Bovine Major Histocompatibility Complex Research Articles

Cloning of cDNAs and the Molecular Evolution of a Bovine MHC Class II DRA Gene

Two overlapping cDNA clones coding for bovine major histocompatibility complex (MKC) class II antigen DRA chain were isolated and characterized. The full-length cDNA clone, MR1, encoded a primary translated product of 253 amino acids, 24 of which were deduced to be a signal peptide and 229 which formed a mature polypeptide. The amino acid sequences deduced from this clone resembled those of class II A molecules from other species in both size and structure, but no potential consensus site of N-linked glycosylation comparable to those in the human, mouse, rat and swine proteins was found in the α2 domain, as well as ovine and equine DRA molecules. Comparison of amino acid sequences encoded by class II A genes among several species and a dendrogram constructed from these data places the DRA gene and the DQA/DYA genes on two distinct branches of a phylogenetic tree, with bovine DRA and ovine DRA being most similar on the DRA branch.

Host genetic mechanism of resistance and susceptibility to a bovine retroviral infection

Abstract The bovine leukemia virus (BLV) has approximately 50% nucleotide sequence similarity to HTLV types I and II, and shares common regulatory mechanisms, but differs in host cell tropism, with B‐cells comprising the primary target of infection. We have shown that progression of BLV infection to persistent lymphocytosis (PL) is under the control of the bovine major histocompatibility complex, mapping closest to BoLA class II genes. We recently narrowed the association to exon 2 of the BoLA‐DRB3 gene. This exon was cloned from animals with BoLA haplotypes previously found to be associated with resistance and susceptibility to PL. Sequence analysis revealed the presence of the amino acids Glu‐Arg (ER) at putative antigen binding residues 70 and 71 only in BoLA haplotypes associated with resistance. This correlation was confirmed in a case‐control study using an allele‐specific polymerase chain reaction for the detection of the ER motif at residues 70‐71. Analysis of infection in peripheral blood leukocytes by a new single cell detection methodology revealed that BLV‐infected PL‐resistant animals have fewer infected peripheral cells than age‐matched susceptible cows. These results provide a molecular basis for Ir gene control of resistance and susceptibility to PL and suggest that the cellular immune response is important in preventing the in vivo spread of BLV infection.

Complexity of the bovine MHC class-II specificity DW3 as defined by alloantisera.

Alloantisera related to the bovine major histocompatibility complex (MHC) class-II specificity Dw3 were investigated by cross-absorption experiments and by application of the monoclonal antibody-specific immobilization of lymphocyte antigen assay (MAILA). The absorption study revealed antibodies specific for an antigenic determinant shared by all Ds03 (Dw3)-positive animals, and several other antibody populations recognizing the locally defined specificities Ds10, Ds11 and Ds15, that are closely associated with Ds03. The results of the MAILA-assay indicate that the Ds03 specificity is probably encoded by DQ, whereas specificities Ds10 and Ds 11 are more closely associated with DR molecules. The data presented here provide the first evidence that bovine DR and DQ specificities can be identified separately by serological methods using alloimmune antisera.

Improved discrimination of bovine class II DRβ‐chains polymorphisms using immunoblotting

An immunoblotting technique is reported that reveals electrophoretic variants in the beta-chains of class II antigens of the bovine major histocompatibility complex. One monoclonal antibody, mAb VPM57, reacted on immunoblots with an epitope present in approximately half of the haplotypes investigated. This reagent is especially useful in discriminating electrophoretic variants that have similar isoelectric points.

Microsatellite DNA variation within and among European cattle breeds

Microsatellite markers offer great potential for genetic comparisons within and between populations. We report the analysis of 12 microsatellite loci in six breeds of European cattle. This yielded a wide spectrum of variability with observed heterozygosities ranging from 0.00 to 0.91. Deviations from Hardy-Weinberg equilibrium were noted for some locus-population combinations, particularly at a microsatellite located within the prolactin gene. Also, significant linkage disequilibrium was detected between two microsatellite loci located within the bovine major histocompatibility complex, and this association was maintained across breeds, providing evidence for marker stability during short-term evolution. The mode of mutation was investigated by comparing the observed data with that expected under the infinite alleles model of neutral mutation, and six of the microsatellite loci were found to deviate significantly, suggesting that a stepwise mutation model may be more appropriate. One indication of marker utility is that, when genetic distance estimates were computed, the resultant dendrogram showed concordance with known breed histories.

