牛MHC(BoLA)基因的研究进展

牛MHC(BoLA)不仅与移植排斥反应和免疫反应有关,而且还与一些经济性状有关.该文简述了BoLA的分布、结构、功能和基本特征,并且介绍了BoLA基因与主要经济性状如生产性能和疾病的关系,并对其作为动物育种遗传标记的应用前景进行了展望.认为MHC作为遗传标记,通过标记辅助选择进行抗病育种可以培育出高产、抗病力强的家畜品种.     

BoLA在牛抗病育种中的研究进展

BoLA基因同牛多种疾病和生产性状相联系,因此如何利用BoLA基因进行牛抗病育种是育种学家关心的问题,作者综述了BoLA基因同牛一些疾病的关系,并对BoLA基因进行牛抗病育种的现状及发展方向作了探讨。     

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.     

Homologies between the major histocompatibility complex of man and cattle: consequences for disease resistance and susceptibility

The major histocompatibility complex (MHC) of mammals contains a large number of mostly duplicated genes. In the HLA system (the MHC of man), which is by far the best-studied major histocompatibility system so far, roughly 20 genes have been defined and mapped. They code for three classes of proteins: HLA-A, -B and -C (Class I), HLA-DP, -DQ and -DR (Class II) and serum complement components C2, C4 and Bf (Class III). Furthermore, the region contains genes for 21-hydroxylase (21-OH) and tumor necrosis factor (TNF). The MHC thus forms a chromosomal segment containing several clusters of genes of only partially defined biological significance, but ondoubtedly playing a role in disease susceptibility. In view of the recently obtained structural information on BoLA, the MHC of cattle, it is hypothesized that susceptibility to diseases in cattle is associated with BoLA in the same way as human diseases. Finally, new technical and conceptual developments in the field of MHC research and their application to the BoLA system are discussed.     

BoLA class I polymorphism and in vitro immune response to M. bovis antigens

SUMMARY: From a sample of 119 Friesian calves, serologically typed for BoLA class I, 47 subjects were chosen expressing 9 different MHC types (A6, A6.9, A10, A11, A14, A15, A30, W16, M103) with the same age and reared in the same farm conditions. The animals were s.c. injected with a water in oil suspension of killed M. bovis and the treatment was repeated two days later. Before the treatment and 21 days later, calves were bled and on PBM (peripheral blood mononuclear leucocytes) were performed the following tests: 1. Lymphocyte Stimulation with bovine and avian PPDs (Purified protein derivative of Mycobacterium bovis and Mycobacterium avium, respectively). 2. Phagocytic activity towards M. bovis. 3. Class II molecules expression on cell surface. 4. Percentage of leucocyte populations and subpopulations. In the in vitro Lymphocyte Stimulation test, all the animals and classes were responders. Animals bearing A10 BoLA class I presented c.p.m. (counts per minute) and index values higher than the other cattle; these values were significantly positively related both to bovine and avian PPDs (P < .01). By variance analysis A14 BoLA type showed a slight positive significant correlation with more efficient phagocytic activity. BoLA class I type did not seem to significantly affect percentage of class II positive cells and leucocyte percentages on PBM. ZUSAMMENFASSUNG: Der BoLA Klasse I Polymorphismus und in vitro immunologische Antwort gegen die Antigene von M. bovis Aus einer Stichprobe von 119 für BoLA Klasse I serologisch typisierten Friesian K?lber, wurden 47 Subjekte ausgew?hlt, die 9 verschiedene MHC Typen ausdrückten (A6, A6.9, A10, A11, A14, A15, A30, W16, M103). Alle waren gleich alt und in gleichen Haltungsbedingungen. Die Tiere wurden mit einer Wasser-in-Ol Suspension abget?teter M. bovis subkutan injiziert und die Behandlung nach zwei Tagen wiederholt. Vor und 21 Tage nach Behandlung wurden die folgenden Tests ausgeführt: 1. Lymphozyten-Stimulationstest mit bovinen und Geflügel PPDs. 2. Phagozyten Aktivit?t gegen M. bovis. 3. Zeil-Oberfl?chen, Expression der Klasse II Moleküle. 4. Anteile der Lymphozyten Populationen und Subpopulationen. Im in vitro Lymphozyten-Stimulationstest waren alle Tiere und Klassen responder. Tiere mit A10 BoLA I zeigten h?here c.p.m. und Indexwerte als die anderen; diese Werte waren in signifikant positiver Beziehung mit der PPD von M. bovis und auch mit M. avium (P < .01). BoLA Typ A14 zeigte leicht signifikant positive Korrelation mit wirksamerer Phagozyten Aktivit?t. BoLA Klasse I Typ scheint nicht den Prozentsatz der positiven Zellen der Klasse II und der Leukozyten der PBM signifikant zu beeinflussen. RESUMEN: Polimorfismo de BoLA clase I y immunidad a los antigenos del M. bovis Se escojeron 47 novillos dentro de un grupo de 119 animales que segun analisis previamente hecha tenian BoLA de clase I. Estos 47 novillos fueron escojidos de manera que tuvieran 9 distintos tipos de MHC (A6, A6.9, A10, A11, A14, A15, A30, W16, M103), la misma edad, las mismas condiciones de cria. Estos animales fueron inoculados subcutaneo con M. bovis matados en una suspension oleosa y la misma inoculacion fue repetida una secunda vez despues de dos dias. Por cada animal se tomaron muestras de sangre antes y 21 dias despues de la inoculacion de arriba. Las muestras de sangre fueron pruebaoas con: 1. Stimulacion Lymhocitaria con PPD bovina y avicola. 2. Actividad phagocitaria a M. bovis. 3. Expresion sobre la superficie celular de moleculas de clase II. 4. Porcentaje de poblaciones y de subpob-laciones de leucocitos. Todos los animales y todos los tipos de MHC dieron respuestas positivas en las pruebas de Stimulacion Lymphocitaria. Los animales que tenian la BoLA A10 presentaron valores de c.p.m. y indices mas altos de los demas animales. Estos valores se encontraron significativamente y positivamente relacionados sea a la PPD bovina que a la PPD avicola. Por medio de la analisis de varianzas se encontro que el tipo BoLA A14 muestraba una correlacion significativa y algo positiva con una mejor actividad fagocitaria. Los tipos de clase BoLA I no parecieron que influenzaran de manera appreciable el porcentaje de positividad por la clase II y el porcentaje de leucocitos en la sangre PBM.     

