NZ330201A - Diagnosis of animals resistant to nematode parasite infection - Google Patents

Abstract

A method of testing a farmed animal to determine resistance or susceptibility to nematode parasites to which that animal is or could be exposed, comprising the step of testing nucleic acid obtained from the animal for the presence or absence of a conservative mutation in the gene encoding interferon gamma. The method is particularly useful in selection of breeding grazing animals where resistance to parasite infection is important.

Description

PATENTS FORM 5 PATENTS ACT 1953 COMPLETE SPECIFICATION Number Dated IDENTIFICATION OF ANIMALS RESISTANT TO NEMATODE PARASITE INFECTION We, NEW ZEALAND PASTORAL AGRICULTURE RESEARCH INSTITUTE LIMITED, a New Zealand company of Invermay Agricultural Centre, Puddle Alley, Mosgiel, New Zealand do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement. - 1 -(followed by 1A) RECF ! v E D 112108 vl WGN * iA IDENTIFICATION OF ANIMALS RESISTANT TO NEMATODE PARASITE INFECTION TECHNICAL FIELD The invention relates to identification of animals resistant or susceptible to nematode parasite infection.

BACKGROUND ART A number of commercially important animals species are prone to infection by nematodes. This particularly applies to farmed grazing animals. Sheep, cattle, goats and deer are among the commercially important animals affected.

The consequences of nematode infection include decreased yields of meat, fibre and milk, and may include premature death.

One animal in which nematode infections are a particular problem is the sheep. Many types of nematode parasite infect sheep but four of these: Trichostrongylus spp., 20 Nematodirus spp., Ostertagia spp. and Haemonchus contortus, are considered to be of greatest economic significance. Currently most farmers rely heavily on anthelmintic drenches to control parasite infection, but nematode resistance to one or more of the anthelmintic drench families is becoming increasingly common with more than 60% of sheep farms affected (McKenna et al 1995).

Sheep show variation in their susceptibility to nematode infection which has been the basis for breeding for host resistance. At present this involves subjecting animals to nematode challenge usually with determination of a faecal egg count (FEC) to measure the degree of resistance. Many breeders do not wish to conduct testing involving 30 infection of animals.

It is an object of the invention to provide an alternative test to pre-determine whether an animal will be more or less resistant to parasites and/or materials for such tests, or at least provide the public with a useful choice. 111533 WGN" 2 SUMMARY OF THE INVENTION The present invention provides a method of testing a farmed animal to determine resistance or susceptibility to nematode parasites to which that animal is or could be 5 exposed, comprising the step of testing nucleic acid obtained from the animal for the presence or absence of a conservative mutation in the gene encoding interferon gamma.

Preferably, the nucleic acid tested is DNA, most preferably genomic DNA.

Alternatively, the nucleic acid tested is mRNA.

| Conveniently, the animal tested is bovine, caprine, cervine, or ovine.

Most preferably, the animal is ovine and said nucleic acid tested is genomic DNA.

Preferably, in testing an ovine animal, the presence of DNA having the nucleotide sequence of Figure 10 is indicative of resistance to nematode parasites.

In the alternative, when testing an ovine animal, the presence of DNA having any one of the mutations shown in Figure 11 is indicative of susceptibility to nematode parasites.

More particularly, the presence of any one of the following mutations to the sequence of Figure 10 is indicative of susceptibility to nematode parasites: (i) a ^ g at position 188; («) a —> g at position 529; (iii) c t at position 538; (iv) g -» a at position 905; (v) t-^cat position 1149 (Vi) c t at position 1270 (vii) a t at position 1294 (viii) c^aat position 1349 (ix) a -» g at position 1620 (x) a -> g at position 1686 (xi) a —> c at position 1717 111533 WGN * (xii) g a at position 1923 (xiii) t. c at position 1989 (xiv) c —> t at position 2118 (xv) g t at position 2173 (xvi) a -» g at position 2252 (xvii) c t at position 2456 (xviii) a - > g at position 2457 (xix) g -> a at position 2458 (XX) t -» a at position 2461 (xxi) t c at position 2816 (xxii) t ->■ g at position 2978 (xxiii) c -» g at position 2979 • (xxiv) c —> t at position 3041 (xxv) c t at position 3359 (xxvi) c -> t at position 3488 (xxvii) g -» t at position 3516 (xxviii) c -» g at position 3540 (xxix) a g at position 3955 (xxx) c -» g at position 4051 (xxxi) t c at position 4059 (xxxii) a g at position 4120 (xxxiii) a -» g at position 4370 (xxxiv) c -> t at position 4379 ®25 (xxxv) a g at position 4441 (xxxvi) a -» g at position 4519.

Preferably, the presence of any one of the following in the sequence of Figure 10 is indicative of resistance to nematode parasites: (i) g 111 at positions 853-856; (ii) t at position 1524; and (iii) g a t g g g g g at positions 2448-2455. 111533 WGN" 4 BRIEF DESCRIPTION OF THE DRAWINGS While the invention is broadly as defined above, it will be appreciated that it is not limited thereto and that it includes embodiments of which the following description 5 provides examples. In particular, a better understanding of the invention will be gained from reference to the accompanying drawings in which: Figure 1 shows a diagrammatic representation of sheep chromosome 3 showing the location of all the markers used in the genomic scan of the five outcross pedigrees. The 10 five vertical lines of filled circles to the right of the chromosome ideogram indicate which sires were heterozygous for which markers, and hence had the 22 most resistant and 22 most susceptible progeny genotyped for that marker.

Figure 2 shows a graph of the -logio(p) score (suggestive linkage = 2.80p=0.0016; 15 significant linkage = 4.30 p=0.0000505) along chromosome 3 for abomasum Trichostrongylus nematode numbers. The peak of 2.38 for sire 1 is located 3 cM centromeric to OarVH34 and the estimated magnitude of the effect at this location is 3300 Trichostrongylus nematodes.

Figure 3 shows a graph of the LOD score (suggestive linkage =1.9; significant linkage = 3.3) along the q arm of chromosome 3 for small intestine Trichostrongylus nematode numbers. The centromere is located on the left hand side. The peak of LOD 0.931 for sire 1 is located 15 cm centromeric to OarVH34 and the estimated magnitude of the effect at this location is 12,700 Trichostrongylus nematodes. Corresponding figures for ^^5 sire 2 are LOD 0.994, 19cM and 7050 nematodes.

Figure 4 shows a graph of the LOD score (suggestive linkage = 1.9; significant linkage = 3.3) along the q arm of chromosome 3 for log(mean FEC1+50). The centromere is located on the left hand side. The peak of LOD 2.448 for sire 3 is located 6 cM 30 telomeric to OarVH34 and the estimated magnitude of the effect at this location is a 52% reduction in faecal strongyle egg numbers per gram of faeces.

