The eosinophil leucocyte, a phenotypic marker of resistance to nematode parasites, is associated with calm behaviour in sheep.

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.

The PCR typing of MHC-DRB genes in the sheep using primers for an intronic microsatellite: application to nematode parasite resistance.

The strong association between polymorphisms in an intronic microsatellite and the coding sequences for (BoLA)-DRB3 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 primes 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 polymerase chain reaction (PCR) products amplified from the DRB microsatellite region. Incomplete amplification of both alleles 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-variable region alleles. Three families of Merino sheep with different antibody responses to intestinal nematode parasites were examined. The sire group with the highest antibody levels possessed two microsatellite 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.

Variation in immune responsiveness of sheep to the antigens of intestinal nematodes and blowfly larvae.

The total and IgG1 antibody responses to the intestinal nematode parasites Haemonchus contortus and Trichostrongylus colubriformis were measured in the serum of 160 lambs, 4 months of age. These antibodies had developed as the result of natural exposure to the parasites on pasture. Three sires were examined and strong sire effects on half-sib progeny were found. Plotting of ELISA antibody results in two dimensions revealed clustering of responses within sire groups. Bimodal antibody distributions were also observed within sire groups and the whole population for T. colubriformis. A bimodal distribution of antibodies to H. contortus was found for one sire group but not for the whole population. The injection of blowfly larvae (Lucilia cuprina) extract into 42/160 lambs at a later age (12 months) was followed by increased antibodies to L. cuprina and an apparent increase in antibodies to T. colubriformis. A bimodal distribution for antibodies to L. cuprina was found in one sire group and in the whole population. These bimodal distributions of antibodies to L. cuprina did not coincide with the distribution of antibodies to T. colubriformis or H. contortus, measured on the same serum samples. It was concluded that high and low responder sire groups could be differentiated in lamb populations for all three parasites. These effects persisted during lamb maturation and appeared to be genetic effects. Finally, cross-reacting antibodies between L. cuprina and T. colubriformis appear to be stimulated by injection of L. cuprina antigens.

The immunogenetics of resistance to Trichostrongylus colubriformis and Haemonchus contortus parasites in sheep.

Three possible immunogenetic markers for resistance to intestinal parasites in sheep have been studied. Allotypes of the major histocompatibility complex (MHC) of the sheep have been investigated as markers, using serological typing or gene probes, for associations between allotypes and resistance to parasites in selected high responder and low responder lines of sheep. Only the serologically-determined class I ovine leucocyte antigen (OLA) types SY 1a and SY 1b have been found to be consistently associated with increased resistance to Trichostrongylus colubriformis, but this association has not extended to the immunologically distinct Haemonchus contortus parasite. Gene probes of the sheep DRB, DQB and DQA MHC class II loci have detected animals with increased susceptibility to T. colubriformis. Eosinophilia was investigated as a marker and found to be associated with increased resistance to parasites in lines of Australian Merinos and New Zealand Romneys selected for resistance on the basis of low faecal egg count. Blood eosinophilia was distinct from eosinophil infiltration of the gut which was poorly associated with resistance. The mechanism of parasite resistance appeared to involve the release of vasoactive amines and leukotrienes into intestinal mucus, since the selected high responder sheep to T. colubriformis and H. contortus had significantly increased amounts of these agents in their gut mucus, compared with selected low responder or random-bred sheep. Antibodies to T. colubriformis and H. contortus have also been used as markers to select high responder sire groups of lambs in contact with the parasites, for the first time, at weaning. This assay had the advantage of detecting distinct antigens for the two parasites, which would allow resistance to the species of parasite to be selected in the lambs. Vaccines have been developed against H. contortus using 'novel' gut antigens from the parasite, but variable responsiveness of the host sheep seemed to result in varying degrees of protection which were stimulated by these vaccines.