AU2379399A - Identification of animals resistant to nematode parasite infection - Google Patents

Description

AUSTRALIA

Patents Act 1990 New Zealand Pastoral Agriculture Research Institute Limited

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Identification of animals resistant to nematode parasite infection The following statement is a full description of this invention including the best method of performing it known to us:-

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1/ 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., 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).

a 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 I 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.

o 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 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.

25 More particularly, the presence of any one of the following mutations to the sequence 0$ 25 of Figure 10 is indicative of susceptibility to nematode parasites: a- g at position 188; (ii) a -g at position 529; S* (iii) c t at position 538; 30 (iv) g a at position 905; t c at position 1149; (vi) c t at position 1270; (vii) a t at position 1294; (viii) c a at position 1349; (ix) a g at position 1620; a g at position 1686; (xi) a c at position 1717; (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; Sa (xxxv) a g at position 4441; 25 (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: 30 g t t t at positions 853-856; S(ii) t at position 1524; and (iii) g a t g g g g g at positions 2448-2455.

S a 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 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 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; 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 S, 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 Seffect at this location is 12,700 Trichostrongylus nematodes. Corresponding figures for 25 sire 2 are LOD 0.994, 19cM and 7050 nematodes.

a* 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 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 along the q arm of chromosome 3 for the trait log(mean FEC2 The centromere is located on the left hand side. The peak of LOD 2.459 for sire 1 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.

1) _1 _1 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 Strongyloides (LSISTR); log(mean X+50) transformed, strongyles and Nematodirus at the end of the first challenge (LFEC1,SNEM1) 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 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 25 cM centromeric of OarVH34. Corresponding figures for 920066 is 8cM telomeric of VH34.

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

a 30 Figure 11 is the sequence of Figure 10 showing mutations which are indicative ofsusceptibility 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 a.

a 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.

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25 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.

a a The focus of the analysis of nucleic acid obtained from the animal is to determine thepresence 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 S.I: 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, I 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 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 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 reactions the products of the reaction may be distinguished according to their size using polyacrylamide 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 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

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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 shownunderlined. 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 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 individually or in combination to develop tests to identifyi individuals resistant or susceptible to nematode parasites.

TTGCTTTCTGGTCATTTGCAAGAAAATTTTAAGAGGCACCCCOTCTGTA AAGGTTTGAGAGCCCCTAGAATTTCCTTTTTCACTTGCTTCAACCACAA 100 AGAATGATCAATGTGATCTCTGGATGAGGAGTCAACATTTTACAAGGGC 150 AAAGGAGGAGGTGTACAAAAATTTCCAATCCCTGGACaGTGTGAGTGAA 200 g AGTGTTTTCAAAGGATCCCACAAGAATGGCATGGGTGGGCATAATGGGTC 250 TGTCTCCTCGTCAAGACCCAAGGAGTTGAGGAACTCTAACTACAA 300 GACCAAATGCCACAAACCATAGTTATTATACCAACTAACTAGCATCT 350 CTGTCTATCTGTCACCATCTCATCTTAAACTTGTGAATACGTAAT 400 CTTGATGAGACTTCAATTAGGTATAAATACCAGCACCAAGGAGACACA 450 GCACATTCTTCTGATCATCTGCAGATCAACAATTAGAAAGAGATCAG 500 CTACCTCCTTGGGACCTGATCATAACGCaGGAGCTACcGATTTCAACTAC 550 g t TCCGGCCTAACTCTCTCCTAAACGATGAAATACACAAGCTCCTTCTTAGC 600 TTTACTGCTCTGTGTGCTTTTGGGTTTTTCTGGTTCTTATGGCCAGGGCC 650 CATTTTTTAAAGAATAGAAACTTAAAGGAGTATTTTGTAGTATGATC 700 TTTTAATGAGACTTTCTTGTGATTGACTTGATGTTTGATGTTGACTTGGA 750 ATTCTATTTGTGATGGGCTCTCATCTCTAGTCTTCAGTCATTTTGAGAAG 800 :ACTTGGTGTTATTGTGACTGTTGGCTAGCTGTGTTTGTTTGTTTGTTTGT 850 TTgt ttGACTAAACGATCTCTGCTCAGTTTGCTACAGAGATTTAGGAGGG 900 ATTCgTGAATCCTCCAAAAGATGGGCATAATATGGGTATATATCATAGTG 950 a :TTGATATTTTGTGATGAG-TCCATTTACTATGATGTTAGTAGTAAGC 1000 ATACTGAAAAGACAGCTGAGTGTTA.ATTTTTGATTTCTCATGTTACTCTA 1050 30 ATGGTGACAGTCAAAAGTCAACATCACATTAATGGTCATTTGAT 1100 :AATTGATCAATTAAAGAAGATGAGGTCCTTATATTTGGCTTA?.tA 1150 AAAACACCTATAGGTGTTTTTCTCTAAATTTATTTTAAAATTTTACTA 1200 :**:TCCCATCTAGATTGTCTGTGAATTACTCTTGGACTCAGTCACTTGCTGAG 1250 1300 a t t GTCTTGTTTTATGAATTTCTTAAAAAATTATTTATGGTTAATTAAAATcG 1350 a CTTGTGCATTTTAAATATTTTCTCATATGTCC "AAATTTAGCTATTATAAT 1400 TATAGCTGGAGCTATCTTCTAAAGGCGATATAATATAGGAGGACACAGGT 1450 AGAGGTTACAGAAACCTATACCATAGGGGGGCAGTATTTTATAGTGAGAT 1500 GGTGATGTTGATTTTTTTTTTTTtAACTATACCTTTTTCTTACCATAGTA 1550 ATTTGGGACAGCTGAAGTATCATCAOTOTTGAATGAGTTCATTGTGATGT 1600 TATACTAGTAAATAAGAAAaCAAAAATCACATGATAGTCTTTAGCTATTA 1650 9 TCCAACAGGATAGCTTTTGAGACTATTTTTTGTTAaAACTACAGTGTT 1700 9 GAAAAGAAAATCAGTAaATATTGCCAACATCCAAGTTCATAACTTGA 1750

