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.

The use of embryo genotyping in the propagation of genes involved in the immune response.

Multiple ovulation and embryo transfer (MOET) now enables researchers to produce identical twin animals, to obtain progeny from pre-pubertal females and to obtain more offspring from valuable animals. MOET and sexed semen have produced genetic progress of up to 60% of milk production. The oestrous cycles of animals are synchronized with progestagens before superovulation with gonadal hormones, pregnant mare serum gonadotrophin and follicle stimulating hormone. Surgical, non-surgical and laparoscopic methods are applied to recover and transfer embryos. Sexing and genotyping of the pre-implantation embryos is a key step in improving the management and breeding programmes for livestock, as well as in the human for the prenatal diagnosis of genetic disorders. Several serological and physiological methods have been used to determine the sex of the pre-implantation embryos; none has had satisfactory results in terms of time and accuracy. Sexing by polymerase chain reaction (PCR) using male-specific chromosome sequences alone or with female-specific chromosomal DNA probes simultaneously has been sufficient to identify the sex of the embryos with 100% accuracy. However, caution should be taken against sources of the contamination. The MHC class I, class II and background genes have been implicated in resistance to internal parasites in animals. Biotechnological methods such as screening of embryos prior to transfer using PCR and primer extension pre-amplification have already made it possible to detect transgenic or genetically disordered embryos and could be applied to select those embryos bearing immunological genotypes of interest, such as resistance to internal parasites. Ultimately, cloning and nuclear transplantation would provide the possibility of isolating these resistance genes and to transfer them to livestock pre-implantation embryos to propagate these desirable traits.

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.

The major histocompatibility complex and peptide vaccines in domestic animals.

Three hypotheses are suggested to explain the phenomenon of low responsiveness in domestic animals after injection of peptide vaccines. The first hypothesis proposes involvement of MHC haplotype and the special case in livestock breeding, where inheritance of the sire's haplotype can be closely examined by injection of antigen into a large number of paternal half-sib progeny. The second hypothesis examines the effect of repeated antigen injections in overcoming age and MHC haplotype effects and distinguishing these effects from those caused by deficiencies in the T cell repertoire. The third hypothesis concerns non-MHC effects that influence the expression of MHC haplotype effects and enable the host to mount an effective immune response. It is suggested that the antigen recognition signal from T cell receptor/MHC interaction is amplified to a varying extent in animal populations. Deficiency in this amplification through myeloid cell or cytokine responses may be yet another factor limiting immune responsiveness.

High and low responsiveness to vaccines in farm animals.

Low responsiveness in some farm animals is emerging as a problem in the application of newly developed vaccines which operate at skin surfaces and mucous membranes. Breeding for resistance to specific diseases seems to be associated with breeding for specific immune responsiveness in farm animals and very likely this involves selection for major histocompatibility complex (MHC) haplotype. However, other factors contribute to low responsiveness and these include poor nutrition, sire effects, antigenic competition and inadequate effector mechanisms. The future of newly developed vaccines will rest on the solution to the low responder problem, and once solved, the application of these vaccines will be fully utilized for disease control in farm animals.

Histamine and leukotriene concentrations in duodenal tissue and mucus of lambs selected for high and low responsiveness to vaccination and challenge with Trichostrongylus colubriformis.

Lambs selectively bred for high responsiveness or low responsiveness to vaccination with irradiated Trichostrongylus colubriformis were vaccinated and challenged. Duodenal tissue histamine concentrations in both high and low responder lambs were lower at 3 days than at 28 days after challenge. At 3 days after challenge, histamine concentrations were higher in both male and female high responder lambs than in low responder lambs whereas at 28 days concentrations were increased only in high responder females. At 3 days after challenge, histamine concentrations were generally lower in mucus than in tissues, but levels were again higher in mucus from high responder groups. In duodenal tissue at 3 days after challenge, leukotriene C4 and B4 concentrations were similar in high and low responder animals. At the same time, concentrations of both leukotrienes were higher in mucus than in tissues, with high responder female lambs having the highest concentration. It is suggested that increased levels of histamine and leukotrienes in mucus and tissue are associated with larval rejection or exclusion.

A positive association between resistance to ovine footrot and particular lymphocyte antigen types.

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.(ABSTRACT TRUNCATED AT 250 WORDS)

The relationship between ovine lymphocyte antigens and parasitological and production parameters in Romney sheep.

The occurrence of ovine lymphocyte antigen (OLA) types in two flocks of New Zealand Romney sheep was examined in relation to resistance to nematode parasites [as judged by faecal egg counts (FEC)], plasma pepsinogen levels, liveweight and weight gains. A panel of OLA-typing antisera (SY 1-16) which determine class 1 MHC antigens of the sheep was used. The OLA combination SY 1a + 1b was found exclusively in Romney sheep of both flocks having below average FEC, but was present in low frequency (5%). In one flock, sheep possessing this antigen combination had consistently lower FEC from weaning to 1 year of age than sheep without this combination. SY 6 occurred significantly more frequently in above average FEC sheep and was associated with significantly higher FEC during secondary challenge infection. Plasma pepsinogen levels were significantly lower in those sheep possessing SY 2 or SY 3 but these OLA types were not associated with lower than average FEC. No OLA type was associated with above average weight gain but in one flock sheep with SY 1b and SY 1a + 1b had significantly lower weight gains between weaning and 6 months of age than sheep without these OLA types. A similar association was not found in the other flock. Sheep in one flock with SY 16 were significantly heavier than those without this antigen. No other associations between OLA types and liveweight were found.