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

SUMMARY: 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: M?gliche Assoziation zwischen dem bovinen Haupthistokompatibilit?tskomplex und subklinischer Mastitis In dieser Studie wurde die Beziehung zwischen dem bovinen Histokompatibilit?tskomplex (BoLA) und der Pr?valenz 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 Spezifit?ten typisiert. Die statistische Auswertung für den UHS erfolgte mit einem logistischen Regressionsmodel. Der Einflu? 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 Einflu? 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 Einflu? des Laktationsstadiums festgestellt (P < 0.0001 resp. P < 0.13).     

Homologies between the major histocompatibility complex of man and cattle: Consequences for disease resistance and susceptibility

Summary

The major histocompatibility complex (MHC) of mammals number of mostly duplicated contains a large genes. In the HLA system (the MHC of man), which is by far the best‐studied major histocompatibility system so far, roughly 20 genes have been defined and mapped. They code for three classes of proteins: HLA‐A, ‐B and ‐C (Class I), HLA‐DP, ‐DQ and ‐DR (Class II) and serum complement components C2, C4 and Bf (Class III). Furthermore, the region contains genes for 21‐hydroxylase (21‐OH) and tumor necrosis factor (TNF).

The MHC thus forms a chromosomal segment containing seva‐al clusters of genes of only partially defined biological significance, but ondoubtedly playing a role in disease suscepti‐ bility. In view of the recently obtained structural information on BoLA, the MHC of cattle, it is hypothesized that susceptibility to diseases in cattle is associated with BoLA in thesame way as human diseases.

Finally, new technical and conceptual developments in the field of MHC research and their application to the BoLA system are discussed.     

Association of the bovine leukocyte antigen major histocompatibility complex class II DRB3*4401 allele with host resistance to the Lone Star tick, Amblyomma americanum

The MHC of cattle, known as the bovine leukocyte antigen (BoLA) complex, plays an integral role in disease and parasite susceptibility, and immune responsiveness of the host. While susceptibility to tick infestation in cattle is believed to be heritable, genes that may be responsible for the manifestation of this phenotype remain elusive. In an effort to analyze the role that genes within the BoLA complex may play in host resistance to ticks, we have evaluated components of this system within a herd of cattle established at our laboratory that has been phenotyped for ectoparasite susceptibility. Of three microsatellite loci within the BoLA complex analyzed, alleles of two microsatellite loci within the BoLA class IIa cluster (DRB1-118 and DRB3-174) associated with the tick-resistant phenotype, prompting further investigation of gene sequences within the DRB3 region. DRB3 is a class IIa gene, the second exon of which is highly polymorphic since it encodes the antigen recognition site of the DR class II molecule. Analysis of the second exon of the DRB3 gene from the phenotyped calves in our herd revealed a significant association between the DRB3*4401 allele and the tick-resistant phenotype. To our knowledge, this is the first report of a putative association between a class IIa DRB3 sequence and host resistance to the Lone Star tick. Elucidation of the mechanism involved in tick resistance will contribute to improving breeding schemes for parasite resistance, which will be beneficial to the cattle industry.     