Possible association between a serologically detected haplotype of the bovine Major Histocompatibility Complex and subclinical mastitis

The incidence of subclinical mastitis in Simmental and Simmental-Red Holstein cattle in relation to the bovine major histocompatibility complex (BoLA) was investigated. Quarter milk samples of 166 cows consisting of fifteen halfsib groups of different ages and lactation stages were analysed for somatic cell counts (SCC) and bacteriological infection. From these data the udder health status (UHS) of the animal was determined. Each cow was typed serologically for BoLA class I and class II specificities. The statistical evaluation for UHS was performed using a logistic regression model. The effect of BoLA on SCC was estimated by least square analysis. Animals carrying the BoLA class II haplotype "b" were significantly more affected by subclinical mastitis than those without this haplotype. Breed group showed a significant influence on both UHS and corrected mean logSCC (P < 0.0001 resp. P < 0.05). Lactation stage had a significant effect on SCC but only a weak influence on UHS (P < 0.0001 resp. P < 0.13). ZUSAMMENFASSUNG: Mögliche Assoziation zwischen dem bovinen Haupthistokompatibilitätskomplex und subklinischer Mastitis In dieser Studie wurde die Beziehung zwischen dem bovinen Histokompatibilitätskomplex (BoLA) und der Prävalenz subklinischer Mastitis bei Simmentaler und mit Red Holstein eingekreuzten Simmentaler Kühen untersucht. Bei 166 Kühen, aus 15 Halbgeschwistergruppen, unterschiedlichen Alters und in verschiedenen Laktationsstadien, wurden Milchproben entnommen und auf Zellzahl (SCC) und bakterielle Infektion untersucht. Aus diesen Daten wurde ein Eutergesundheitsstatus (UHS) definiert. Jede Kuh wurde serologisch für BoLA Klasse I und Klasse II Spezifitäten typisiert. Die statistische Auswertung für den UHS erfolgte mit einem logistischen Regressionsmodel. Der Einfluß von BoLA-Haplotypen auf SCC wurde mit der Least Square Analyse ermittelt. Tiere mit dem Klasse II Allel "b" zeigten mehr Euterprobleme als Kühe ohne dieses Allel. Die Rassegruppe übte sowohl auf den UHS wie auch auf den korrigierten Mittelwert der Zellzahlen einen signifikanten Einfluß aus (P < 0.0001 resp. P < 0.05). Der Effekt des Laktationsstadiums auf die Zellzahl war signifikant, aber für den UHS wurde nur ein schwacher Einfluß des Laktationsstadiums festgestellt (P < 0.0001 resp. P < 0.13).

Improved single-strand conformation polymorphism analysis by asymmetric polymerase chain reaction with end-labeled primers

An improved form of single-strand conformation polymorphism (SSCP) assay has been developed for the analysis of bovine major histocompatibility complex class II alleles. The method uses asymmetric polymerase chain reaction (PCR) amplification from each of two end-labeled primers to generate individual single-stranded products that are analyzed by electrophoresis in nondenaturing polyacrylamide gels. This technique gives good resolution of labeled single strands derived from 392-bp bovine DRB exon-2 PCR products, without interference from double-stranded products, and enables assignment of SSCP bands to the individual strands of the template DNA. The allelic groupings defined by this method in a panel of test animals were confirmed by independent typing by restriction fragment-length polymorphism.

Differential expression of bovine MHC class II antigens identified by monoclonal antibodies

Four monoclonal antibodies (mAbs), UC-A4, UC-D3, UC-H9, and IL-A21, specific for bovine major histocompatibility complex class II proteins are described. Sequential immunoprecipitation experiments using biotin-labeled peripheral blood mononuclear cells suggested, but did not conclusively establish, that each of these antibodies recognized a different epitope. The epitope identified by IL-A21 appeared to be common to all of the class II proteins precipitated by the four mAbs, and UC-D3 and UC-H9 each appeared to react with distinct epitopes on separate subsets of these class II proteins. Monoclonal antibody UC-A4 appeared to identify an epitope on a subset of the class II molecules identified by UC-H9. Differences found in the expression by lymphoid cells of class II proteins identified by the four mAbs were indicative of each mAb recognizing a different epitope. UC-H9 and IL-A21 class II proteins were detected on all surface immunoglobulin (S'Ig) positive cells in peripheral blood, but UC-A4 and UC-D3 class II proteins were not. Expression of UC-A4 class II proteins, detected at low density on a strikingly reduced number of S'Ig+ cells from the blood of some bovine leukosis virus-infected cattle, could be increased by culturing these B cells with lipopolysaccharide. All peripheral blood monocytes expressed UC-H9 and IL-A21 class II proteins, but only a proportion of monocytes expressed detectable UC-A4 and UC-D3 class II proteins. Almost all mitogen-stimulated BoCD4+ and BoCD8+ T cells expressed UC-H9 and IL-A21 class II proteins, whereas fewer stimulated T cells of both subsets expressed UC-A4 and UC-D3 class II proteins. All gamma/delta receptor (gamma/delta R) T cells expressed UC-D3, UC-H9, and IL-A21 class II proteins, but no cells (of gamma/delta R+ or CD4+/CD2+ phenotype) from gamma/delta R+ T cell-enriched cultures expressed UC-A4 class II proteins.