Analysis of C3 and C4 in ovine plasma.

The distributions of plasma concentrations of complement proteins C3 and C4 were studied in sample populations of merino and Suffolk sheep. No differences between the breeds or the sexes were observed. The distribution for ovine C4 was polymodal and very disperse relative to that for C3. It was found, however, that C3 concentrations were elevated in specimens from 20 merino sheep bred as high responders to a Trichostrongylus vaccine. Significantly decreased plasma C4 concentrations were observed in representatives of both merino and merino X Border Leicester cross-bred sheep affected with congenital progressive ovine muscular dystrophy. Agarose gel electrophoretic variants of ovine C3 were not detected. Evidence for electrophoretic variants of ovine C4 in agarose gels was found although individual allotypes could not be reliably identified. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) did not reveal size heterogeneity for the alpha and beta chains of immunoprecipitated ovine C3. Analysis of reduced immunoprecipitated ovine C4 by SDS-PAGE revealed considerable size heterogeneity in the alpha chain consistent with isotypic and/or allotypic variability. The data presented strongly suggest the presence of two C4 loci in sheep, each of which exhibits polymorphism.

Antibodies to ovine lymphocytes exist in bovine alloantisera.

Forty-six percent of 149 reagents against class 1 antigens of the bovine major histocompatibility system reacted with lymphocytes from 1 or more sheep in a microlymphocytotoxicity test. The reactions fell into six groups. The majority of antisera defined an antigen present on both erythrocytes and lymphocytes. This antigen is probably the R blood group antigen and the reactions were unrelated to the presence of anticlass I activity and are probably due to the additional presence of antibodies of R specificity. The other antigens are not identical to any of the 14 red blood cell factors or seven lymphocyte antigens examined, nor did they correspond to any of the class I bovine lymphocyte antigens defined by the alloantisera.

Development of cell-mediated immunity in young ruminants.

Development of cell-mediated immunity in young ruminants appears to be under the influence of the thymus. In sheep, prenatal removal of the thymus has little effect on postnatal growth of lambs. However, lambs are immunodeficient compared with normal controls, and they exhibit depressed delayed hypersensitive skin responses to antigens such as tuberculin purified protein derivative. Lymphopenia accompanying the immunodeficiency appears to be due to depletion of a particular population of T-cells (thymus derived) that have reduced response to mitogens and decreased numbers as the lamb matures. Young lambs are less responsive than adult sheep to vaccination with irradiated Trichostrongylus colubriformis larvae. The basis of this lowered responsiveness appears to be not only the immaturity of the cell-mediated immune response but also the segregation of the lambs into high and low responders. This immune responsiveness is possibly under the genetic control of the ovine major histocompatibility complex. It may be possible to select and breed sheep and cattle for responsiveness to vaccination against parasitic, viral, and bacterial diseases.

Studies on the bovine lymphocyte antigens and the production of lymphocytotoxic antibodies by parous cattle.

Studies were carried out to determine the time of appearance, frequency, titre and specificity of lymphocytotoxic antibodies in the plasma of parous Hereford cattle. Cytotoxic antibody was first detected in a small proportion (3/62 = 4.8%) of primigravid cattle during the last third of pregnancy. Titres were low (neat or 1 in 2) at this time and decreased in one animal so that antibodies were not detectable in samples obtained on the day of calving or 9 days beforehand. Following parturition, the proportion of primiparous cattle producing lymphocytotoxic antibodies increased markedly and reached a maximum value (8/19 = 42.1%) during the third month post partum. Antibody levels also rose over the same period. An increase in the parity of the dam also resulted in an increase in the proportion of cattle with lymphocytotoxic plasma. These antibodies appeared earlier in pregnancy, were at a higher titre and had a wider specificity than those found in primigravida. Non-foetally stimulated antibody was detected in 4 cattle. In one plasma sample, lymphocytotoxic activity was present prior to mating, and in the 3 others it was not directed against cells from either the bull to which the dam was mated or the calf produced by the sire and dam.

Subpopulations of pig blood E-rosette-forming lymphocytes and thymus-dependent null cells: separation by nylon wool columns, rosette formation and macrophage-dependent mitogen and antigen responsiveness.

Pig peripheral blood lymphocyte subpopulations were characterized by their adherence to nylon wool. Thus whilst B cells, defined by surface Ig and Fc receptor, were predominantly adherent, thymus-dependent Null cells and the T cells forming rosettes with sheep red blood cells in dextran (DS+) were predominantly non-adherent. There was a direct relationship between the size of the non-adherent fraction and the proportion of the total DS+ cells which were in the non-adherent fraction. Using nylon wool and/or DS rosette separation, lymphocytes from normal and BCG sensitized animals were stimulated in vitro with phytohemagglutinin (PHA), concanavalin A (Con A) and tuberculin (PPD). The cells responding to PHA and Con A (T cell mitogens in other species) were DS+ lymphocytes. Non-rosetting, adherent (B cells) and non-adherent (thymus dependent Null cells) lymphocytes were unresponsive, even in the presence of homologous alveolar macrophages at levels which greatly enhanced the PPD response. Cells responding to PPD were present in the DS+ and DS-, adherent and non-adherent fractions but their response was apparently controlled by macrophage dependence and suppression. Techniques are described for the preparation and storage in liquid nitrogen of alveolar macrophages resulting in high numbers of viable cells, capable of helping in vitro responses, even after 3 years of storage.