Immunology and Cell Biology (1996) 74, 330-336 Research Article The PCR typing ofMHC-DRB genes in the sheep using primers for an intronic microsatellite: Application to nematode parasite resistance PM OUTTERIDGE,' L ANDERSSON,^ PGC DOUCH,^ RS GREEN,^ PS GWAKISA,- MA HOHENHAUS'' and S MIKKO- ^Department of Farm Animal Medicine and.Froduction. University of Queensland. Queensland. Australia, ^Swedish University of Agricultural Sciences. Department of Animal Breeding and Genetics. Uppsala. Sweden. ^AgResearch. Wallaceville Animal Research Centre. Upper Hutt. New Zealand and '^Department of Animal Froduction. Gatton College. University of Queensland. Queensland. Australia Summary The strong association between polymorphisms in an intronic microsatellite and the coding sequences for (BoLA)-/)/^5i genes, previously described for demonstrating alleles of class II major histocom- patibility complex (MHC) in the cow, was examined in sheep to see if similar polymorphisms could be demonstrated in the DRB region of the MHC. The bovine primers LA53 and LA54, previously used to amplify the bovine DRB3 microsatellites. were used with DNA from Australian sheep, eight DRB alleles were identified by length polymorphisms of poiymerase chain reaction (PCR) products amplified from the DRB microsatellite region. Incomplete amplification of both aileles was sometimes found for sheep DNA samples using bovine primers, so a modified primer (LA53b) was used, and found to amplify the microsatellite next to intron 2 of the MHC more reliably than the LA53 primer. Two additional primers {LA31 and LA32), used in amplification of the exon 2 region of bovine DRB3, were used in the sheep, and the PCR products were analysed by single-stranded conformation polymorphism (SSCP). These primers successfully amplified the variable region of the ovine DRB region coded by exon 2, and the SSCP technique demonstrated polymor- phisms with sheep DNA. Family studies demonstrated the segregation of alleles, by amplification both of intronic microsatellites and of the exon 2 variable region. Close correspondence was found between the two regions for several alleles. suggesting that the intronic microsatellites were closely linked to DRB-\av\ab\e region alleles. Three families of Merino sheep with ditferent antibody responses to intestinal nematode parasites were examined. The sire group with the highest antibody levels possessed two mierosatellite alleles of closely similar length (alleles 3 and 4) inherited from the sire and present in high frequency in the lambs. In contrast, the other two sires did not possess these two alleles and the alleles were in low frequency in their progeny. Further studies are required in unrelated sheep to confirm whether these two alleles are associated with resistance to nematode parasites. Key words: MHC-DRB, parasites, resistance, sheep. Introduction The major histocompatibility complex (MHC) in the sheep and other mammals is important because it has a vital role in the presentation of antigen to thymus-derived (T) cells. The molecules of the MHC are expressed in cell membranes and are highly polymorphic. They are en- coded by several gene loci, divided into class I and class II regions, for molecules which have distinct functions in the immune response.' The class I molecules present peplide antigens to cytotoxic CD8 * T cells which are involved in the elimination of virus-infected cells. The class II mole- cules present peptide antigens to CD4* helper T cells. Correspondence: PM Outtcridge, Department of Farm Animal Medicine and Production, University of Queensland, St Lucia 4072, Australia. Received 10 November 1995; accepted 9 April 1996. which in turn stimulate bone marrow-derived (B) cells to differentiate into plasma cells producing specific anti- bodies. The class II region of the MHC in humans has three isotypic forms, HLA-DQ, -DR and DP, while in the sheep and the cow, only DQ and DR homologues have been found to be expressed.- The highly polymorphic DR class II molecules are expressed in higher concentration than the DQ molecules in the cell membranes of mac- rophages and B cells, and it is thought that the DR locus, with its extensive allelic polymorphism, is likely to have a strong influence on genetic restriction of humoral im- mune responses to antigen. Considerable progress has been made in the character- ization of the highly polymorphic bovine DRB3 gene locus,^ and the DNA sequences of seven microsatellite alleles associated with different class II haplotypes have been determined."* This work has suggested that a PCR-