The relationships among ecto- and endoparasite levels, class I antigens of the bovine major histocompatibility system, immunoglobulin E levels and weight gain

Natural infestations of the cattle tick Boophilus microplus, levels of the buffalo fly Haematobia irritants exigua and faecal nematode egg concentrations (Bunostomum phlebotomum, Cooperia spp., Haemonchus placei, Oesophagostomum radiatum and Trichostrongylus axei) were assessed in 221 Belmont Red calves during the post-weaning period, when the animals were between 9 and 18 months of age. In addition, the 98 males of this group were challenged with B. microplus larvae on two separate occasions. There were strong positive correlations among replicate assessments of the same parasite. Field tick counts and tick counts following deliberate challenge were strongly correlated, and both showed negative correlations with post-weaning weight gain. There was a weak positive correlation between buffalo fly counts and post-weaning weight gain. There was a negative correlation between total worm egg count and weight gain. Among worm species, only the effect of O. radiatum on weight gain was significant. Cattle with bovine major histocompatibility (BoLA) antigens W6.1 and W7 had significantly fewer ticks than cattle lacking these antigens. Cattle with BoLA antigens W7 and CA36 had lower concentrations of nematode eggs in their faeces than cattle lacking these BoLA antigens.     

BoLA-DRB3 exon 2 mutations associated with paratuberculosis in cattle

A single nucleotide polymorphism at the antigen recognition site of the bovine leucocyte antigen (BoLA) DRB3 gene was assessed in healthy and Mycobacterium avium subsp. paratuberculosis (MAP) - infected cattle, in order to determine if there was a correlation between mutations and altered susceptibility to infection. Of a sample of 200 animals, 19.6% were found to be infected with MAP. PCR - single strand conformational polymorphism analysis of the BoLA DRB3 gene found 19 genotypes (16 in the heterozygous and three in homozygous state, respectively). Four mutations, Val53Glu (OR 453.7), Val53Leu (OR 453.7), Asp57His (OR 1.944) and Arg84Gly (OR 1.458), were linked with increased susceptibility to infection, whereas, Asp57Asn (OR 0) and Phe60Tyr (OR 0.453) were associated with increased resistance. The findings indicate potentially important mutations in the protein-binding site of DRB3, which may be crucial to the activation of an appropriate immune response against MAP.     

Studies on the major histocompatibility complex of indigenous cattle in the Ivory Coast

The MHC (BoLA) type has been determined for cattle from three breeds in West Africa. Seventy Baoule, 50 N'Dama and 30 Zebu cattle from the centre and north of the Ivory Coast were tested. Lymphocytes from these cattle were tested in a lymphocytotoxicity test with alloantisera detecting all of the internationally recognised BoLA sera. 78 sera prepared in Edinburgh and 57 in Jouy-en-Josas were used in the study. The results showed that sera prepared in Europe detect similar specificities in West Africa. Although with some specificities the frequencies differ from those seen in Europe. The frequency of null alleles is higher than in Europe in the Ndama and Zebu animals indicating the existence of additional specificities which will require the production of alloantisera in these breeds. However in the Baoule the null allele frequency is lower even then in some European breeds. The population data in which no animals have more than two workshop specificities is consistent with a single locus control in West Africa as in Europe.     

