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Flystrike is a problem for most sheep industries worldwide as it affects animal welfare and production. The cost of flystrike is multifactorial, and includes production losses resulting from reductions in wool and body growth, morbidity, and treatment and control costs. Flystrike incidence is environmentally dependent and its prevalence is variable. However, several factors predispose individual sheep to flystrike, such as variation in the fleece and skin of the sheep, and variation in their immune response. Identifying genes that are involved with these factors and the predisposition to parasitic disease, will potentially allow for flocks to be genetically improved for flystrike resilience, thus reducing its impact in the NZ sheep industry. This study aimed to identify variation in five candidate genes associated with phenotypic factors that do, or may, predispose sheep to flystrike, and determine whether the genetic variation (if any) was associated with susceptibility to disease. The genes chosen had either previously been implicated in resistance to fleecerot (FBLN1 and FABP4), or potentially affected the wax content of the fleece (ABCC11), or were involved in immune response (TLR4 and RASGRP1). In the sheep studied, variation in ovine FABP4, FBLN1 and RASGRP1 was associated with susceptibility to flystrike. Sheep with the A1 variant of FABP4 were found to be less likely (P = 0.014, with an odds ratio of 0.689) to have flystrike than those without A1. The likelihood of flystrike occurrence continued to decrease as copy number of A1 increased from one copy to two (P= 0.002, with an odds ratio of 0.674). The FBLN1 B1 variant was found to be associated with reduced flystrike occurrence (P = 0.029, odds ratio = 0.286), while the FBLN1 C1 variant was associated with increased occurrence (P = 0.002, odds ratio = 1.687). The RASGRP1 A (P = 0.015, odds ratio = 1.557) and C (P = 0.047, odds ratio = 0.733) variants had a significant association with flystrike occurrence. This study showed identified FABP4, FBLN1 and RASGRP1 as good candidate’s genes for flystrike resilience in sheep. Further research is required to verify these genes as markers for flystrike resilience.

Introduction: Fly-strike is a major economic and animal welfare issue in both the New Zealand and Australian sheep industry. There are several factors that predispose sheep to fly-strike, such as fleece-rot, urine staining of wool from the crutch and dags. The FABP4 gene has previously been associated with variation in fleece-rot resistance in sheep. There is a strong genetic correlation (r=0.9) between fleece-rot and fly-strike. In this context, this study set out to determine if there is an association between a gene that has previously been linked to fly-strike susceptiblity (FABP4) and susceptibility to disease. Methods and materials:Blood samples were taken from sheep with and without fly-strike at shearing time and from different properties through Canterbury. These samples were collected onto FTA cards for DNA typing. PCR-SSCP analysis was used to genotype a portion of the ovine FABP4 gene. Results: Four variants of FABP4 gene were found (A₁, B₁, C₁ and D₁). there was a difference between sheep with and without fly-strike and the presence/absence of the A₁and C₁ variant, (P=0.0073) and (P=0.0154) respectively. Sheep with the A₁ variant are less likely to get fly-strike than sheep with the C₁ variant. The overall Chi-squared test was insignificant, indicating that it cannot be determined from the genotype whether sheep will or will not get fly-strike. Discussion: Based on these findings the development of a gene marker test for selecting sheep that are genetically resistant to fly-strike is a possibility. However further studies need to be done, with a larger sample of sheep. The exact nature of FABP4 causes sheep to be resistant or susceptible to fly-strike also needs to be determined.