Figure 5 shows a graph of the LOD score (suggestive linkage = 1.9; significant linkage = 3.3) along the q arm of chromosome 3 for the trait log(mean FEC2 +50). The 35 centromere is located on the left hand side. The peak of LOD 2.459 for sire I is located 19 cM centromeric to OarVH34 and the estimated magnitude of the effect at this location is a reduction of 74% in faecal egg count numbers. 111533 WGN* Figure 6 shows an autoradiograph of a Southern blot of genomic DNA from sheep that has been restricted with Taql and separated according to size on agarose, Southern blotted and subsequently hybridised with a 780bp DNA probe derived from ovine cDNA for IFNG (Mclnnes et al. 1990). The probe was labelled with 32P using the random prime method.

Figure 7 is a graph of the transformed probability score for all sires combined (suggestive linkage = 2.8; significant linkage = 4.3) along chromosome 3q for log(X+100) transformed; abomasal Trichostrongylus (LATRICH), small intestine Nematodirus (LSINEM), , small intestine Trichostrongylus (LSITRI), small intestine Strongyloid.es (LSISTR); Iog(mean X+50) transformed, strongyles and Nematodirus at the end of the first challenge (LFECl.SNEMl) and the end of the second challenge, (LFEC2,SNEM2) The centromere is located on the left hand side.

Figure 8 is a graph of the transformed probability score (suggestive linkage = 2.8; significant linkage = 4.3) along the q arm of chromosome 3 for the trait log(mean FEC2 +50) for individual sires. The centromere is located on the left hand side. The peak for sire 92066 is located 4 cM centromeric to OarVH34, corresponding figures for 920153 and 930124 are 32 and 22cM telomeric of VH34.

Figure 9 is a graph of the transformed probability score (suggestive linkage = 2.8; significant linkage = 4.3) along the q arm of chromosome 3 for log(mean FEC1+50). The centromere is located on the left hand side. The peak of for sire 920155 is located 10 cM centromeric of OarVH34. Corresponding figures for 920066 is 8cM telomeric of VH34.

Figure 10 is the complete sequence of the ovine interferon gamma gene.

Figure 11 is the sequence of Figure 10 showing mutations which are indicative of susceptibility to nematode parasites. Differences from the sequence of Figure 10 are shown as bold lower case letters below the nucleotide from which they differ. Absent nucleotides are shown as 111533 WGN * 6 DESCRIPTION OF THE INVENTION As described above, the invention provides in its primary aspect, a method for testing a farmed animal for resistance or susceptibility to nematode parasites. The method is particularly useful in selection of breeding grazing animals where resistance to parasite infection is important. It may also be used to identify animals suitable or unsuitable for particular types of farming. For example, highly susceptible animals would be unsuitable for grazing at high density with minimal anti-parasite drenching.

Sheep, cattle, goats and deer are included among suitable subjects for the method of the invention. The method is particularly useful for sheep.

The method of the invention involves in vitro analysis of nucleic acid obtained from the animal being tested. The nucleic acid will be that which codes for the protein interferon gamma for the animal. By "codes for", it is meant, inclusively, DNA which encodes the protein interferon gamma (the exons); the non-transcribed intervening regions (the introns); the regulatory elements; and the transcribed mRNA.

The term "gene encoding interferon gamma" will therefore be understood to mean the DNA sequence present in the genome of the animal including the exons, introns and regulatory elements.

The nucleic acid to be tested can be extracted from the animal in any convenient manner. Blood samples are currently preferred for this, although this is by no means critical. Typically, genomic DNA is then extracted from these by known methods, such as those exemplified below.

The focus of the analysis of nucleic acid obtained from the animal is to determine the presence or absence of a conservative mutation (alteration) in the gene encoding interferon gamma. By "conservative mutation" it is meant that the mutation does not prevent expression of the protein interferon gamma and that the protein as expressed has its wild-type amino acid sequence. This means that the mutations can be, for example, in the non-transcribed regions and be insertion, deletion or substitution mutations. The mutations can also be in the exons which code for the protein, 111533 WGN » 7 provided that each codon in-frame codes for the same amino acid post-mutation as pre-mutation.

There are numerous in vitro methods for analysing the nucleic acid sample which are 5 useful in the present invention. Included among these are analysis of microsatellites, of single nucleotide polymorphisms (SNPs), Taqman detection of SNPs and restriction fragment length polymorphisms (RFLP's).

Microsatellites are regions of DNA sequence that contain a simple sequence of 2, 3 or 4 10 nucleotides in length repeated tandemly up to 30 times. The number of repeats varies and analysis involves the detection of changes in the number of tandemly arrayed simple sequence repeats. In one method of analysis DNA primers are designed to amplify the region of DNA containing the microsatellite. One of the primers is labelled with 33P and incorporated into PCR reactions. Following completion of the PCR 15 reactions the products of the reaction may be distinguished according to their size using polyaciylamide gel electrophoresis followed by autoradiography of the gel to detect products of the PCR reaction.

The sequences of ruminant interferon gamma genes are available from Genbank. For 20 example, coding sequences for capra hircus and bovine interferon gamma (mRNA) are available under Genback Accession No. U34232 and M29867, respectively.

A complete sequence of the ovine interferon gamma gene is shown below. The position of all single nucleotide polymorphisms (SNP's) found to differ between highly selected 25 resistant sheep and susceptible sheep are shown. The sequence of a resistant sheep is shown in full. Differences discovered in the susceptible animals are shown as bold lower case letters below the nucleotide from which they differ. To the right hand side of each line the cumulative total of the number of nucleotides in the sequence is given. The start codon, ATG is shown in bold (nucleotides 575-577); the stop codon TAA is 30 also in bold (nucleotides 4780-4782). The coding sequence (in 4 exons) is shown underlined. In addition to the 36 SNP's at nucleotides 188, 529, 538, 905, 1149, 1270, 1294, 1349, 1620, 1686, 1717, 1923, 1989, 2118, 2173, 2252, 2456, 2457, 2458, 2461, 2816, 2978, 2979, 3041, 3359, 3488, 3516, 3540, 3955, 4051, 4059, 4120, 4370, 4379, 4441, 4519. There is a tetranucleotide microsatellite (repeat unit GTTT) at position 35 853, (Schmidt at al 1997) an insertion / deletion of a single T nucleotide at position 1524 and an insertion / deletion of an 8 nucleotide sequence, GATGGGGG, beginning at position 2448. All of these DNA sequence differences can be utilised either 111533 WGN* 8 individually or in combination to develop tests to identify individuals resistant or susceptible to nematode parasites.