Genetic variation and responses to vaccines

Disease is a major source of economic loss to the livestock industry. Understanding the role of genetic factors in immune responsiveness and disease resistance should provide new approaches to the control of disease through development of safe synthetic subunit vaccines and breeding for disease resistance. The major histocompatibility complex (MHC) has been an important candidate locus for immune responsiveness studies. However, it is clear that other loci play an important role. Identifying these and quantifying the relative importance of MHC and non-MHC genes should result in new insights into host-pathogen interactions, and information that can be exploited by vaccine designers. The rapidly increasing information available about the bovine genome and the identification of polymorphisms in immune-related genes will offer potential candidates that control immune responses to vaccines. The bovine MHC, BoLA, encodes two distinct isotypes of class II molecules, DR and DQ, and in about half the common haplotypes the DQ genes are duplicated and expressed. DQ molecules are composed of two polymorphic chains whereas DR consists of one polymorphic and one non-polymorphic chain. Although, it is clear that MHC polymorphism is related to immune responsiveness, it is less clear how different allelic and locus products influence the outcome of an immune response in terms of generating protective immunity in outbred animals. A peptide derived from foot-and-mouth disease virus (FMDV) was used as a probe for BoLA class II function. Both DR and DQ are involved in antigen presentation. In an analysis of T-cell clones specific for the peptide, distinct biases to particular restriction elements were observed. In addition inter-haplotype pairings of DQA and DQB molecules produced functional molecules, which greatly increases the numbers of possible restriction elements, compared with the number of genes, particularly in cattle with duplicated DQ genes. In a vaccine trial with several peptides derived from FMDV, BoLA class II DRB3 polymorphisms were correlated with both protection and non-protection. Although variation in immune responsiveness to the FMDV peptide between different individuals is partly explainable by BoLA class II alleles, other genetic factors play an important role. In a quantitative trait locus project, employing a second-generation cross between Charolais and Holstein cattle, significant sire and breed effects were also observed in T-cell, cytokine and antibody responses to the FMDV peptide. These results suggest that both MHC and non-MHC genes play a role in regulating bovine immune traits of relevance to vaccine design. Identifying these genes and quantifying their relative contributions is the subject of further studies.     

The relationships of birth weight, preweaning gain and postweaning gain with the bovine major histocompatibility system

A total of 739 cattle from nine breeds maintained at the Roman L. Hruska U.S. Meat Animal Research Center, Clay Center, Nebraska were tested for 42 class I antigens of the bovine major histocompatibility system (BoLA). Each antigen appears to be the product of a distinct co-dominant allele of the BoLA-A locus. The number of antigens present in each breed ranged from a minimum of 10 in Hereford to a maximum of 21 in Charolais cattle. There were large differences among breeds in the frequencies of antigens. The effect of each antigen on birth weight, preweaning weight gain and postweaning weight gain was estimated in a gene substitution model. Each breed was analyzed separately. There were significant effects of some BoLA antigens on birth weight, preweaning weight gain and postweaning weight gain, which is consistent with previous reports showing associations between the major histocompatibility system and growth parameters in mice, rats and pigs. However, further research is necessary to confirm these findings and to determine the biological mechanisms underlying these associations.     

Association of class I bovine lymphocyte antigen complex alleles with in vitro blood neutrophil functions, lymphocyte blastogenesis, serum complement and conglutinin levels in dairy cattle

Ninety-eight lactating Holstein cows from two genetic lines selected for high and average milk production were used in the study. Five peripheral blood samples were collected over a 60-day period from each cow for evaluation of neutrophil function, lymphocyte blastogenesis, leukocyte count, and serum complement and conglutinin levels. Blood samples were typed for antigens encoded by alleles at the bovine major histocompatibility complex (BoLA) A locus. Alleles w14(w8), w20A, and w19(w6) were the most frequent of 14 alleles present in this herd. Association of BoLA type with immune function results was examined by using gene substitution models including and ignoring sire effects. Alleles w15(w8) and w16 were associated with greater circulating mononuclear cell and total leukocyte numbers, while w27(w10), w11, and w20A were associated with lower numbers of these cell types. Alleles EU28D and w20A were positively and negatively associated with granulocyte percentage, respectively. Allele w16 was associated with greater antibody-independent neutrophil cytotoxicity, unstimulated lymphocyte proliferation, serum conglutinin activity, and with lower antibody-dependent neutrophil cytotoxicity. Allele w19(w6) was associated with decreased conglutinin activity and decreased neutrophil iodination. Increased antibody-dependent neutrophil cytotoxicity was observed for animals bearing allele w14(w8), and decreased neutrophil iodination, serum conglutinin, and nonstimulated lymphocyte blastogenesis were observed in individuals carrying w20A or EU28D. Significance of both sire and BoLA complex effects suggests that both major histocompatibility complex genes and background genes of the sire significantly affect immune function. This research suggests BoLA-A locus genes may be major genes or markers for closely linked major genes involved in regulation of nonspecific immune function.     