The major histocompatibility complex (MHC) consists of a long array of genetic loci which are central to the immune response. Numerous studies have demonstrated associations between the genes in this complex and economically important traits, such as disease resistance in farm animals. However, in several species including the sheep, little is known of the organisation and structure of this genetic region. The main aim of this study was to investigate polymorphism within the ovine MHC class II region by Southern blot hybridisation using sheep-specific probes. The DQA, DQB, DRA and DRB loci were investigated, resulting in identification of thirty-two alleles (8 DQA1, 16 DQA2, 5 DQB, and 3 DRA). Little information was derived from the DRB subregion due to the poor quality of the autoradiographs generated with the DRB probe and extensive cross-hybridisation with the DQB locus. Allele frequencies were found to vary both between breeds and within different flocks of the same breed. Recombination within the ovine DQ-DR subregion was investigated in twelve families (9 paternal half-sib and 3 full-sib) by linkage analysis. Lod scores were derived for a range of recombination fractions (θ) between 0.5 and 0.000001 for linkage between pairs of markers within the DQ-DR subregion. For each comparison, maximum lod scores were identified at the recombination fraction closest to zero (θ= 0.000001) of those evaluated, indicating the absence of recombination and the close linkage of genes in the DQ-DR subregion. Individuals were identified which did not generate hybridising bands with the DQA1 exon 2 probe, suggesting a deletion in these sheep. This postulate was supported by a similar finding when hybridisation was performed with a DQA1 probe which covered the third and forth exons. The apparent deletion of the DQA1 gene was termed the DQA1 null allele (DQA1-N). The frequency of DQA1-N homozygotes was comparable between distantly related breeds, suggesting that the deletion occurred before the establishment of present breeds of sheep. The usefulness of PCR-RFLP analysis for the identification of polymorphisms within the DQA1 and DQA2 genes was demonstrated. With the DQA1-specific primer pair DQA1(top)/DQA1(bot) a conserved exon 2 BglII restriction site and a polymorphic BglII site in the 5' non-coding region of the DQA1 gene were identified. However, this primer pair selectively amplified certain DQAl alleles, indicating that additional primers would be required to enable PCR-RFLP analysis of all DQA1 types. PCR-RFLP analysis of animals of known DQA2 genotype with the primer pair DQA1(oz)/DQA2(oz) revealed association of polymorphic and conserved restriction sites with different DQA2 alleles. One allele (DQA2-E) appeared to be associated with extensive polymorphism compared to the other DQA2 alleles. Two of the four restriction endonucleases used for RFLP analysis of the PCR product from DQA2-E homozygotes identified polymorphic restriction sites within the second exon. Southern blot hybridisation and PCR-RFLP analysis were utilised to provide evidence for the detection of extra DQA2-like genetic material in individuals which possessed the DQA1 null allele. Banding patterns were consistent with the detection of a second DQA2 gene, suggesting that this gene may have been duplicated in individuals which lost the DQA1 gene. The composition of the ovine DQA region therefore appears to vary between haplotypes, some possessing DQA1 and DQA2 while others possess DQA2 and a second DQA2-like gene. Evidence was also obtained for the presence of extra DQB-like genetic material in individuals which lack the DQA1 gene. Whether the extra DQA2 and DQB genetic material constitute a third DQA/DQB locus remains to be determined. Association between genetic variation within the ovine MHC class II region and resistance to footrot was investigated. Half-sib families of the Broomfield Corriedale Breeding Group were subjected to footrot challenge by the introduction of footrot infected animals into the flock. Based on their response to challenge, the progeny of the different families were classified as resistant, susceptible or self-curing. Each sire's two MHC haplotypes, comprising alleles at the DQA1, DQA2, DQB and DRA loci, were inferred from the alleles present in his offspring. The progeny in each family were divided two groups based on the haplotype inherited from the sire and associations between paternal haplotype and disease status investigated by chi-square analysis. A highly significant association (P= 0.005) was observed within one family sired by ram 20/88 following challenge conducted in 1992. The significance of this association increased (P= 0.002) when the self-curing animals were combined with the resistant animals and became even more significant (P= 0.001) when the self-curing animals were excluded from analysis. No associations were observed in the other three half-sib families subjected to challenge in 1992. In a second challenge conducted in 1993 the association of MHC haplotype with resistance to footrot in the half-sib family sired by ram 20/88 was not reproduced. However, this challenge was adversely affected by weather conditions which did not favour the transmission of footrot resulting in unreliable assignments of disease status. This study, while not conclusive, augurs well for future investigations of association between the ovine MHC and footrot resistance or other bacterial infections.

Dichelobacter nodosus is the causative agent of ovine footrot. The Type IV multi-subunit fimbriae of D.nodosus represents the key immunogenic component of footrot vaccines (Stewart 1989). Variation within the gene (jimA) for the fimbrial protein suQ.unit is currently the basis for the serotype classification of these bacteria. To date, no sequencing of the fimA gene has been conducted for New Zealand isolates. This study describes the development of a PCR specific for the variable region of the fimA gene. Cloning and sequencing of a single field derived amplimer and eight cultures currently being used in commercial footrot vaccines (Mallinckrodt Veterinary Ltd), is described. The rimA sequences exhibited varying degrees (89-100%) of homology to the prototype Australian strains (Mattick et ai., 1991). Serotype B sequences of New Zealand origin showed the greatest variation at the protein level. Two of these sequences predicted 11 and 13 amino acid substitutions (compared to the prototype B 1 isolate) in the hypervariable region of the protein between cysteine residues, that is thought to comprise the B-cell epitope. Such sequence variation may account for the ineffectiveness of vaccines which currently employ a majority of isolates of Australian origin. These findings indicate that further characterisation of Jim A gene heterogeneity is required within New Zealand to allow optimisation of footrot vaccines. AfimA based PCR-RFLP typing system was described and potentially represents a quick and cost effective means of typing D.nodosus from lesion material. The development of molecular techniques to rapidly "type" D.nodosus may enable strain-specific vaccines to be formulated, thereby circumventing the problems associated with antigenic competition in polyvalent formulations.