European Journal of Immunogenetics (1992), 19, 253-262

C H A R A C T E R I Z A T I O N OF BOVINE MHC CLASS I1 POLYMORPHISM USING T H R E E TYPING M E T H O D S : S E R O L O G Y , RFLP AND IEF

C . J . D A V I E S , * ’ ~ L . A N D E R S S O N , ~ I . JOOSTEN,* P . M A R I A N I ~ ’ ~ L . C . G A S B A R R E * & E . J . HENSEN*

*Helniinthic Disease Laboratory, Livestock and Poultry Sciences Institute, Agricultural Research Service, United States Department of Agriculture, Beltsville, MD, USA, ‘Department

of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsaia, Sweden, and ‘Institute of Infectious Diseases and Immunology, Department of Immunology,

Faculty of Veterinary Medicine, University of Utrecht, Utrecht, The Netherlands

(Received 5 February 1992; accepted 29 February 1992)

S U M M A R Y

Various methods. with different strengths and weaknesses, are currently used to define polymorphism of the bovine major histocompatibility complex (MHC) class I1 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 I1 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 I1 typing methods recognized polymorphism associated with the same or very tightly linked genes in the DQ-DR class I1 subregion. In total 25 BOLA-A locus (class I) - DQ-DR subregion (class 11) haplotypes were defined. Three of the serological class I1 specificities, DxI, Dx3. and Dx4, were associated with more than one RFLP defined DQ-DR haplotype. The other 4 class I1 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.

Correspondence: Dr Christopher J . Davies, Department of Animal Breeding, Wageningen Agricultural

‘Present address: Departmcnt of Animal Breeding, Wageningen Agricultural University, Wageningen,

”Permanent address: lnstituto di Zootecnica, Facolta di Medicina Veterinaria. Universith degli Studi di

University, P.O.B. 338,6700 A H Wageningen, The Netherlands.

The Netherlands.

Milano, Milano, Italy.

253

254 C. J. Davies et al.

I N T R O D U C T I O N

The major histocompatibility complex (MHC) contains two groups of highly polymorphic genes, the class I and class I1 genes. The proteins encoded by these genes play a central role in the regulation of T-cell immune responses. The bovine MHC is known as the bovine lymphocyte antigen (BoLA) complex. Alleles of the class I, BOLA-A, locus can be readily identified serologically (Caldwell et al . , 1977; Amorena & Stone, 1978; Spooner et a l . , 1978; Bernoco et a l . , 1991). Characterization of BOLA complex class I1 polymorphism has proved more difficult. Five different methods of BOLA class I1 typing are currently in use: mixed lymphocyte culture (MLC) typing (Davies & Antczak, 1991a); T-cell typing with T-cell lines and clones (Teale & Kemp, 1987; Rothel et at., 1990); restriction fragment length polymorphism (RFLP) typing (Sigurdar- dottir et al . , 1988); typing by immunoprecipitation and isoelectric focusing (IEF) (Joosten et a l . , 1989); and serology (Mackie & Stear, 1990; Arriens et a l . , 1991; Davies & Antczak, 1991b; Williams et al . , 1991).

At the DNA level two BoLA class I1 subregions have been identified (Anderson et al., 1988). The first subregion contains one DRA, three to five DRB, one to two DQA, and one to two DQB genes (Anderson et al., 1986a,b; Anderson & Rask, 1988) and encodes the DR and DQ gene products that are detected on the cell surface (Lewin et al., 1985; Joosten et al . , 1990; Davies & Antczak, 1991b). The second subregion contains the DOB, DNA, DYA, and DYB genes and is separated from the class I region and the DQ-DR subregion by a high recombination frequency (estimated at 0.17 k 0.07; Anderson et a l . , 1988). The recently described class I1 gene DZB is most likely located in the DO-DY subregion since it is separated from the DQ-DR subregion by a similar high recombination frequency (Stone & Muggli-Cockett, 1990). To date no gene products encoded in the second class I1 subregion have been identified. Consequently, in this paper the term BoLA haplotype will refer to genes in the class I region and the DQ-DR class I1 subregion.

The three most frequently used methods for class I1 typing are RFLP, IEF, and serology. These class I1 typing methods have different strengths and weaknesses. The methods are highly complementary: serological class I1 typing is ideal for population studies; RFLP typing is very useful for the characterization of BOLA haplotypes; and IEF is a powerful technique for studying expressed polymorphism. In the studies presented here we have compared these three methods and have used them to characterize a number of BoLA haplotypes in two different breeds of cattle.