The use of binding-prediction models to identify M. bovis-specific antigenic peptides for screening assays in bovine tuberculosis

The identification of MHC class II-restricted antigenic peptides for inclusion into vaccines and/or as diagnostic test reagents for mycobacterial infections remains a high research priority. To expedite discovery of such peptides, numerous bioinformatic tools have been developed to predict whether a given peptide is likely to form a stable binding interaction with MHC class II molecules. However, no prediction tool dedicated to the identification of bovine MHC (BoLA) class II-restricted peptides is currently available. Using experimental immunogenicity data derived from the stimulation of whole blood of Mycobacterium bovis-infected cattle with 105 individual M. bovis-derived peptides, we have compared the ability of a novel BoLA DRB3 structure-based prediction method (Hepitom) with the human MHC class II binding predictor model ProPred in predicting peptides that induce bovine T-cell activation. When a stringent cut off for considering peptide antigenicity was applied, the sensitivities of Hepitom and ProPred in detecting immunogenic peptides were 62% and 77%, respectively. In contrast, the Hepitom model showed greater specificity, with values of 66% and 34% for Hepitom and ProPred, respectively. Using all peptides, seven out of eleven M. bovis proteins were identified as being highly immunogenic. All but one of these antigens were also identified when just the Hepitom predicted peptides were used, while only four of the seven were identified using the ProPred predicted peptides. In conclusion, we demonstrate that the Hepitom model is a useful pre-screening tool to select peptides for further immunogenicity studies in cattle without major impact on the identification of antigenic M. bovis proteins.     

Study on the association of BoLA-DRB3.2 alleles with clinical mastitis in Norwegian Red cows

Genotyping of bovine leucocyte antigen DRB3.2 (BoLA-DRB3.2) in a total of 523 Norwegian Red (NR) cows from two groups selected for high protein yield and low clinical mastitis, respectively, identified 27 previously reported BoLA-DRB3.2 alleles across the groups. Significant differences in BoLA-DRB3.2 allele frequencies were found between the selection groups. Alleles *13, *18, *22 and *27 had a significantly higher frequency in cows selected for low clinical mastitis, while alleles *3, *9, *11 and *26 had a higher frequency in cows selected for high protein yield. Associations between BoLA-DRB3.2 alleles and clinical mastitis were analysed based on mastitis data from 741,072 first-lactation NR cows, of which 452 were genotyped. Alleles *22 and *26 were found to be associated with increased clinical mastitis, while alleles *7, *11, *18 and *24 had a favourable effect on mastitis resistance. Contradictory results from different studies investigating associations between BoLA-DRB3.2 alleles and mastitis indicate that future studies should focus on associations of mastitis with BoLA haplotypes rather than with single BoLA genes.     

Use of bovine lymphocyte antigens in diagnosis of parentage

Lymphocyte antigens controlled by alleles at the BoLA-A locus were used to indicate which of two bulls could be eliminated as the sire in 18 cases of disputed parentage. The use of bovine lymphocyte antigens (BoLA) significantly increased the exclusion probability over that of the standard red cell and electrophoretic techniques.     

Recombinant bovine interferon-gamma enhances expression of class I and class II bovine lymphocyte antigens

Recombinant bovine interferon-gamma augments expression of class I and class II histocompatibility antigens on the surface membrane of bovine lymphocytes. Immunofluorescence techniques using a series of monoclonal anti-HLA antibodies demonstrate that this enhancement is detectable as early as 24 h after incubation with rBoIFN, while maximum surface expression is obtained within 3-5 days. A concentration as low as 10 units of rBoIFN is effective. Such results may be useful for characterizing the BoLA gene products.     

The major histocompatibility complex of West African cattle. Typing of lymphocyte antigens (BoLA) of Baoulé cattle (Bos taurus) and Sudanese zebus (Bos indicus) of Burkina Faso (West Africa)

Lymphocyte antigen (BoLA) typing on 247 Baoule cattle (Bos taurus) and 106 Sudanese zebus (Bos indicus), allowed us to determine gene frequencies of 43 class 1 specificities, as international "W" and European "EU", as African local from Kenya "KN" and Burkina Faso "BF". In comparing these frequencies, it appears that some specificities could be considered as significant breed markers for Baoule cattle and zebus.     

Selected phenotypic and cloning properties of a bovine lymphoblastoid cell line, BL20

A bovine lymphoblastoid cell line, BL20, was shown to express a BoLA antigen and surface IgM, implying that it was probably of B-cell origin. At low density (less than 10(5)/ml), the cells failed to grow. Inclusion of growth factor-containing supernatants from concanavalin A or pokeweed mitogen (PWM)-activated bovine lymphocytes or from thymus fibroblast-like cells did not improve cloning of the cells. Feeder cells also did not enhance cloning of the cells except for bovine thymus fibroblast-like cell monolayer.