TTGCTTTCTGGTCATTTGCAAGAAAAATTTTAAGAGGCACCCCCTCTGTA 5 0 AAGGTTTGAGAGC C C C TAGAATTTC C TTTTTCAC TTGC TTCAAAC CACAA 10 0 AAGAATGATCAATGTGATCTCTGGATGAGGAGTCAACATTTTACAAGGGC 15 0 AAAGGAGGAGGTGTACAAAAATTTC CAATC C CTGGAC aGTG TGAAGTGAA 2 0 0 g AG TGTTTTCAAAGGATC C CACAAGAATGG CATGGGTGGG CATAATGGGTC 2 50 TG TC TC C TCGTCAAAAGAC C CAAGGAGTTGAAAGGAAAC TCTAACTACAA 3 0 0 GACCAAAATGCCACAAAACCATAGTTATTAATACCAACTAACTAGCATCT 3 5 0 CTGTCTATCTGTCACCATCTCATCTTAAAAAACTTGTGAAAATACGTAAT 400 CTTGATGAGACTTCAATTAGGTATAAATACCAGCACCAAAAGGAGACACA 450 GCACATTCTTCTGATCATCTGCAGATCAACAATTAGAAAAGAAAGATCAG 500 C TAC C TCCTTGGGAC C TGATCATAACG C aGGAGC TAC C GATTTCAACTAC 550 g t TC CGGC CTAACTCTCTC C TAAACGATGAAATACACAAGCTC CTTC TTAGC 600 TTTACTGCTCTGTGTGCTTTTGGGTTTTTCTGGTTCTTATGGCCAGGGCC 650 CATTTTTTAAAGAAATAGAAAACTTAAAGGAGTATTTTGTAAGTATGATC 700 TTTTAATGAGACTTTCTTGTGATTGACTTGATGTTTGATGTTGACTTGGA 7 50 ATTCTATTTGTGATGGGCTCTCATCTCTAGTCTTCAGTCATTTTGAGAAG 800 acttggtgttattgtgactgttggctagctgtgtttgtttgtttgtttgt 850 TTg111GACTAAACGATC TCTGCTCAGTTTG C TACAGAGATTTAGGAGGG 900 ( ) ATTCgTGAATCCTCCAAAAGATGGGCATAATATGGGTATATATCATAGTG 950 a TTGATATTTTGTGATGAGAATCCATTTAACTATGATGTTAAGTAGTAAGC 1000 ATACTGAAAAGACAGCTGAGTGTTAATTTTTGATTTCTCATGTTACTCTA 1050 ATGGTGACAGTCAAAAGTCAACATCAACATTAAAAATGGTCATTTGAAAT 1100 AATTGATC AATTAAAG AAGATGAGGTC C TTAAATATTTGGC TTAAAAA t A 1150 c AAAACACCTATAGGTGTTTTTCTCTAAAAATTTATTTTAAAATTTTACTA 12 00 TCCCATCTAGATTGTCTGTGAATTACTCTTGGACTCAGTCACTTGCTGAG 12 50 ATTTTTTTTGTAGGAGTTAcAATAATCTAGTTTATATTTAAAAaTTTTTT 13 00 t t GTCTTGTTTTATGAATTTCTTAAAAAATTATTTATGGTTAATTAAAATcG 1350 a CTTGTGCATTTTAAATATTTTCTCATATGTCCAAATTTAGCTATTATAAT 1400 111533 WGN* 9 TATAGCTGGAGCTATCTTCTAAAGGCGATATAATATAGGAGGACACAGGT 145 0 AGAGGTTACAGAAACCTATACCATAGGGGGGCAGTATTTTATAGTGAGAT 15 0 0 GGTG ATGTTGATTTTTTTTTTTT t AAC T ATACC TTTTTC TTAC C ATAGT A 15 5 0 (-) ATTTGGGACAGCTGAAGTATCATCAGTCTTGAATGAGTTCATTGTGATGT 1600 TATACTAGTAAATAAGAAAaCAAAAATCACATGATAGTCTTTAGCTATTA 1650 g TCCAACAGGATAGCTTTTGAGACTATTTTTTGTTAaAACTAAACAGTGTT 1700 g GAAAAGAAAATCAGTAaATATTGCCAACATCCAAGTTCAATAAAACTTGA 17 50 c GGTACATTTTTACAGCAATTTATGGACTAATTTTAAGCCAATTTTTTCTT 1800 GTGTTGTTTTATTTTCCAGAATGCAAGTAACCCAGATGTAGCTAAGGGTG 1850 GG C CTC TTTTCTC AGAAATTTTGAAGAATTGGAAAGAGGTAAGC TG AAC A 1900 ^15 TTTCCATTTGGCTGATTTTCCTgTTGCTTTTTTTCTGATGGATAAATTCA 1950 a CATCAACCTCTCTTTGTGCTCTTTTCTCCCAAGGAGAGtGACAAAAAGAT 2 000 C TATTCAGAGCCAAATTGTCTCCTTCTACTTCAAACTCTTTGAAAACCTCA 2 050 AAGATAACCAGGTCATTCAAAGGAGCATGGATATCATCAAGCAAGACATG 2100 TTTCAGAAGTTCTTGAAcGGCAGCTCTGAGAAACTGGAGGACTTCAAAAG 2150 t G C TGATTC AAATTCCGGTGAGAqGATC TTAATG C TTTCTTTGGT ATC ATT 2200 t GCAGAGACTCTTACAAAGCACTTCATTCCCAGAAAATAGAAATTAGATAC 22 50 CaGGTATAAAGCTCTGAAGTGAAGTTGTAGTCAAAATGCCTCTTTGCTAG 23 0 0 g TTCTCTTTTGCCTTGGGTGACTTTGCAGAGTCAGTTATTGGAGGCACTTA 23 5 0 GGTGAATGGAC AAATC AGTGAGGAGGAAGC AG AG AAGGAGTTGGGGTga t 245 0 ( —- gggggc agAGt attgaaaac caattctccc attgcc c cttgtctctgttg 2500 tga a) gaaattctcttgctttgaatagcttgcatgtcttaatggaaagagcagtg 2550 GGAGGGAGAGGGAAGATGTGTGCTCCGCCCAGCTTAGCCACCAAAGAAAT 2600 GTGTGACTTCAGATGAATCACAGGCCTGGCTGGGGCTGTTTCCTCATCTT 2650 aaaag aagc c tgttgag ttc ac tgtaatttct atggtc tc ctttg c tcta 2700 aaattc tacaatgc cgtag atag aaaatg acaatg agatagg ag aaagg a 2750 at agc c tttgaggagac aaaggccc tc cc ac tgtcg aac tctgg tc aac c 2800 40 ATGCAGATTGTGTAGtCCAAGGAGAGGTGATTGCATTTGTTTTTAGAAAG 2850 c aaaataggtagaattgtc c c atc aatgaatctttaaagtttttagaagc t 2900 111533 WGN * GAAAGATAACTTTGAAG CATAAGGACAAG TCACATTTTC CAAAATTTC C T 2 9 5 0 CTAAGGGACAAAGG C C TC CAGATTTTTt C GGGGGGAGAGTTGGC TTTTG T 3 000 gg ATAAATAGGCTTGGGTGAGAAGAGGAGGGGTGCTTGTGTTcTCAGGATTT 3 050 5 t GTACCAGCTCTTGCTCTAACCCTGTCTGCCTCTCAACAATCTCGTATAAA 3100 CTC C CATGAACTGAAAGAAGTGATTGC TCTTCAAGATTTC TCTAGTCTC C 3150 ATATATAAGATGGTATGAGAGTGGC TTGGAAAAATTATG C GTAATGTTTT 32 00 CCATGTTCAGCAGATCTCCATTGGTTGCAGGCTAGAGAATAAAAAAGCAC 32 50 TGTTCCTAACCCAGTGCTGCCTCTGGCTGCTGTTCAGTCCTAGAGAAGTC 33 00 CCTTAGTTTCTTGGTGATGCTTAGAAAACTGGAAAGTGAAATTTATCACC 33 50 TGTAAACAcACAGCTGGTAGAAATTTTTCTACTCTGTTGTAACTGGGCTT 3400 t ) TGAAAACAATAGGTTGGTCCCATCCTTGTGGAAGCTCCAAGCCTCCTGCT 3450 TCACAATGTGCAATC TTGAGTGAGTGC TCAAC CATTGcCGTCTTTTGCTC 3 500 t AAATG TGAGCATATTgAAGGCATG TTTTGC C TTTTTGTCc TGTAAC TTAG 3 550 t g AATCTCCTAGTCATTCTCAGAAATGTGGCAAGGTGGCTGCAGTGTTTCCA 3 600 G CTTTAGAAAGAAATGTAG TGGCAGAG CAAAGATGAAGCAAATGGTCCT C 3 650 attttatgaagtgtgaattcacctatatggtcctactgggaagagtgaga 37 00 AAATAGTTCAGTTTGCTGGAATGGAAGGCAGGGTACAGGAAGATTGCTTT 3750 CATTACTAATTTTATTATATAATGCCAGTAATTGATTATTACCATTTAAA 3 800 TAAGATCAGTGTTTATTTTTAATCCCATCATGGAAATGATGAGACTGACC 3 850 TAATTTGGAGGAAATTAAAAATTATGGTTTTCCTCCTTCTAGAACTCGGT 3 900 GTTTATAAAGTAC TGAAC C TATAC CAGTGG TTTTCAAACTTGGGTGGTG C 3 950 CTAAaTCACCTGGTGATGTTGGTAACCACACAATGGCTGAGGCCTAGTCC 4000 g GATTTCATCCACCTGGGTGTCAGAGTTCTTCATTAGGTCCTCAGGTTACT 4050 c TCTTACAtAC CAAGCATGAGAGC CATTGGCTATAGTAACTCATCTGATA 4100 g c CTTATAGCAAAGACTAAGTaCTAAGCGATAGAAGTGACAGAGACCCTGGG 4150 g GATCATCTAAACAATCTCATACTTAGTGCAGAAAACAGAGATCTGGAGAG 4200 3 5 CTTC AGTGACTGGCTTAAAGTCAG ATGTATTGC ACAC AC AGAATTCAC AC 4250 CCACATTTTTCTGTCACTCCACTTCTGGGTTTTTTTCACTATATAATTTT 43 00 CAGATCCCTGAAGTGATAAATTTATGGATATGTGATGTGCTCCATCCTGT 43 5 0 TACAGCACAACTGTCAATTaAATTGTTAcAATTTTAGTCTTTATCACTGA 440 0 g t 40 AGAAACCAACATTACATATTAAAGTTCATTACTGCTAGTGaAAATAATTT 445 0 11L533 WGN * 11 3 GTTTTAAAGAATACTTTGATTTTCTTGGGCCTAGACAGCAGAATATCCTA 4500 ATGAC TCATATG CTTGAAaTTAATTTTG C TGTTTTC TTTC CCAATAGG TG 4550 g GATGATCTGCAGATCCAGCGCAAAGCCATCAATGAACTCATCAAGGTGAT 4600 GAATGACCTGTCGCCAAAATCTAACCTCAGAAAGCGGAAGAGAAGTCAGA 4650 ATCTCTTTCGAGGCCGGAGAGCATCAATGTAATGGTTCTCCTGCCTGCAA 4700 TATTTGAATTTTAAAATCTAAATCTATTTATTAATATTTAATATTTTACA 4750 TTATTTATATAGGGAATATATATTTAGACTTATCAAAGTATTTATAATAG 4800 TAACTTTTATGTCATGAAAATGAGTATCTATTAATATATGTG 4842 An example of such a typing system is shown below: This demonstrates the typing of two SNP's at positions 529 and 538. Shown above or below their sequence are all 15 the primers used to type the two SNP's.