M A T E R I A L S A N D M E T H O D S

Animals Populations were screened using class I and class I1 serology and individuals expressing an array of well defined haplotypes were selected for characterization by RFLP and IEF. Twenty five Holstein-Friesian cattle from the Cornell Teaching and Research Center dairy herd in Harford, New York; fifteen Angus cattle from the Wye Research and Education Center in Queenstown, MD; and one BoLA complex homozygous Holstein-Friesian steer from MD were studied. This group of animals included BoLA complex homozygotes representing five Angus and six Holstein haplotypes.

Serology The complement mediated lymphocyte microcytotoxicity assay was used for class I and class I1 BoLA typing (Terasaki et al., 1978). The antisera and methods used for the serological BoLA

Characterization of bovine M H C class II polymorphism 255

typing have been described previously (Davies & Antczak, 1991a,b). Antisera defining seven class I1 specificities ( D x l , Dx2, Dx3, Dx4, Dx5, Dx8, and Dx9) were used. However, the antisera for Dx8 and Dx9 were not available when the studies involving the Cornell Holstein cattle were conducted. Class I1 typing was performed on plastic adherent B lymphocytes isolated by the panning method (Davies & Antczak, 1991b). The terminology used for the BoLA class I antigens is that presented in the report of the Fourth International BoLA Workshop (Bernoco et a i . , 1991). The terminology used for the BoLA class I1 antigens has been described by Davies & Antczak (1991b).

RFLP analysis

DNA isolation, restriction enzyme digestion, and Southern blot analysis were carried out as previously described (Andersson et al., 1986a; Andersson, 1988). Two restriction enzymes, PvuII and TaqI, were used. For the group of 25 Holsteins from New York, human cDNA probes for DRA (Larhammar etal., 1982a); DRB (Gustafsson etal., 1984), D Q A (Schenningetal., 1984) and DQB (Larhammar etal., 1982b) were used. For the 15 Angus and 1 Holstein from Maryland, bovine genomic probes for DRB, D Q A , and DQB were used (Groenen et al., 1990; van der Poel et al., 1990). It has been shown that the same D Q and DRB alleles can be defined with human or bovine probes (Sigurdardottir et al., 1991b). The nomenclature proposed by Sigurdardottir et al. (1998) has been used for the D R and D Q RFLPs.

Isoelectric focusing

Cell lysates from biosynthetically labelled lymphocytes were immunoprecipitated with a mouse anti-bovine class I1 monoclonal antibody, IL-A21 (a gift of A. J. Teale), and analysed by one-dimensional IEF as previously described (Joosten et al., 1988,1989). The D-region Focusing ( D F ) nomenclature proposed by Joosten etal. (1989) has been used for the IEF banding patterns.

R E S U L T S

Typing results

Typing results for the Cornell Holstein cattle and the Wye Angus cattle plus one Holstein steer are presented in Tables 1 and 2, respectively. Serologically defined class I-class I1 haplotypes for the Cornell Holsteins (Davies & Antczak, 1991b) and the Wye Angus (C. J . Davies, unpublished) were established in family studies.

The cattle selected for the comparison studies included BoLA complex homozygotes and individuals with different combinations of haplotypes. The cattle were originally characterized using class I and class I1 serology. The genetic make up of the panel selected for RFLP and IEF analysis, especially the inclusion of BOLA complex homozygotes, made it possible to establish the RFLP and IEF patterns that were associated with particular haplotypes. A summary of the haplotypes defined is presented in Table 3. The mixed lymphocyte culture (MLC) designation assigned to some of these haplotypes (Davies & Antczak, 1991a) is also given.

Three of the serological class I1 specificities, D x l , Dx3, and Dx4, behaved as public specificities. Each of these specificities was associated with more than one RFLP defined DQ-DR haplotype. The remaining class I1 specificities, Dx2, Dx5, Dx8 and Dx9, were associated with a single RFLP defined haplotype or in the case of Dx8 with two closely related haplotypes. These two haplotypes had identical DQ RFLP patterns and identical IEF patterns but different DRB RFLP patterns.