Association between the bovine major histocompatibility complex and chronic posterior spinal paresis--a form of ankylosing spondylitis--in Holstein bulls.

A highly significant association was found between the bovine MHC class I antigen BoLA-A8 and a form of vertebral osteophytosis/ankylosing spondylitis known as chronic posterior spinal paresis (PSP) in Holstein bulls (P < 0.001). In a population study, restricted to unrelated bulls, BoLA-A8 was significantly associated with PSP (P = 0.0015) with a relative risk of 34.6. In a family study, one PSP bull, BoLA A8/A20, sired 13 offspring. BoLA-A8 was significantly associated with PSP (P = 0.0008). All five PSP sons inherited the A8 allele and the eight healthy sons each inherited the A20 allele. In three other families a complete association of BoLA-A8 and PSP was observed. Lod score analysis, using all available families, indicated a significant linkage between BoLA and PSP (lod score = 6.9). Based on clinical observation, pathology, age/sex predilection, and a strong association with a class I MHC molecule, this inflammatory disease appears analogous to the human condition known as ankylosing spondylitis.

Order of bovine DRB3, DYA, and PRL determined by sperm typing.

The order and recombination fractions (theta) between the bovine major histocompatibility complex DRB3, DYA, and prolactin (PRL) genes were determined by typing of 254 sperm from a triply heterozygous bull. A recently developed method, primer extension preamplification (PEP), was used to amplify the bovine sperm genome prior to amplification of specific loci by the polymerase chain reaction (PCR). At least 28 copies of the DRB3, PRL, or DYA gene were obtained from 50 cycles of PEP. For sperm typing, alleles of each locus were discriminated by restriction endonuclease cleavage of PCR products and polyacrylamide gel electrophoresis of the restriction fragments. The most likely gene order is PRL-DRB3-DYA, with theta = 0.025 (+/- 0.012) and theta = 0.150 (+/- 0.024), respectively. The odds are 128:1 in favor of this order in comparison with the second most likely order DRB3-PRL-DYA. Our results demonstrate the power of sperm typing in concert with PEP for multilocus gene mapping.

BoLA‐DIB: species distribution, linkage with DOB, and Northern analysis

The bovine major histocompatibility complex differs from that of other species in that it has a second group of class II genes that are separated from the classical class I/class II complex by 15 to 20 centimorgans. Two class II bovine genes, BoLA-DYA and BoLA-DIB, seem to be unique based on sequence analysis. To investigate the occurrence of one of these genes, BoLA-DIB, in other species, a DIB 2nd domain exon probe was hybridized to Southern genomic blots containing DNA from representatives from a variety of mammalian families. The probe hybridized only with DNA from representatives of the Cervidae, Giraffidae and Bovidae families, indicating that significant sequence similarity to DIB may be confined to these families. Analysis of the linkage relationship between DIB and DOB in cattle half-sib families did not yield any recombinants among 39 informative offspring, indicating that DIB maps within the second group of class II bovine genes; DOB has previously been mapped to the second group of class II genes. A 1st domain exon probe from BoLA-DQB hybridized to RNA from either stimulated or unstimulated peripheral bovine lymphocytes, while 1st domain exon probes from BoLA-DIB and HLA-DOB gave no hybridization signals, indicating that transcription in peripheral lymphocytes of these genes is at least very minor compared to BoLA-DQB.

A study on association 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.

Production, immunoanalysis and selection of monoclonal antibodies for studies of polymorphism of bovine MHC class I antigens.

Two monoclonal antibodies (MoAbs) were raised against bovine lymphocyte antigens of the major histocompatibility complex (MHC) for studies of the polymorphism of MHC class I antigens of African cattle. Immunoanalysis of lymphoid tissues and molecular weight data were used to characterize the epitopes seen by the MoAbs. Competitive binding assays indicated specificity of the MoAbs for two distinct epitopes. Application of the MoAbs for MHC typing of a cattle population showed that the epitopes were co-expressed on the target MHC antigen but occurred independently on non-target MHC antigens. It is concluded that, to obtain MoAbs for studies of MHC polymorphism, it is important to conduct large-scale analysis using immunoassays and population studies.