'GATCCCACAAGAATGGC3' (SNP1F) 'GATCCCACAAGAATGGC3' (SNP2F) 20 5 ' GGATCCCACAAGAATGGCATGGGTGGGCATAATGGGTCTGTCTCCTCGTCAAAAGACC CAAGGAGTTGAAAGGAAACTCTAACTACAAGACCAAAATGCCACAAAACCATAGTTATTA ATACCAACTAACTAGCATCTCTGTCTATCTGTCACCATCTCATCTTAAAAAACTTGTGAA AATACGTAATCTTGATGAGACTTCAATTAGGTATAAATACCAGCACCAAAAGGAGACACA GCACATTCTTCTGATCATCTGCAGATCAACAATTAGAAAAGAAAGATCAGCTACCTCCTT 25 GGGACCTGATCATAACGC (G/ A) GGAGCTAC (C / T) GATTTCAACTACTCCGGCCTAACT 3'C CCTCGATG A CTAAAGTTGATG5' (SNP1G) 3'T CCTCGATG G CTAAAGTTGATG5' (SNP1A) { SNP2C)3'GCTAAAGTTGATGAGGCCG5' (SNP2T)3'ACTAAAGTTGATGAGGCCG5' 30 CTCTCC TAAACG ATGAAA TA CA CAAGCTCCTTCTTAGCTTTA CTGCTCTGTGTGCTTTTG GGTTTTTCTGGTTCTTATGGCCAGGGCCCATTTTTTAAAGAAATAGAAAACTTAAAGGAG 11L533 WGN ' 12 TATTTTGTAAGTATGATCTTTTAATGAGACTTTCTTGTGATTGACTTGATGTTTGATGTT GACTTGGAATTCTATTTGTGATGGGCTCTCATCTCTAGTCTTCAGTCATTTTGAGAAGAC 3'GAGTAGAGATCAGAAGTCAGTAA5'(SNPlR) 3'GAGTAGAGATCAGAAGTCAGTAA5'(SNP2R) 5 TTGGTGTTATTGTGACTGTTGGCTAGCTGTGTTTGTTTGTTTGTTTGTTT(GTTT}GACT AAACGATCTCTGCTCAGTTT Another method that can be used for detection of SNP's uses the "Taqman" system (Holland et al., 1991). This system allows the typing of SNP markers without needing ^^10 an electrophoresis step. Instead the different alleles are detected by a change in the colour of the PCR reaction.

Using a fluorogenic probe complementary for the target DNA sequences being amplified the system detects and quantifies cycle by cycle increases in the level of PCR 15 products. The probe consists of an oligonucleotide with a reporter and quencher dye attached. Uncoupling of the two dyes, which occurs when the probe, bound to the internal sequence of the PCR product, is cleaved by the nucleolytic activity of the Taq polymerase, results in an increase in the fluorescence intensity of the reporter dye.