256 C. J . Davies et al. TABLE 1. Serological and RFLP typing results for Cornell Holstein cattle

RFLP typing

ID no. Serological typing DQ DRA DRB

Boll A1 1 - D ~ 3 l A l I -0x3 I A I I A Ill l A l l A B281 A6-Dx l IA l l - D x ~ 9AI lA 211 9ai1a B502 A6-DxIIA6- D x ~ 9Al2 212 9ai2a B505 A6- DxIIA6-Dx3 9AI lA 211 9a11a B563 A6- D x l l A I l -DxS 9Al13C 212 9a14a B575 A6- DXllAlO- D x ~ 9Al6A 215 9a16 B576 A6-DxIIA6- Dx l 9Al9A 212 9a19a B578 A6- DxllAlO- D x ~ 9Al6A 21s 9a16 B579 A6-DxIIA6-Dx2 9Al2 212 9a12a B580 A ~ - D x ~ I A I O - D X ~ 2l6A 21s 2a16 B581 A6- Dx2IAlO- Dx4 2I6A 215 2 A16 B.582 A6- Dxl lA6- D x ~ 9Al2 212 9a12a B595 A6- DxllAlO- Dx4 9Al6A 21s 9a16 B602 A I O - D X ~ I A ~ O - D X ~ 6Al6A 515 616 B641 AlO-Dxl lwl- D x ~ I l ' I 7A 214 11'17A B678 A6-Dxllwl-Dx3 9Al7A 214 9a17a B695 A6- Dxl lA l4- D x ~ 9Al1 B 214 9All B B766 AZO-Dxl f Dx3lA31-DxS 12113C 212 12Bl4A B768 AIO-DX~IA I 0- D x ~ 6Al6A 515 616 B773 A ~ I - D X ~ I A ~ ~ - D X S 13Cl13C 2 12 4a14a B868" A6110 Dxl,Dx3 121118 212 12BI l lA 8897 A20- DxI f Dx3IA20- Dxl f D x ~ 12112 212 12 8112 B 8927 A6- Dxllw44- D x ~ 10l2 312 1012a B928 ~ 4 4 - DxZIA31 - D x ~ 2113C 212 2a14a B933 ~ 4 4 - D ~ 2 l A l l - D x ~ 2 l lA 211 2a11a

'' The class I-class I1 haplotypes for this cow are not known.

TABLE 2. Serological, RFLP and IEF typing results for Wye Angus cattle and one BOLA complex homozygous Holstein

DRB RFLP IEF DQ RFLP ID no. Breed Serology

B0957 Angus A9-Dx4lA30-Dx9 1617 Sl4A 5 B l l l A B0966 Angus A35-Dx9IA3S- Dx9 71 7 4Al4A 11 A l l I A

B0976 Angus A9- Dx4IA 9- D x ~ 16/16 51.5 5 B15 B B1056 Angus A3-Dx2IA35- D x ~ 216 2 l l l C 2 A l l l C B 1083 Angus A2-DxZIA35- D x ~ 216 2l11C 2Al l IC

B1103 Angus A24- Dx2IBlank- Dx2 212 212 2A12A B1106 Angus A1 1 - D X ~ / A ~ O - D X ~ 416 I A I I I C l A l l l C B1232 Angus A1 1 - Dx3IA23- Dx l 4113 1Al9A 1a19c B1233 Angus A2-Dx2IA3S-Dx9 217 214A 2 A l l l A B1245 Angus A30- D x ~ I A ~ O - D X ~ 71 7 4Al4A I1 A l l I A B1307 Angus A24-Dx2IA30-Dx9 217 214A 2 A I I l A B1309 Angus A2- D~3lA3-Dx2 412 I A12 I A R A B1351 Angus A1 1- Dx3IAl I-Dx3 414 I A I I A I A I I A B 1684 Hol. A12-Dx8IA12-Dx8 616 I lCI l lC l l A l l l A

B0968 Angus A3S- Dx8IA3.5- Dx8 616 I 1 CII I c I l C I I l C

BllOl Angus A3O- Dx8IA23- Dx l 6/13 11 Cl9A l lCI9C

Characterization of bovine M H C class 11 polymorphism 257 TABLE 3 . Summary of BoLA haplotypes defined in Holstein and Angus cattle

Serological typing RFLP typing

Class I Class I1 MLC IEF DQ DRA DR B Breed

A6 A6 A10 A23 A 2 A 3 A6 A24 w44 Blank A 2 A6 A l l A l l A14 wl A 9 A10 A l l A31 A20 A12 A30 A35 A30 A3S

~~

Dx1 Dxl Dxl Dx1 Dx2 Dx2 Dx2 Dx2 Dx2 Dx2 DX3 Dx3 Dx3 Dx3 Dx3 Dx3 Dx4 Dx4 Dx5 Dx.5

Dxl+ Dx3 Dx8 Dx8 Dx8 Dx9 Dx9

- 0 4 D5 D.5 0 7 - -

0 8 D9 D9

I " 6"

13 2 2 2 >' 2 2 <' 2 4 4'' 4" 4 7.'

