 workshop animals analysed. Using PCR-RFLP, 11 DRB3 alleles were identified in 18 workshop animals for which DRB RFLPs were not informative. PCR-RFLP typing of additional animals revealed five new DRB3 alleles, of which three contained a putatively located three basepair deletion in the identical position as found for the sequenced allele DRB*2A. PCR-RFLP was shown to be a rapid and sensitive method for the detection of polymorphism in a functionally relevant domain of the BoLA-DRB3 gene and should be useful for studying the evolution of DRB polymorphism in cattle and other Bovidae.

Alleles

Anti-BoLA-w8 monoclonal antibody: production of a tissue typing reagent after blocking monomorphic sites on bovine mononuclear cells.

A murine monoclonal antibody, WIM-8 with specificity for BoLA-w8, was generated after using an immunization protocol designed to reduce the response to bovine monomorphic determinants. BoLA-w8/w9 peripheral blood mononuclear (PBM) cells were incubated with an antiserum from mice immunized with BoLA-w10/w20 PBM cells. Splenocytes from a mouse that received three intravenous injections of the treated cells were fused with murine myelomas. A hybridoma supernatant with alloreactivity in ELISA against BoLA-w8 positive but not BoLA-w8 negative PBM cells was tested for lymphocytotoxicity in parallel with parous alloantisera. WIM-8, an IgG2b monoclonal antibody, identified the 44,000 dalton protein of the bovine MHC class I molecule present on Bos taurus and Bos indicus animals.

Animals

Preliminary report on BoLA polymorphism in Guernsey cattle.

A total of 95 registered Guernsey cows and heifers sired by 34 bulls were typed for class I antigens encoded by the bovine major histocompatibility complex (BoLA). A panel of alloantisera was used to detect 21 of the 33 internationally recognized BoLA specificities. Fourteen BoLA specificities were detected in the herd using a standard lymphocyte microcytotoxicity test. The most frequent BoLA specificity detected was w6; but in 85% of these animals, a subtype of w6, either w17 (13.7%) or UR6.2 (34.7%), could be assigned. The next most common BoLA specificities, by frequency, were w12, w15, UR1, and w21. Specificities not detected were w2, w3, w5, w7, w11, w16, w19, w20, and w24.

Animals

Possible association of antibody responses to human serum albumin and (T,G)-A--L with the bovine major histocompatibility complex (BoLA).

Antibody responses to human serum albumin (HSA) and (T,G)-A--L were determined in 130 young bulls in Norway and the BoLA types of the bulls were defined. Significant associations of some BoLA antigens with immune responsiveness were shown, indicating the likely existence of an immune response (Ir) region linked to the BoLA class I antigens. High response to HSA seems to be a dominant trait. BoLA w2 showed an association with low response to HSA. This may reflect the effect of a specific MHC-associated immune suppressor gene.

Aging

Genetic analysis of the antigens defined at the third international BoLA workshop.

A comparison of lymphocyte antigens showed that 32 of the 33 BoLA antigens defined at the third international BoLA workshop (Bull et al., 1989) corresponded to previously defined local antigens (Stear et al., 1988). The third workshop antigen w18 had no locally defined equivalent. All 32 antigens were shown in family studies to be expressed by autosomal co-dominant genes, and all 32 workshop antigens were shown to be products of the BoLA system. After excluding the supertypic antigens, nearly all animals tested possessed only one or two antigens and there were no observed recombinants in family studies. These results do not exclude the possibility that the 32 workshop antigens are the products of one locus (BoLA-A).

Alleles

Bovine alloreactive cytotoxic cells generated in vitro detect BoLA w6 subgroups.