For SNP analysis, competition between oligonucleotides differing only at the point mutation is used. The two allele specific oligonucleotides are labelled with different dyes so that binding by the "correct" oligonucleotide is detected by increased fluorescence of the particular dye. With a heterozygote a mixture of the two dyes is released and detected.

Another method that may be used is restriction fragment length polymorphisms (RFLP's). This method uses restriction endonuclease digestion of genomic DNA, its separation by size using agarose electrophoresis and detection and analysis of the DNA sequence by Southern blotting (eg. Sambrook et al, 1989) using eg. a 780bp DNA probe 30 derived from ovine cDNA for gamma interferon (Mclnnes et al 1990).

Other methods are also suitable for detecting these and other SNPs as is well known in the art. The critical feature is that the method used directly or indirectly detects the 11L533 WGN • 13 presence or absence of a DNA sequence associated with resistance or susceptibility to nematode parasites.

EXAMPLES The following Examples further describe practice of the invention. It will be appreciated that these Examples are illustrative only and are not intended to limit the invention in any way.

EXAMPLE 1 GENOTYPING OF SUSCEPTIBLE AND RESISTANT ANIMALS WITH MICROSATELLITE MARKERS FROM CHROMOSOME 3.

Overview of the experimental approach.

To identify polymorphic loci affecting the development of host resistance the following progressive "screening" strategy was used. Firstly, alleles conferring host resistance, or susceptibility, were initially concentrated by the use of divergent selection lines. The principal selection criteria being strongyle faecal egg count in lambs. After a number 20 of generations (4-5 max) extreme individuals from these lines were then crossed and a small number of F1 sires were then mated to unrelated and unselected ewes to create large half-sib populations. The resulting outcross progeny were evaluated for host resistance to internal parasites. Then the F1 sires and their outcross progeny, which should be segregating for presumptive alleles affecting host resistance, were genotyped with microsatellite markers evenly spaced along the genome. The resulting data was analysed by a variety of statistical techniques to identify regions of the genome where there was evidence of segregation. Subsequently, these results can be independently tested in additional flocks or on the parental flocks.

To identify the likely polymorphic gene involved, an alternative approach was used. The previously localisation, described above would only identify a 10 to 25 cM region of the genome, so likely known candidate genes in this region affecting the trait of interest were then identified by the use of database searches and comparative mapping using the scientific literature. In this case one of the candidate genes, 35 interferon gamma had already been cloned and sequenced and a microsatellite marker identified within intron 1 of the gene (Radford et al 1991; Schmidt et al. 1996). Using this published information, DNA from a small number of animals from the parental 111533 WGN* 14 selection lines were collected and genotyped with the published microsatellite marker. If the marker was close to (<0.1 to 5cM depending on previous population history prior to commencement of selection) the actual polymorphic gene affecting host resistance, and given a restricted set of initial conditions, a marked difference in allele frequencies 5 between the parental lines should be observed. The significance of these differences can be tested statistically, based on the pedigrees of the sampled individuals using the computer program PEDRIFT (Dodds et al 1997). These results being considered definitive independent evidence that the gene, its control sequences, or a closely linked gene were selected out by the breeding process used to create selection lines and 10 hence will have an effect on resistance to parasitic nematodes.

MATERIALS AND METHODS Breeding the Wallaceville selection lines and establishing the outcross 15 segregation pedigrees.

The Wallaceville divergent FEC selection lines of Romneys commenced in 1979 (Baker et al. 1991; Bisset et al, 1996). Selection is currently based on FEC levels after natural challenge, measured several times during the first 8 months of life, in both ram arid ewe lambs. At present, the lambs in the high line (susceptible animals) have an 8 fold 20 higher FEC than the low line (resistant animals) during the autumn. On a logarithmic scale this translates to a 2.6 cfp divergence between lines. As the selection lines involved a relatively small number of animals outcross rather than backcross pedigrees were initially used to search for regions of the genome segregating with resistance or susceptibility to parasites. F1 Rams derived from extreme reciprocal crosses of the ^^25 selection lines were mated with between 100 and 300 unselected Coopworth ewes to generate 5 half-sib pedigrees segregating for parasite resistance (Table 1).

Table 1: Half-sib outcross sheep families used to detect QTL for disease resistance Sire identification Number of progeny Sire 1 92/0066 225 Sire 2 92/0153 175 Sire 3 92/0154 348 Sire 4 92/0155 111 Sire 5 93/0124 101 1 i 1 533 WGN * Phenotype measurements of parasite resistance in outcross progeny.

Two natural field challenges of infective nematode larvae (Baker et al. 1991) were given to all outcross lambs. Faecal egg counts (FEC) were determined using the McMaster method (Whitlock and Marsden, 1957) with 3 separate samples taken over 5 days at 5 the end of each challenge and results averaged. Mean strongyle and Nematodirus infection levels (epg) of 1091 (FECI) and 70 respectively were achieved for the first challenge and 1462 (FEC2) and 36 respectively for the second challenge. Live weight measurements were taken at birth, weaning, and after each challenge. T colubriformis L3 antibodies were measured by ELISA (Douch et al. 1994) from serum samples taken 10 at 5 times; at the start, middle and end of each challenge. Dagginess was scored at weaning, and after each challenge using the scale described by McEwan et al. (1992). At slaughter, which took place approximately a week after the end of the second I challenge, the first 10m of small intestine and the abomasum were collected and adult parasites in the lumen were identified to genus and counted.

DNA Purification Cloning and Manipulation.

Blood was collected from animals by venipuncture of the jugular vein and DNA was purified from leukocytes using standard methods (Montgomery and Sise 1990). The AgResearch Molecular Biology Unit where all DNA manipulations were performed 20 has a CO containment categorisation from the University of Otago Safety Committee.

DNA samples were collected from all the parents of the F1 rams, the rams themselves and all their progeny. In addition DNA from randomly selected individuals from the parental selection lines was also obtained. ^^5 Ge no typing.

The microsatellite markers were amplified using the polymerase chain reaction following the method described by Crawford et al. (1991). The amplified products were separated according to their size by polyacrylamide gel electrophoresis. The separated 30 products were detected by autoradiography of the dried gel. A list of all the microsatellites used for the chromosome 3 analysis is shown in Figure 1. The IFNG microsatellite of Schmidt et al (1996) was amplified and analysed using the published method DNA Sequencing.