10.' 16 9 "

-

- - -

6 6 6 7 7

9A I0 11' 9A 2 2 2 2 2 2

I A I A 1A I A I B 7A 5

6 A 13C 13C 12

I I C I I C I IC 4A 4A

2 3 2

- 2

4 4

5 2 2 2

-

9A Hol 10 Hol 11' . Hol 9 c Ang 2A 2A Ang 2A Hol 2A Ant? 2A Hol 2A Ang 1A A % IA Hol I A Hol

1B Hol 7A Hol

6 Hol 4A Hol 4A Hol

128 Hol I I A Hol

IA Ang

5 B Ang

1IC Ang IIC *ng IIA Ang I I A Ang

'I Inferred from the RFLP defined DQ-DR haplotype on the basis of a previous study (Joosten el a l . , 1990).

IEF typing was only done on the Angus cattle and the Holstein steer from Maryland. Because these animals exhibited limited polymorphism, the class I1 specific antisera behaved as monomorphic reagents and the ability of the IEF technique to split the public serological specificities could not be assessed. Nevertheless, IEF typing information inferred from the earlier study by Joosten et al. (1990) implies that the IEF technique would split the three public specificities: Dxl, Dx3 and Dx4 (Table 3).

New R FL P types

DQ and DRB types have previously been described by Sigurdardottir et al. (1988), Joosten et al. (1990) and Bernoco et al. (1991). Two new DRB types were documented in the present study. DRBYC, associated with DO9*, consisted of TaqIfragments of6.0+4.1(?)+1.6+1.3+0.9kb; the question mark for the 4.1 kb fragment indicates that its presence could not be determined with confidence. DRB"', associated with DQ"', consisted of TaqI fragments of 6.0+4.1+3.3+1.6kb. Furthermore, although the DQ'3c RFLP pattern was described in the recent BoLA workshop report (Bernoco et al., 1991), the associated DRB pattern could not be determined as only a few heterozygous animals were available. The present study included one BoLA complex homozygous Holstein cow with this haplotype and the DRB RFLP pattern was

258 C. J . Davies et al. determined to consist of TaqI fragments of 5.4+3.7+2.8+2.1 kb. This RFLP type is identical to the DRB4A type previously found in the Swedish Red and White breed (Sigurdardottir et al., 1988). Thus, the haplotype in question was designated DQ'3"DRB4A. The DRB'" RFLP type ascribed to two Angus cattle in the present study, was associated with DQ' and consisted of TaqI fragments 8.4+2.8+1.9+1.6. This DRB RFLP type is most likely identical to the DRB'" type previously found at a very low frequency in the Swedish Red and White breed (Sigurdardottir et al., 1988). A new subtype of the DQ",DRB" haplotype was also found. Unfortunately, this haplotype was present in a single heterozygous Holstein cow and the fragment pattern could not be determined with confidence. Consequently, it was designated DQ"',DRB"'. In addition to the new DQ-DR haplotypes containing novel alleles, one 'recombinant' haplotype, DQ4A- ,DRB1IA, was identified in the Angus breed. This haplotype may have arisen by a recombination event between the haplotypes DQ4A,DRB4A and DQllA.B,CorD ,DRB1IA previously found in other breeds (Sigurdardottir e ta / . , 1988).

New IEF types

Two new IEF types were found. These IEF types were associated with RFLP DRB types not previously analysed by IEF. The new IEF types have been provisionally designated DF13 and DF16. Final acceptance of DF13 and DF16 is contingent on confirmation of their banding patterns in an independent study.

D I S C U S S I O N

The present study provides strong evidence that the three class I1 typing methods, cerology. IEF and RFLP, define polymorphism associated with the same or very tightly linked genes, i.e. the DQ and DR genes. Evidence that none of the methods defines polymorphism encoded in the class I region is also provided.

Three associations between IEF and RFLP types demonstrated in the current study (DF? with D@,DRBZA; DF4 with DQIA,DRBlA; and DF6 with DQ"C,DRB"A) confirm associations established previously (Joosten et al., 1990). In addition many of the associations demonstrated in the current study, or inferred on the basis of the study by Joosten eta/ . (1990), were identified in the Fourth International BoLA Workshop (Bernoco et al . , 1991). For the 54 animals from 5 breeds tested in the BoLA Workshop the following associations identified in the current study had correlation coefficients equal to or greater than 0.50: Dx2 with D@, DRB2A and EDF9 (equivalent to D n ) ; Dx4 with D@ and DRB'"; Dx4 with DQ6A and DRB6; Dx5 with DQIiC; Dx8 with DQ"' and EDF4 (DF6); EDFI (DF9) with DQbA and DRB6; EDF? (DFIO) with DQ7A and DRB7A; EDF7 (DF4) with DQ'" and DRBIA.