Alloreactive cytotoxic T cells (CTL) were generated in mixed lymphocyte culture against cells bearing subgroups of BoLA w6, as well as in BoLA w4, w10 and w16. Primary cultures were restimulated at weekly intervals with irradiated stimulator cells and tested in a 51Cr-release assay with target cells derived from Theileria annulata-infected cell lines. Generation of CTL was accelerated in animals that had been previously primed in vivo by skin grafting. CTL were generated that were specific for BoLA w6 subgroups and not w6. With w6.1 the specific killing was significant at the 5% level, and with w6.2, 6.3 and 6.4 it was significant at the 0.1% level. Where CTL were potentially generated against two BoLA-A locus allele products at the same time (i.e. with heterozygous stimulator cells), one of which was a w6 subgroup, there was similar CTL activity against both products when w6.4 and w16 or w6.1 and w4 were the combinations involved. In two generations against w10 and either w6.1 or w6.2 there was significantly more killing of w10-bearing targets than those with the w6 subgroup.

Animals

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

The results and agreements of the 1 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 microlymphocytotoxicity 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).

Animals

Serological relationships among antigens of the BoLA and the bovine M blood group systems.

Alloimmunizations with either lymphocytes or red cells from donor cows positive for BoLA w16 and blood group M' antigens into recipients negative for these antigens produced antisera reactive in the cytotoxic test with w16-positive lymphocytes and in the haemolytic test with M'-positive erythrocytes. Similarly, alloimmunizations of blood group M1-negative recipients with either lymphocytes or red cells from donor cows possessing the M1 blood group factor produced antisera specifically reactive with lymphocytes and erythrocytes from M1-positive cattle. Absorptions with either lymphocytes or erythrocytes from individual animals of the same M antigenic type as the donor removed all haemolytic and cytotoxic reactivity. The results indicate that blood group M' and BoLA w16 share a similar antigenic structure. Likewise, blood group M1 has an antigenically similar counterpart which is also part of the BoLA system.

Animals

Relationship between the bovine major histocompatibility complex (BoLA), erythrocyte markers and susceptibility to mastitis in Icelandic cattle.

Milk and blood samples were obtained from three Icelandic dairy herds. The herds were monitored regularly for mastitis incidence. Cell counts, adenosine triphosphate (ATP) and antitrypsin levels of the milk samples were recorded. In addition, red cell and BoLA typing were performed on the blood. Although cell counts and ATP levels showed significant associations with mastitis, antitrypsin levels did not. Red blood cell antigens N'2 and S1 and the lymphocyte antigen detected by the monoclonal antibody M7 were associated with low cell counts, whilst BoLA w6 and w6.1 were associated with high cell counts. BoLA w6.2 and w11 showed significant association with high antitrypsin levels. Only ED116 showed a significant association with mastitis.

Animals

Immunomagnetic isolation of cells for serological BoLA typing.

This paper describes a totally new immunomagnetic (IM) technique adapted to serological BoLA typing. The basic technique has recently been developed by Vartdal et al. (1986) for serological HLA typing. The main advantage is that bovine mononuclear cells (e.g. T-cells and possibly their subsets, B-cells and monocytes) can be quickly and specifically isolated with high yield and viability from whole blood in a one-step procedure. This is achieved by magnetic separation of rosettes formed between the cells and superparamagnetic monosized polystyrene microspheres (Dynabeads TM) coated with cross-species reactive monoclonal antibodies (MAbs) specific for various human T-cell antigens or for HLA class II monomorphic epitopes. The cells are isolated within 5 min after a 5-min incubation at 4 degrees C. Magnetic separation of rosettes with a strong cobalt-samarium magnet eliminates all the laborious centrifugation steps necessary with conventional procedures. The isolated cells, still attached to the particles, are available for microcytotoxic assay. This is carried out within 55 min, including a two-step application of alloantiserum and complement and addition of acridine orange/ethidium bromide for the staining of viable (green) and dead (red) cells. The high viability of isolated cells gives a very low background kill compared with the conventional cytotoxic assay. The IM typing technique is also superior in sensitivity to the conventional technique as standardized for the international BoLA comparison test. The IM technique is likely to have its greatest impact on class II typing; class II positive cells being separated very efficiently. Polymorphic HLA class II MAbs detected likely polymorphic BoLA class II epitopes.

Animals