The commercial service operated by the University of Otago, Centre for Gene Research (ABI 373 automated sequencer) was used to sequence all subclones and PCR products. 111533 WGN * 16 Data Analysis: Outcross pedigrees. Initially the 5 sires were genotyped at 178 loci spaced at approximately 20 cM intervals throughout the genome. Whenever a sire was 5 heterozygous at any of these loci (Figure 1) the 22 most susceptible and the 22 most resistant progeny were genotyped to determine, where possible, which allele they had received from the sire. Preliminary analyses were also conducted for each trait so as to identify what fixed effects (year, farm, sex, birthday, birth and rearing rank, dam age) and transformations (generally logarithmic) were appropriate for each trait. We then 10 used single marker regression methods (SAS, PROC GLM) for initial analyses with inherited sire allele nested within sire and the appropriate fixed effects to determine which regions of the genome were associated with host resistance to internal parasites, ^ Subsequently, selected chromosomes showing evidence of marker linkage to host resistance traits were reanalysed using a maximum likelihood technique, Animap 15 (Georges et al 1995). This technique allows all the genotype information from all markers on the chromosome from all sires and progeny to be analysed simultaneously for a single host resistance measurement. The results provide estimates of both the allele substitution size, genomic position and probability of any QTL affecting the trait. The data, however, needs to be adjusted prior to analysis for any other fixed effects 20 (see above) and when only extreme individuals are genotyped, as in this experiment, the allele substitution effects are overestimated and provide only an upper bound on the magnitude of the likely effects. Based on this information chromosome 3 was selected to be genotyped at additional markers and also a subset of these markers were genotyped for all informative half sib progeny groups. This revised information was then mapped using the regression technique described by Knott et al. (1996). Prior to analysis, the data was checked and any anomalies resolved using: genotype exclusion, allele frequency disequilibrium in progeny, and comparison of estimated and published (de Gortari et al. 1998) map distances for the sheep markers used. Map distances used in the final analysis were from de Gortari et al. (1998) as these were 30 considered more accurate than those estimated from the widely spaced markers used in this experiment.

Genotype differences between lines The analysis technique and the assumptions used have been described Dodds et al 35 (1997). 11L533 WGN* 17 RESULTS AND DISCUSSION Outcross pedigrees.

The 22 most susceptible and 22 most resistant animals from the 5 outcross pedigrees 5 (see Figure 1) were genotyped with 15 microsatellite markers from chromosome 3. The pedigree was genotyped whenever the sire was heterozygous at the locus . The approximate position of each marker used and for what sire each marker was hetero2ygous is shown in Figure 1.

Initial single marker regression techniques identified a region on chromosome 3q to resistance/susceptibility to parasites using 4 different phenotypic measurements: log(FECl + 50), log (FEC2 + 50), abomasum Trichostrongylus numbers, and small | intestine Trichostrongylus numbers. Subsequent analysis using maximum likelihood analysis (Animap) on the chromosome 3q region provided evidence for segregation in 2 15 of the 5 families (see Figures 2-5 Based on the criteria of Lander and Kruglyak (1995) "suggestive linkage" occurs in sheep half-sib experiments when P<0.0016 i.e. when the LOD score exceeds 2.17and "significant linkage" occurs when P<0.0000505 i.e. when the LOD score exceeds 3.60. These results demonstrate that "suggestive linkage" occurred for three host resistance traits and in every case the effect was located close 20 to marker OarVH34. Figures 7 and 8 provide information after the additional genotyping was undertaken. In both cases presented the magnitude of the peaks declined. Estimates of the allele substitution effects from this analysis were derived at the IFNG locus (216cM or 6cM telomeric of VH34) for sires 920066 (0.33ap, 5% difference in FEC) and 920155 (0.66aP) 29%) for log(mean FEC 1+50). Corresponding 25 figures were derived for log(meanFEC2 +50) for sire 920066 (0.438op, 16%) and 930124 (0.534op, 31%). To accurately estimate the likelihood that these observations could occur by chance is difficult, particularly given the lack of genome wide significance. In these cases it is better to either repeat the experiment or confirm the results independently using a different approach. The latter approach was used here where 30 linkage disequilibrium or allele association studies showed that there had been significant selection for particular alleles of the IFNG gene in the resistant and susceptible selection line as described below. 111533 WGN * 18 Parental line allele frequency differences.

A microsatellite marker for the human interferon-gamma (IFNG) gene has previously been developed (Schmidt et al 1996). We used the method described by Schmidt et al (1996) to genotype sheep for this marker.

The microsatellite marker was first used to place the IFNG gene on the sheep genetic linkage map. (Crawford et al 1995). Two-point linkage analysis of the IFNG genotypes in the AgResearch International Mapping Flock with all the other markers that had been typed in the same flock (in excess of 500 markers) placed the gene on sheep 10 chromosome 3q approximately 7 cM centromeric of the microsatellite marker OarVH34.

The IFNG microsatellite was then examined to determine whether there was any significant association of a particular marker allele with either the resistant or 15 susceptible selection lines using PEDDRIFT. The results are presented in Table 2. The result shown for the IFNG microsatellite is clearly significant (P<0.05). This result is evidence that this marker and other markers in and around the IFNG gene can be used to identify sheep resistant to nematode parasites. This evidence is reinforced by the "suggestive linkages" for resistance traits and their localisation from the outcross 20 experiment, which used rams generated from the same source.

Table 2: The number of animals with various genotypes in Wallaceville Parasite selection line progeny for a microsatellite located within intron 1 of the INFG gene.

Marker Selection line Marker Alleles (No. of animals) Significance1 AA AB BB IFNG microsatellite Susceptible 1 15 34 P= 0.021 Resistant 17 25 8 significance was determined using PEDRIFT analysis (Dodds and McEwan 1997) which simulates inheritance of these alleles in the actual selection line pedigrees. This methodology is much more conservative than traditional Chi square analysis. 40 111533 WGN* 19 EXAMPLE 2 SINGLE NUCLEOTIDE POLYMORPHISMS.

Sequencing of the promoter region, the 5' untranslated region, exon 1 and part of intron 1 of the IFNG gene has revealed the following sequence. The sequence coding for the amino terminus of the IFNG protein is underlined with the signal peptide (which is cleaved off the protein prior to being secreted by the cell) sequence shown in italics. The Upstream end of intron one is shown in bold and the GTTT sequence whose presence or absence is the basis of the microsatellite marker is bracketed.

'GGATCCCACAAGAATGGCATGGGTGGGCATAATGGGTCTGTCTCCTCGTCAAAAGACC CAAGGAGTTGAAAGGAAACTCTAACTACAAGACCAAAATGCCACAAAACCATAGTTATTAA TACC AACT AACTAGC AT CTCTGTCTATCTGT C AC C ATCTC ATCTTAAAAAACTTGTG AAAAT AC GTAATCTTG ATG AG ACTTC AATT AGGTAT AAATAC C AGC ACC AAAAGG AG AC AC AGC AC 15 ATTCTTCTGATCATCTGCAGATCAACAATTAGAAAAGAAAGATCAGCTACCTCCTTGGGAC CTG ATC ATAACGC (g/ a) GG AGCTAC (c /1) GATTTC AACTACTCCGGCCTAACTCTCTC CTAA AC GATGAAATA CACAA GCTCCTTCTTAGCTTTACTGCTCTGTGTGCTTTTGGGTTTTTCTGG TTCTTATGGCC AGGGC C C ATTTTTTAAAG AAAT AGAAAACTT AAAGGAGT ATTTTGTAAGTA TGATCTTTTAATGAGACTTTCTTGTGATTGACTTGATGTTTGATGTTGACTTGGAATT 2 0 CTATTTGTGATGGGCTCTC ATCTCTAGTCTTC AGTCATTTTGAGAAGACTTGGTGTTA TTGTGACTGTTGGCTAGCTGTGTTTGTTTGTTTGTTTGTTT(GTTT)GACTAAACGATCT CTGCTCAGTTT 3' The sequence was derived from three sheep from the parasite susceptible line and 25 three sheep from the parasite resistant line. Parts of this sequence have been published previously (Mclnnes et al. 1990 Genbank Accession # X52640; Schmidt,P et al. 1997 Genbank Accession # Z92887) but this is the first time that the complete 5' untranslated sequence has been identified. Two SNP's were identified in the 5' untranslated region of the IFNG gene. The two SNP's are shown in brackets with the 30 two alternative nucleotides at each position within the bracket. The A/G polymorphism we have called SNP1 and the C/T polymorphism we have called SNP2.