Exceedingly strong linkage disequilibrium exists in the DQ-DR class I1 subregion. Evidence for this is provided by the fact that 9 of the 15 DQ-DR haplotypes identified in the current study have also been found in other breeds (Sigurdardbttir eta!. , 1988; Joosten et al . , 1990; Bernoco et al., 1991). This very strong linkage disequilibrium makes it difficult to determine if particular class I1 specific antisera recognize the products of the DQ or DR genes or are anti-haplotype reagents. Nevertheless, because of the very strong linkage disequilibrium, even DQ-DR haplotype specific antisera are useful typing reagents.

Both class I and class I1 typing are needed to define BoLA haplotypes. This has been shown previously (Lindberg & Anderson , 1988; Davies & Antczak, 1991a,b) and is demonstrated again in the present study. The existence of haplotypes with different combinations of class I , BOLA-A

Characterization of bovine MHC class I I polymorphism 259 locus, alleles and class I1 DQ-DR haplotypes provides evidence that over time there has been significant recombination between the class I and class I1 regions. Nevertheless, certain common BoLA haplotypes are found in several breeds. The present study provides evidence that the BOLA-A11 ,Dx3,DQlA,DRBiA,DF4 haplotype exists in both Holstein and Angus cattle. The identity of this haplotype in Holsteins and Angus has also been confirmed with the MLC assay (C. J. Davies, unpublished). In addition there is evidence that this haplotype exists in Ayrshire cattle (Bernoco er al. , 1991). Two other BoLA haplotypes described here have been identified in more than one breed: BoLA-A2,Dx2,D(22,DRBA,DF2 in Angus, Swedish Red and White, Norwe- gian Red and Ayrshire cattle; and BoLA-AIO,DX~,DQ~~,DRB~,DF~ in Holstein and Swedish Red and Whitecattle (Lindberg & Anderson, 1988; Bernoco eta/. ,1991; Lewinetal., 1991). The presence of identical BoLA haplotypes in several breeds suggests that these haplotypes exist in linkage disequilibrium.

Three of the serological class I1 specificities, DxZ, Dx3, and Dx4, behaved as public specificities. Interestingly, the DQ-DR haplotypes that share public specificities have similarities at the DNA level. These similarities were initially recognized in the RFLP banding patterns (Anderson et al., 1986a, 1986b; Sigurdardottir et al. , 1988) and more recently have been confirmed by sequence analyses (Sigurdardottir et al., 1991a, 1991b). Dxl was associated with the haplotypes DQYA,DRBYA and DQ",DRB'" which show quite similar DQ RFLP patterns, share identical DQBl first domain sequences (DQBI refers to one of the four DQB subtypes defined by Sigurdardottir et al. (1992), apparently all BoLA haplotypes contain a DQBI allele while haplotypes with duplicated DQ genes also contain a DQBz, DQB.?, or DQB4 allele) and have very similar DQBz first domain sequences (Sigurdardottir et al., 1992). Dx3 was associated with haplotypes DQIA,DRBIA, DQ'",DRB'", and DQ7A,DRB7A which share several polymor- phic DQ restriction fragments and have similar DRB3 [DRB3 is the third bovine DRB gene that was described (Groenen et al., 1990) and apparently it is the only DRB gene that is expressed at a high level (Burke et a / . , 1991)) sequences in the carboxy terminal part of the first domain (Sigurdardottir et al., 1991a). The DQBI first domain sequences of haplotypes DQIA ,DRBIA, and DQ7A,DRB7A are also very similar. The Dx4 specificity was associated with haplotypes D@,DRB5B, and DQ6*,DRB6 which show DQ RFLP similarities and share an identical DQB3 first domain sequence (Sigurdardottir et al., 1992). The present study and the earlier study by Joosten eta/. (1990) have demonstrated a very close

