SummaryThe 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 histocompatibility 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 polymorphisms 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.

The hypothesis that behaviour in sheep is influenced by resistance to infections with Trichostrongylus colubriformis and Haemonchus contortus was explored. Sheep were assessed phenotypically as resistant and susceptible in four ways and thereafter, the effect of resistance on several behavioural traits was measured in an arena test. The behavioural parameters recorded for each sheep were: approach/avoidance distance; travel; number of moves; and spread. Four phenotypic groups of sheep were set up each with two subgroups: ovine lymphocyte antigen (OLA) type (subgroups SY1a type vs other types); blood eosinophil leucocyte counts (high vs low); T. colubriformis and H. contortus serum antibodies (high vs low titres) and faecal egg count (FEC) (high vs low). Only the behavioural comparison between sheep with high versus low eosinophil leucocyte count showed consistent differences that were statistically significant, although other comparisons favoured associations between OLA type SY1a, low FEC and reduced approach/avoidance distance and/or locomotor behaviour. The eosinophil leucocyte association was established on two non-overlapping test populations of sheep, with mean eosinophil leucocyte counts of 3.19 x 10(5)/mL blood for high and 0.83 x 10(5)/mL blood for low subgroups; results were reproduced on five separate occasions over 6 weeks to detect differences as approach/avoidance distance (P < 0.01), travel (P < 0.01), number of moves (P < 0.01), and spread (P < 0.05). High eosinophilia correlated with low FEC for the infected sheep, which identified sheep that were resistant to parasites. We concluded, therefore, that the resistant sheep were more at ease with the environment of the test than were the sheep with low eosinophil leucocyte counts. The strong association between high eosinophil leucocyte counts and behaviour is consistent with other reports which suggest that retention of normal levels of circulating eosinophil leucocytes is associated with resistance to stress.

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SUMMARY The distribution of 12 Class I ovine lymphocyte antigens (OLA) was examined in 4 flocks of sheep vaccinated against and/or challenged with Bacteroides nodosus, the transmitting agent of footrot. In a flock of 47 Corriedales in New Zealand, which had been specially bred for resistance to footrot, a higher frequency (70.2%) of OLA type SY6 was found compared with 42.9% in 49 unselected Corriedale sheep (P = 0.001) The serum antibody response of 12 selected Corriedale ewes was compared with that of 12 unselected ewes of the same age after vaccination with a multivalent footrot vaccine and the selected ewes had significantly (P = 0.01) higher agglutinin titres than the unselected ewes, 7 weeks after vaccination. In 3 trials involving 108, 120 and 135 Australian Merinos in Victoria, SYlb was associated with a reduction in the number of feet affected with severe footrot (P = 0.05, P = 0.01, P = 0.02) and in 2 of the trials there was a relationship between SY6 and high vaccinal agglutinin titres. This SY6 effect was evident in the first trial 31 days after primary vaccination (P = 0.05) and again 20 days later after secondary vaccination (P = 0.01). In the second trial, when the sheep were vaccinated 49 days after challenge, an association was again found between SY6 and high agglutinin titres (P = 0.05) after primary but not after secondary vaccination. Exposure of 157 vaccinated Merino rams to B. nodosus during a footrot outbreak in New South Wales also showed an association between low infection and SY6 and SYlb. Field challenge of 130 Merino wethers in another independent trial again showed significant associations between low footscore and SYlb at 4 weeks (P = 0.05), 7 months (P = 0.02) and 12 months (P = 0.05) after challenge. The results suggest that lymphocyte antigen type is a significant factor in the susceptibility or resistance of sheep to footrot and the evidence reported here implies that SY6 is associated with high responsiveness to vaccination and SYlb with resistance to footrot.

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A method for MHC DRB typing in cattle based on two closely linked and highly polymorphic microsatellites is described. The two microsatellites DRBP1ms and DRB3ms are located in intron 2 of the corresponding DRB gene. The very strong linkage disequilibrium between the two loci made it possible to establish DRB microsatellite haplotypes. The typing results with this method on reference samples followed closely that obtained with RFLP and direct sequence analysis of DRB3 exon 2. The method is well suited for large scale genotyping and was successfully applied for typing more than 600 unrelated animals representing 23 breeds. The data were used to test whether the observed DRB allele frequency distributions were consistent with that expected for selectively neutral alleles in populations at mutation-drift equilibrium. A significant heterozygosity excess was detected and there was an obvious trend across breeds towards a more even allele frequency distribution than expected. The deviation may be due to balancing selection acting on the DRB locus or by recent population bottlenecks.