Ill S33 WGN ' The typing method of Xhu and Clarke (1996) was used to type the SNP2 polymorphism. In the diagram of the sequence below all the primers used to type SNP2 are shown above or below the DNA sequence they are derived from. 5'GATCCCACAAGAATGGC3' (SNP2 F) 'GGATCCCACAAGAATGGCATGGGTGGGCATAATGGGTCT GTCTC C TC GTCAAAAGAC CCAAGGAGTTGAAAGGAAACTC TAAC TACAAGACCAAAATG C CACAA AACCATAGTTATTAATACCAACTAACTAGCATCTCTGTCTATCTGTCACCATCTCATCTTAAAAA ACTTGTGAAAATACGTAATCTTGATGAGACTTCAATTAGGTATAAATACCAGCACCAAAAGGAGA 10 CACAGCACATTCTTCTGATCATCTGCAGATCAACAATTAGAAAAGAAAGATCAGCTACCTCCTTG GGACCTGATCATAACGC(G/A)GGAGCTAC(C/T)GATTTCAACTACTCCGGCCTAACT (SNP2C)3'GCTAAAGTTGATGAGGCCG5' (SNP2T)3'ACTAAAGTTGATGAGGCCG5' C TCTC C TAAACGA TGAAATACACAAGCTCCTTCTTAGCTTTACTGCTCTGTGTGCTTTTGGGTTT 15 TTCTGGTTC TTATGGCCAGGG C C CATTTTTTAAAGAAATAGAAAAC TTAAAGGAGTATTTTGTAA GTATGATCTTTTAATGAGACTTTCTTGTGATTGACTTGATGTTTGATGTTGACTTGGAATTCTAT TTGTGATGGGCTCTCATCTCTAGTCTTCAGTCATTTTGAGAAGACTTGGTGTTATTGTGACTGTT 3'GAGTAGAGATCAGAAGTCAGTAA5'(SNP2R) GGCTAGCTGTGTTTGTTTGTTTGTTTGTTT(GTTT)GACTAAACGATCTCTGCTCAGTTT..3' As shown in the sequence above the 4 primers used to type the SNP2 polymorphism were: SNP2F 5' GATCCCACAAGAATGGC SNP2R 5' AATG ACTG AAG ACT AG AGATG AG SNP2C 5' GCCGGAGTAGTTGAAATCG SNP2T 5' GCCGGAGTAGTTGAAATCA Using primers SNP2F, SNP2R and SNP2C, a CC animal will give 2 PCR products whereas TT animals will give only 1.

Using primers SNP2F, SNP2R and SNP2T, a TT animal will give 2 PCR products whereas CC animals will give only 1. 111533 WGN* 21 A heterozygote animal, CT, will give 2 PCR products with both reactions. In this way the selection line animals were typed.

The results are presented in Table 3.

Table 3: Genotypes in Wallaceville selection line progeny for a single nucleotide polymorphism located within the INFG gene on sheep chromosome 3q Marker Selection line Marker Alleles (No. of animals) Significance TT TC CC IFNG Point mutation Susceptible Resistant 28 1 12 7 4 32 P < 0.001 JO EXAMPLE 3 USE OF RFLP MARKER DNA was digested with Taql and separated according to size on agarose gels. The gels were then Southern blotted and then hybridised with a probe containing part of the DNA sequence of the IFNG gene. The probe comprised a 780 bp region of IFNG cDNA cloned into pBluescribe obtained from Dr C,J. Mclnnes, Moredun Research Institute, Scotland. The polymorphism detected (see Figure 6) is the presence (D allele} or absence (A allele) of a Taql restriction endonuclease site somewhere within the IFNG gene.

The D allele was strongly associated with resistance to nematode parasite resistance (see Table 4).

Table 4: Genotypes in Wallaceville selection line progeny for an RFLP located within the INFG gene on sheep chromosome 3q 40 Marker Selection line Marker Alleles (No. of animals) Significance* AA AD DD IFNG Point Susceptible 33 14 1 45 mutation Resistant 8 24 16 P =0.04 * significance determined using PEDRIFT software (Dodds and McEwan 1997) 1L1533 WGN* 22 Other RFLP's have been detected for this gene (Penty and Hill 1991} using Rsal, Mspl and Pstl restriction enzymes. It is likely that these other RFLP's will also prove to be 5 useful in detecting those animals resistant or susceptible to parasites.

CONCLUSION The above results demonstrate that a conservative mutation in the gene encoding 10 interferon gamma is an indicator of susceptibility to nematode parasite infection.

INDUSTRIAL APPLICATION The above results demonstrate the utility of the present method in selecting for 15 animals resistant to nematode parasites, or against animals susceptible to such parasites. The method has particular application to sheep, but also has utility in the selection of other grazing animals such as cattle, goat and deer.

For use in the method, materials useful for testing for resistance of animals to 20 nematode parasites can be provided. Such materials include nucleic acid sequences (usually DNA) useful as primers and/or probes for detecting sequences within the interferon gamma gene associated with high or low resistance to infection by nematode parasites and also to kits containing these materials. The kits may contain other conventional reagents useful for determining the presence or absence of sequences associated with resistance to nematode parasites.

Particularly preferred are primers/ sequences for use in detecting mutations in the ovine interferon gamma gene, including SNP1F, SNP1R, SNP1A, SNP1G, SNP2C, SNP2T; sequences comprising any of the four mentioned sequences having additional 30 nucleotides at the 5' terminal or 3' and 5' terminals for SNP IF and SNP1R; and fragments of these containing at least 15 nucleotides provided that for SNP1A, SNP1G, SNP2C and SNP2T the missing nucleotides are from the 5' terminal. Other primers include those in the form of an oligonucleotide having a 3' terminal sequence corresponding to the 3' sequence of SNP1A, SNP1G, SNP2C, or SNP2T or a sequence 35 having a 5' terminal complementary to that sequence. 111533 WGN * 23 Preferred kits for use in the invention contain probes/primers as described above, such as those of the preferred embodiment of the invention together with conventional reagents used in PCR or other amplification protocols.