association between class I1 polymorphism defined by serology, IEF, and RFLP. In both studies the RFLP method distinguished the largest number of allelic variants. In fact, serology or IEF did not reveal any heterogeneity within the RFLP defined DQ-DR haplotypes. Consequently, it is important to know if the additional polymorphism revealed by RFLP typing is associated with expressed polymorphism. Recently sequence analysis has revealed that there is a good correlation between RFLP and sequence polymorphism in the DRB and DQB first domain exons (Sigurdardottir et al., 1991a, 1991b). All of the major DQ-DR haplotypes defined by RFLP analysis (i.e. DQlA,DRBiA, DQ'",DRBlB, and DQ2,DRBzA to DQ'.zA,DRB13A) have different DRB3 first domain sequences and, in most cases, different DQBl first domain sequences. Most, but not all, of these haplotypes can also be distinguished by IEF analysis (Joosten et al., 1990; and above). For those haplotypes denoted as subtypes, sequence differences have been found in some cases (e.g. DRB5A vs. DRB"; associated with IEF types DF!J and DF16, respectively) but not in others (e.g. DRB2A vs. DRB2'; both associated with D E ) (L. Anderson et al . , unpublished).

The need for the detailed characterization of BoLA haplotypes is dramatized by some functional data acquired using animals carrying haplotypes described here. BoCD4 positive T-cell clones specific for a crude worm extract from Oesophagostomum radiatum were generated from B1684, the A I ~ , D X B , D Q " ~ , D R B " ~ , D F ~ homozygote characterized in the present study

260 C. J. Davies et al.

(Canals et al . , 1992). Some but not all of the clones proliferated when exposed to antigen presented in the context of the A35,Dx8,DQ1'",DRB"',DF6 haplotype while none of the clones proliferated in response to antigen presented in the context of the A35,Dx9,DQ4",DRB"",DF7 haplotype (Canals et al. , 1992). In addition, it has been found that despite having different DRB RFLP patterns the A12, Dx8,DQ'", DRB"", DF6 and A3.5, Dx8, DQ"', DRB"", DF6 haplo- types have identical DRB3 first domain sequences (L. Anderson et al . , unpublished). On the other hand, the A12, Dx8. DQ"', DR B"" , DF6 and and A35, Dx9, DQ4A ,DR B1IA, D F7 haplo- types, which share DRB RFLP patterns but have different IEF types, have different DRB3 first domain sequences (L. Anderson et al., unpublished). Hence, the DRB3 sequence data and the I E F typing, believed to define polymorphism at a single DRB locus, suggest that the A12 ,Dx8, DQ"', D D F6 and A35, Dx8, DQ"", DR B"", D F6 haplotypes share at least one DRB product. Despite the extensive BOLA complex characterization that has been performed, the difference between the A I ~ , D x ~ , D Q " " , D R B " ~ , D F ~ and A35,Dx8,DQ"".DRB"",DF6 haplotypes that affects class I1 mediated presentation of 0. radiatum antigens has yet to be identified.

The use of serology, IEF, and RFLP in this study has permitted the definition of BOLA haplotypes with considerable precision. The precise definition of BOLA haplotypes has made it possible to identify identical or very similar BOLA haplotypes in different cattle populations. We believe that the use of these three techniques to characterize BOLA haplotypes in different cattle populations will prove to be important in efforts to understand the diversity of this complex genetic system. Nevertheless, because the number of BOLA haplotypes segregating in most cattle populations is quite limited, most populations can be readily typed using the simplest technique (current serology) provided that the haplotypes are defined using the most powerful technique (presently RFLP) or group of techniques.

A C K N O W L E D G M E N T S

The authors thank the Cornell Teaching and Research Center and the Wye Research and Education Center for access to their animals; Dr D. F. Antczak for advice regarding the development of the class I1 serology; and M. F. Sanders for technical assistance with the IEF analysis. The research was supported by the United States Department of Agriculture and the Swedish Council for Forestry and Agricultural Research.

R E F E R E N C E S

AMORENA, B. & STONE, W.H. (1978) Serologically defined (SD) locus in cattle. Science, 201, 159. ANDERSON, L. (1988) Genetic polymorphism of a bovine t-complex gene (TCPl) linkage to major

histocompatibility genes. Journal of Heredity, 79, 1. ANDERSON, L. & RASK, L. (1988) Characterization of the MHC class 11 region in cattle. The number of DQ

genes varies between haplotypes. Immunogenerics, 27, 110. ANDERSON, L., BOHME, J . , RASK, L. & PETERSON, P.A. (1986a) Genomic hybridization of bovine class I1

major histocompatibility genes: 1. Extensive polymorphism of DQa and DQP genes. Animal Generics, 17, 95.