Transmembrane and cytoplasmic domain sequences demonstrate at least two expressed bovine MHC class I loci.

We have used the polymerase chain reaction to amplify cDNA from expressed bovine major histocompatibility complex class I genes. Sequences obtained from transmembrane and cytoplasmic domains were used to identify the number of expressed alleles. Data from three animals suggest that there are four major expressed alleles, representing the products of two (or more) loci. We have also demonstrated the presence of an alternatively spliced mRNA, which has been observed in five animals. The alternative splicing removes exon 7 (the major site of class I phosphorylation), which predicts a truncated molecule with a cytoplasmic portion 16 amino acids shorter than usual. This phenomenon was detected for only a single class I allele within each individual.

Characterization of bovine MHC class II polymorphism using three typing methods: serology, RFLP and IEF.

Various methods, with different strengths and weaknesses, are currently used to define polymorphism of the bovine major histocompatibility complex (MHC) class II genes. A more complete characterization of bovine lymphocyte antigen (BoLA) haplotypes can be achieved by combining several of these methods. In this study BoLA class II polymorphism was characterized using three typing methods: serology, restriction fragment length polymorphism (RFLP), and isoelectric focusing (IEF). Twenty six Holstein-Friesian and 15 Angus cattle that carried an array of serologically defined BoLA haplotypes were selected for the study. The panel included 12 BoLA complex homozygotes. The three class II typing methods recognized polymorphism associated with the same or very tightly linked genes in the DQ-DR class II subregion. In total 25 BoLA-A locus (class I)--DQ-DR subregion (class II) haplotypes were defined. Three of the serological class II specificities, Dx1, Dx3, and Dx4, were associated with more than one RFLP defined DQ-DR haplotype. The other 4 class II specificities behaved as private specificities. One BoLA haplotype was found in both Holstein and Angus cattle. Two other BoLA haplotypes defined here have previously been described in other breeds. This suggests that these haplotypes exist in strong linkage disequilibrium.

Bovine major histocompatibility complex class I specific monoclonal antibodies characterized by flow cytometry, one- and two-dimensional electrophoresis, Western blot and inhibition of cytotoxic T lymphocyte function

To identify and characterize the bovine major histocompatibility complex (MHC) class I molecules, a panel of 11 monoclonal antibodies (mAbs) were analyzed. The mAbs reacted with bovine MHC class I antigens, as assessed by flow cytometry and immunoprecipitation followed by one- and two-dimensional gel electrophoresis. Analysis by flow cytometry revealed that class I molecules were expressed less on a class I mutant B-lymphoblastoid cell line than on the parent cell line. The relative molecular weights of the proteins identified by these mAbs were similar to those reported previously for cattle and humans. Nonequilibrium pH gradient two-dimensional gel electrophoresis showed that RH16C recognized four different class I gene products, indicating this mAb reacts with a conserved epitope present on different class I molecules. These mAbs effectively blocked cytotoxic T lymphocyte killing of allogeneic lymphoblasts, suggesting the functional importance of β-2m in this process. These mAbs should be useful reagents for studying bovine MHC class I molecules.

Alloreactive T‐cell recognition of bovine major histocompatibility complex class II products defined by one‐dimensional isoelectric focusing

T‐cell recognition of bovine MHC (BoLA) class II antigens was investigated in relation to BoLA class II polymorphisms defined by one‐dimensional isoelectric focusing (1D‐IEF). One‐way mixed lymphocyte reactions (MLRs), and allospecific cell lines and clones were used. In general, T‐cell responses correlated with the 1D‐IEF defined haplotypes (EDF types). However, with MLRs some responses appeared to be associated with BoLA class I differences. All combinations of responder‐stimulator pairs produced alloreactive T‐cell responses both in MLR and in generation of allolines/clones. Thus allospecific lines and clones were generated to all EDF types tested. Splits in the IEF typing were observed with EDF6 and EDF3, indicating that distinct BoLA class II haplotypes are not necessarily distinguished by 1D‐IEF alone. Furthermore, the patterns of reactivity with EDF3 expressing cells were complex with the T‐cell specificities splitting EDF3 into several distinct types. Also, in some cases it was clear that more than one T‐cell specifieity per EDF type was detectable. Thus, allospecific lines and clones provide complementary and additional information to the 1D‐IEF typing for polymorphism of the BoLA class II complex. This extra information is particularly important in terms of the functional significance of the BoLA complex for antigen presentation and immune response gene effects.

Biochemical evidence of the expression of two major histocompatibility complex class I genes on bovine peripheral blood mononuclear cells.