Aspects of the invention have been described by way of example only in the above examples and it should be appreciated that modifications and additions thereto may be made without departing from the scope of the invention. 111533 WGN ■ 24 REFERENCES Baker, R.L., Watson, et al. (1991). In "Breeding for Disease Resistance" pl9 editors 5 G.D. Gray and R.R. Woolaston, Wool Research Development Corporation, Melbourne, Australia Bisset, S. A., A. Vlassoff, et al. (1996). "Nematode burdens and immunological responses following natural challenge in Romney lambs selectively bred for low or 10 high faecal worm egg count." Veterinary Parasitology 61(3-4): 249-263.

Crawford, A.M., F.C, Buchanan, and P.A. Swarbrick, (1991). The use of dinucleotide repeats or microsatellites as genetic markers in domestic animals. Proceedings of the New Zealand Society of Animal Production. SI: 79-83.

Dodds, K. G. and J, C, McEwan (1997). "Calculating exact probabilities of allele frequency differences in divergent selection lines." Proc. AAABG 12: 556-560.

Douch P. G. C., Green R. S. and Risdon P. L. 1994. Antibody responses of sheep to 20 challenge with Trichostrongylus colubriformis and the effect of dexamethasone treatment. International Journal for Parasitology 24: 921-928.

Georges, M., D. Nielsen, et al. (1995). "Mapping quantitative trait loci controlling milk production in dairy cattle by exploiting progeny testing." Genetics 139(2): 907-05 920.

Holland P.M., Abrahamson, R.D., Watson, R. and Gelfand, D.H. (1991) Detection of specific polymerase chain reaction products by utilising the 5' to 3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. 30 Sci. USA 88: 7276 - 7280 Lander, E. and L. Kruglyak (1995). "Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results." Nature Genetics 11(3): 241-247.

McEwan, J. C., P. Mason, et al. (1992). "Effect of selection for productive traits on internal parasite resistance in sheep." Proceeding of the New Zealand Society of Animal Production 52: 53-56. 111533 WGN* MclnneSjC. J., Logan,M., Redmond,J., Entrican,G. and Baird,G.D. (1990). The molecular cloning of the ovine gamma-interferon cDNA using the polymerase chain reaction. Nucleic Acids Res. 18: 4012.

McKenna, P. B., C. M. Allan, et al. (1995). The prevalence of anthelmintic resistance in ovine case submissions to animal health laboratories in New Zealand in 1993. New Zealand Veterinary Journal 42:151-152.

Montgomery, G. W. and J. A. Sise (1990). Extraction of DNA from sheep white blood cells. New Zealand Journal of Agricultural Research 33: 437-441.

Penty J.M, and Hill D.F. (1991) A Rsal restriction fragment length polymorphism at the ovine locus for gamma interferon. Animal Genetics 22: 439 Radford A.J., A.L.M. Hodgson,et al. (1991). Cloning and sequencing of the ovine gamma interferon gene. Australian Veterinary Journal 68: 82-84 Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning a Laboratory 20 Manual. (2nd Edition) CSHL Press. Cold Spring Harbour. NY.

Schmidt,P., Kuehn,C., Pitra,C., Tiemann,U. and Seyfert,H.M. (1997) Molecular cloning and nucleotide sequence of the bovine interferon-gamma gene. Genbank submission only Accession number Z92887 ™5 Schmidt, P., Ludt, C. et al (1996). A dialleleic tetranucleotide repeat (GTs)s or 6 within intron 1 of the ovine interferon gamma gene. Animal Genetics 27: 437-438 Whitlock, J. H. and H. Madsen (1957). "The inheritance of resistance to 30 Trichostrongylidosis in sheep. II. Observations on the genetic mechanism in Trichostrongyldosis." : 134-145.

Zhu, K.Y. and Clarke, J.M. (1996) Addition of a competitive primer can dramatically improve the specificity of PCR amplification of specific alleles. Biotechniques 35 21: 586-587. 111533 WGN* 26

Claims (11)

WHAT WE CLAIM IS:

1. A method of testing a farmed animal to determine resistance or susceptibility to nematode parasites to which that animal is or could be exposed, comprising the step of testing nucleic acid obtained from the animal for the presence or absence of a conservative mutation in the gene encoding interferon gamma.

2. A method of testing according to claim 1 in which the nucleic acid tested is DNA.

3. A method of testing according to claim 1 in which the nucleic acid tested is mRNA.

4. A method of testing according to claim 1 wherein the nucleic acid tested is genomic DNA.

5. A method of testing according to any one of claims 1 to 4 in which the animal is bovine, caprine, cervine, or ovine.

6. A method of testing according to claim 1 in which said animal is ovine and said nucleic acid tested is genomic DNA.

7. A method according to claim 6 wherein the presence of DNA having the nucleotide sequence of Figure 10 is indicative of resistance to nematode parasites.

8. A method according to claim 6 in which the presence of DNA having any one of the mutations shown in Figure 11 is indicative of susceptibility to nematode parasites.

9. A method according to claim 6 wherein the presence of any one of the following mutations to the sequence of Figure 10 is indicative of susceptibility to nematode parasites: (i) a —> g at position 188; (") a g at position 529; (iii) c -» t at position 538; (iv) g -» a at position 905; (v) t -» c at position 1149 (vi) c -» t at position 1270 (vii) a —» t at position 1294 (viii) c-^aat position 1349 (ix) a -» g at position 162C a -> g at position 1686 (xi) a->cat position 1717 111533 WGN *;27;10;15;20;25;(xii);(xiii);(xiv);(xv);(xvi);(xvii);(xviii);(xix);(xx);(xxi);(xxii);(xxiii);(xxiv);(xxv);(xxvi);(xxvii);(xxviii);(xxix);(xxx);(xxxi);(xxxii);(xxxiii);(xxxiv);(xxxv);(xxxvi);g —> a at t ->■ c at c -¥ t at g -» t at a -> g at c —^ t at a —> g at g -» a at t ->■ a at t c at t —> g at c g at c —> t at c -> t at c -> t at g t at c ->■ g at a -> g at c -> g at t -> c at a -» g at a -> g at c t at a -*■ g at a -» g at position 1923; position 1989; position 2118; position 2173; position 2252; position 2456; position 2457; position 2458; position 2461; position 2816; position 2978; position 2979; position 3041; position 3359; position 3488; position 3516; position 3540; position 3955; position 4051; position 4059; position 4120; position 4370; position 4379; position 4441; position 4519. 35020 1 30

10. A method according to claim 6 in which the presence of any one of the following in the sequence of Figure 10 is indicative of resistance to nematode parasites: (i) (a) (iii) g 111 at positions 853-856; t at position 1524; and Satgggggat positions 2448-2455. 35

11. A method as defined in claim 1 of testing a farmed animal to determine resistance or susceptibility to nematode parasites to which that animal is or could be exposed substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings. 40 END OF CLAIMS INTELLECTUAL PROPERTY OFFICE j OF N.Z, 2 1 JUL 2000 RECEIVED 111533 WGN#