ANDERSON, L., BOHME, J . , PETERSON, P.A. & RASK, L. (198hb) Genomic hybridization of bovine class I1 major histocompatibility genes: 2. Polymorphism of DRgenes and linkage disequilibrium in the DQ-DR region. Animal Genetics, 17,295.

ANDERSON, L., LUNDEN, A, , SIGURDARD6TTIR. s., DAVIES, C.J. & RASK, L. (1988) Linkage relationships in the bovine MHC region. High recombination frequency between class I1 subregions. Irnmunogenetics, 27, 273.

Characterization of bovine M H C class I I polymorphism 26 1

ARRIENS, M.A., HESFORD, F., RUFF, G . & LAZARY, S. (1991) Production of alloantibodies against bovine

BERNOCO, D. , LEWIN, H.A. , ANDERSON, L., ARRIENS, M.A., BYRNS, G., CWIK, S . , DAVIES, C.J. , HINES,

STEEWART-HAYNES, J.A., TEALE, A.J., TEMPLETON, J.W. & ZANOTTI, M. (1991) Joint report of the Fourth International Bovine Lymphocyte Antigen (BoLA) Workshop, East Lansing, Michigan, USA, 25 August 1990. Animal Genetics, 22,477.

BURKE, M.G., STONE, R.T. & MuGGLI-COCKER, N.E. (1991) Nucleotide sequence and Northern analysis of a bovine major histocompatibility class I1 DR p-like CDNA. Animal Genetics, 22, 343.

CALDWELL, J . , BRYAN, C.F., CUMBERLAND, P.A. & WESELI, D.F. (1977) Serologically detected lymphocyte antigens in Holstein cattle. Animal Blood Groups and Biochemical Genetics, 8, 197.

CANALS, A , , GASBARRE, L.C., DAVIES, C.J., & ZARLENGA, D. (1992) Oesophagostomum radiatum-specific bovine T cell clones: specificity, MHC restriction and cytokine transcription. Veterinary Immunology and Immunopathology, in press.

DAVIES, C.J. & ANTCZAK, D.F. (1991a) Mixed lymphocyte culture studies reveal complexity in the bovine major histocompatibility complex not detected by class I serology. Animal Genetics, 22, 31.

DAVIES, C.J. & ANTCZAK, D.F. (1991b) Production and characterization of alloantisera specific for bovine class I1 major histocompatibility complex antigens. Animal Genetics, 22, 417.

GROENEN, M.A.M.,VANDERPOEL, J.J., DIJKHOF,R.J.M. &GIPHART, M.J. (1990)Thenucleotidesequence of bovine MHC class I1 DQB and DRB genes. Immunogenetics, 31, 37.

GUSTAFSSON. K . , WIMAN, K . , EMMOTH, E., LARHAMMAR, D . , BOHME, J., HYLDIG-NIELSEN, J .J . , RONNE, H., PETERSON, P.A. 8 RASK, L. (1984) Mutations and selection in the generation of class I1 histocompatibility antigen polymorphism. EMBO Journal, 3, 1655.

JOOSTEN, I., OLIVER, R.A., SPOONER, R.L., WILLIAMS, J.L., HEPKEMA, B.G., SANDERS, M.F. & HENSEN, E.J. (1988) Characterization of class I bovine lymphocyte antigens (BoLA) by one-dimensional isoelectric focusing. Animal Genetics, 19, 103.

JOOSTEN, I., SANDERS, M.F., VAN DER POEL, A., WILLIAMS, J.L., HEPKEMA, B.G. & HENSEN, E.J. (1989) Biochemically defined polymorphism of bovine MHC class I1 antigens. Immunogenetics, 29, 213.

JOOSTEN, I . , HENSEN, E.J., SANDERS, M.F. & ANDERSON, L. (1990) Bovine MHC class 11 restriction fragment length polymorphism linked to expressed polymorphism. Immunogenetics, 31, 123.

LARHAMMAR, D. , GUSTAFSSON, K., CLAESSON, L., BILL, P., WIMAN, K . , SCHENNING, L., SUNDELIN, J., WIDMARK, E., PETERSON, P.A. & RASK, L. (1982a) Alpha chain of HLA-DR transplantation antigens is a member of the same protein superfamily as the immunoglobulins. Cell, 30, 153.

LARHAMMAR, D., SCHENNING, L., GUSTAFSSON, K. , WIMAN, K . , CLAESSON, L., RASK, L. & PETERSON, P.A. (1982b) Complete amino acid sequence of an HLA-DR antigen-like p chain as predicted from the nucleotide sequence: similarities with immunoglobulins and HLA-A, -B, and -C antigens. Proceedings of the National Academy of Sciences, USA, 79, 3687.