For a long time, the bovine major histocompatibility complex (MHC) (BoLA) class I region was characterized, rather uniquely among mammalian species, as having one expressed locus. Recent reports have suggested otherwise. Selective immunoprecipitation and molecular characterization of products enable a decisive answer to the question of whether there is indeed more than one locus expressed. Therefore, we characterized serologically defined w10 encoding haplotypes in European and African cattle by immunoprecipitation of [35S]-methionine-labelled peripheral blood mononuclear cells (PBMC), followed by one- and two-dimensional isoelectric focusing (1D/2D-IEF) of cell lysates. Monoclonal antibodies (mAb) used were directed against either human class I monomorphic determinants (W6/32 and B1.1G6) or bovine polymorphic determinants expressed on products encoded by serologically defined w10 encoding haplotypes of Boran and Friesian cattle. Sequential immunoprecipitations with W6/32 and B1.1G6 using lysates of PBMC of British Friesian cattle, revealed that from this haplotype W6/32 precipitated one product, whereas B1.1G6 precipitated two products. The product precipitated in addition appeared to be the one that was selectively precipitated by the mAb directed against polymorphic determinants on a product of w10 encoding haplotypes. Additionally, peptide maps of protease V8-digested precipitates showed that this particular 'w10' associated product was distinctly different from the product recognized by W6/32. Thus, we suggest that the two products are distinct gene products and that the product with higher pI is associated with the serologically defined A-locus product, whereas the product with lower pI is the putative second locus product. In the African Boran breed, variants of the serologically defined w10 specificity were found on the basis of IEF typing. These variants appeared to be associated with different second locus products. Therefore, we conclude that serologically defined w10 encoding haplotypes encode at least two independent class I locus products, expressed on normal bovine PBMC. In IEF analysis the additional use of mAb recognizing polymorphic determinants on serologically defined A-locus products highly facilitated the detection and typing of second locus products.

Alloreactive T‐cell recognition of bovine major histocompatibility complex class II products defined by one‐dimensional isoelectric focusing

T-cell recognition of bovine MHC (BoLA) class II antigens was investigated in relation to BoLA class II polymorphisms defined by one-dimensional isoelectric focusing (1D-IEF). One-way mixed lymphocyte reactions (MLRs), and allospecific cell lines and clones were used. In general, T-cell responses correlated with the 1D-IEF defined haplotypes (EDF types). However, with MLRs some responses appeared to be associated with BoLA class I differences. All combinations of responder-stimulator pairs produced alloreactive T-cell responses both in MLR and in generation of allolines/clones. Thus allospecific lines and clones were generated to all EDF types tested. Splits in the IEF typing were observed with EDF6 and EDF3, indicating that distinct BoLA class II haplotypes are not necessarily distinguished by 1D-IEF alone. Furthermore, the patterns of reactivity with EDF3 expressing cells were complex with the T-cell specificities splitting EDF3 into several distinct types. Also, in some cases it was clear that more than one T-cell specificity per EDF type was detectable. Thus, allospecific lines and clones provide complementary and additional information to the 1D-IEF typing for polymorphism of the BoLA class II complex. This extra information is particularly important in terms of the functional significance of the BoLA complex for antigen presentation and immune response gene effects.

Impairment of MHC class I transcription in a mutant bovine B cell line.

To better define the regulation and expression of bovine major histocompatibility complex (MHC) antigens, the bovine B lymphoblastoid cell line, BL3, was exposed to gamma-irradiation and surviving cells were immunoselected for MHC class I antigen loss. The resulting class I expression loss variant, BL3.1, was characterized at both the protein and genetic levels to ascertain the nature of the defect. Microfluorimetry analysis revealed a 3--5-fold surface density reduction of all class I products on BL3.1 cells relative to the parental BL3 cells. This decreased surface expression was specific for MHC class I and not for MHC class II or the non-MHC-linked gene product, immunoglobulin (Ig). Northern and quantitative slot blot analyses demonstrated a corresponding diminution of class I RNA in BL3.1 suggesting a transcriptional level defect. Nuclear run-off and transcription inhibition experiments confirmed no post-transcriptional changes while Southern blot analysis provided no evidence for alterations within or near the class I genes. To help elucidate the mechanism of altered class I expression, the parent, BL3, and variant, BL3.1, were cultured with factors known to enhance MHC class I transcription. Interferon (IFN)-gamma, lipopolysaccharide (LPS), and activated peripheral blood lymphocyte (PBL) supernatant cultured with both cell lines induced MHC class I transcription and surface expression 2--3-fold greater than the untreated controls. It is likely, therefore, that a genetic alteration outside of the class I genes has occurred within BL3.1 impairing expression of MHC class I.