LEWIN, H.A., DAVIS, W.C. & BERNOCO, D. (1985) Monoclonal antibodies that distinguish bovine T and B lymphocytes. Veterinary Immunology and Immunopathology, 9, 87.

LEWIN, H.A., ARNET, E.F., LIE, 0., BERNOCO, D . , DAVIES, C.J., NILSSON, P.R., RUFF, G. & LAZARY, S. (1991) First international comparison test of serological reagents for typing class I1 products of the bovine major histocompatibility complex. Animal Genetics, 22, Suppl. 1, 46 (abstract).

LINDBERG, P.G. & ANLIERSSON, L. (1988) Close association between DNA polymorphism of bovine major histocompatibility complex class I genes and serological BOLA-A specificities. Animal Genetics, 19,245.

MACKIE, J.T. & STEAR, M.J. (1990) The definition of five B lymphocyte alloantigensclosely linked to BoLA class I antigens. Animal Genetics, 21, 69.

ROTHEL, J.S., DUFTY, J.H. &WOOD, P.R. (1990) Studieson the bovine major histocompatibilityclass I and class I1 antigens using homozygous typing cells and antigen-specific BoT4+ blast cells. Animal Genetics, 21, 141.

SCHENNING, L., LARHAMMAR, D., BILL, P. , WIMAN, K., JONSSON, A.K., RASK, L. & PETERSON, P.A. (1984) Both alpha and beta chains of HLA-DC class I1 histocompatibility antigens display extensive polymorphism in their amino-terminal domains. EMBO Journal, 3,447.

SIGURDARD6TTIR, s., LUNDEN, A. & ANDERSON, L. (1988) Restriction fragment length polymorphism of DQ and DR class I1 genes of the bovine MHC. Animal Genetics, 19, 133.

SIGURDARD6TTlR, S . , BORSCH, C., GUSTAFSSON, K. & ANDERSON, L. (1991a) Cloning and sequence analysis of 14 DRB alleles of the bovine MHC by using the polymerase chain reaction. Animal Genetics, 22, 199.

B-lymphocyte antigens. Animal Genetics, 22, 399.

H.C., LEIBOLD, W., LIE, 8.. MEGGIOLARO, D. , OLIVER, R.A., OSTERGARD, H., SPOONER, R.L.,

262 C. J. Davies et al.

SlGURDARD6TTIR, s., MARIANI, P. , GROENEN, M.A.M., VAN DER POEL, J.J. & ANDERSON, L. (1991b) Organization and polymorphism of bovine major histocompatibility complex class I1 genes as revealed by genomic hybridizations with bovine probes. Animal Genetics, 22,465.

SIGURDARD6TTIR, s., BORSCH, c . , GIJSTAFSSON, K. & ANDERSON, L. (1992) Gene duplication and sequence polymorphism of bovine class I1 DQB genes. Immunogenetics 35, 205.

SPOONER, R.L., LEVEZIEL, H., GROSCLAUDE, F., OLIVER, R.A. & VAIMAN, M. (1978) Evidence for a possible major histocompatibility complex (BLA) in cattle. Journal of Immunogenetics, 5 , 335.

STONE, R.T. & MUGGLI-COCKETT, N.E. (1990) Partial nucleotide sequence of a novel bovine major histocompatibility complex class I1 P-chain gene, BOLA-DIB. Animal Genetics, 21, 353.

TEALE, A.J. & KEMP, S.J. (1987) A study of BoLA class I1 antigens with BoT4+ T lymphocyte clones. Animal Genetics, la, 17.

TERASAKI, P.I., BERNOCO, D., PARK, M.S., OZTURK, G. & IWAKI, Y. (1978) Microdroplet testing for HLA-A, -B, -C, and -D antigens. American Journal of Clinical Pathology, 69, 103.

VAN DER POEL, J.J., GROENEN, M.A.M., DIJKHOF, R.J.M., RUYTER, D. & GIPHART, M.J. (1990) The nucleotide sequence of the bovine MHC class I1 alpha genes: DRA, DQA, and DYA. Immunogenetics, 31, 29.

WILLIAMS, J.L., OLIVER, R.A. , MORGAN, A.L.G., GLASS, E.J. & SPOONER, R.L. (1991) Production of alloantisera against class I1 bovine lymphocyte antigens (BoLA) by cross-immunization between class I matched cattle. Animal Genetics, 22, 407.