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GGTACATTTTTACAGCAATTTATGGACTAATTTTAAGCCAATTTTTTCTT 1800 GTGTTGTTTTATTTTCCAGAATGCAAGTAACCCAGATGTAGCTAJAGGGTG 1850 GGCCTCTTTTCTCAGAATTTTGAGAATTGGAAGAGGTAGCTGAACA 1900 TTCATGCGTTC~TGCTTTCGTGTATC 1950 a CATCAACCTCTCTTTGTGCTCTTTTCTCCCAAGGAGAGtGACAAGAT 2000

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TATTCAGAGCCAAATTGTCTCCTTCTACTTCAACTCTTTGAACCTCA 2050 AAGATAACCAGGTCATTCAAGGAGCATGGATATCATCAGCAAGACATG 2100 TTTCAGAAGTTCTTGA~cGGCAGCTCTGAGAACTGGAGGACTTCAAG 2150 t GCGTCATCGTA~GTCTAGTTTTGACT 2200 t GCAGAGACTCTTACAAGCACTTCATTCCCAGAAATAGATTAGATAC 2250 :CaGGTATAAAGCTCTGAAGTGAAGTTGTAGTCAAAATGCCTCTTTGCTAG 2300 9 TTCTCTTTTGCCTTGGGTGACTTTGCAGAGTCAGTTATTGGAGGCACTTA 2350 AGTCTGTGATTTTGGGAAAATACTGGCATTATGCCTATCGCACACATG 2400 30 GGTGAATGGACAAATCAGTGAGGAGGAAGCAGAGAAGGAGTTGGGGTgat 2450 gggggcagC9AGtATTGAAAACCAATTCTCCCATTGCCCCTTGTCTCTGTTG 2500 tga a) 35 GGAGGGAGAGGGAAGATGTGTGCTCCGCCCAGCTTAGCCACCAGAT 2600 *GTGTGACTTCAGATGAATCACAGGCCTGGCTGGGGCTGTTTCCTCATCTT 2650 AAAAGAAGCCTGTTGAGTTCACTGTAATTTCTATGGTCTCCTTTGCTCTA 2700 *0.AAATTCTACAATGCCGTAGATAGAAAATGACAATGAGATAGGAGAAAGGA 2750 :~*ATAGCCTTTGAGGAGACAAGGCCCTCCCACTGTCG.CTCTGGTCAACC 2800 ATGCAGATTGTGTAGtCCAAGGAGAGGTGATTGCATTTGTTTTTAGAAAG 2850

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AAAATAGGTAGAATTGTCCCATCAATGAATCTTTAAAGTTTTTAGAAGCT 2900 GAAAGATAACTTTGAAGCATAAGGACAAGTCACATTTTCCAAAATTTCCT 2950 CTAAGGGACAAAGGCCTCCAGATTTTTtcGGGGGGAGAGTTGGCTTTTGT 3000 ATAAATAGGCTTGGGTGAGAAGAGGAGGGGTGCTTGTGTTCTCAGGATTT 3050 t GTACCAGCTCTTGCTCTAACCCTGTCTGCCTCTCAACAATCTCGTATAAA 3100 CTCCCATGAACTGAAAGAAGTGATTGCTCTTCAAGATTTCTCTAGTCTCC 3150 ATATATAAGATCGTATGAGAGTGGCTTGGAAAAATTATGCGTAATGTTTT 3200 CCATGTTCAGCAGATCTCCATTGGTTGCAGGCTAGAGAATAAAAcACAC 3250 TGTTCCTAACCCAGTGCTGCCTCTGGCTGCTGTTCAGTCCTAGAGAAGTC 3300 CCTTAGTTTCTTGGTGATGCTTAGA.AAACTGGAAAGTGAAATTTATCACC 3350 TGTAAACAcACAGCTGGTAGAAATTTTTCTACTCTGTTGTA.ACTGGGCTT 3400 t TGAAAACAATAGGTTGGTCCCATCCTTGTGGAAGCTCCAAGCCTCCTGCT 3450 TCACAATGTGCAATCTTGAGTGAGTGCTCAACCATTGcCGTCTTTTGCTC 3500 t AAATGTGAGCATATTgAAGGCATGTTTTGCCTTTTTGTCcTGTAACTTAG 3550 t 9 AATCTCCTAGTCATTCTCAGAAATGTGGCAAGGTGGCTGCAGTGTTTCCA 3600 GCTTTAGAAAGAAATGTAGTGGCAGAGCAAAGATGAAGCAAATGGTCCTC. 3650 ATTTTATGAAGTGTGAATTCACCTATATGGTCCTACTGGGAAGAGTGAGA 3700 AAATAGTTCAGTTTGCTGGAATGGAAGGCAGGGTACAGGAAGATTGCTTT 3750 CATTACTAATTTTATTATATAATGCCAGTAATTGATTATTACCATTTAAA 3800 *TAAGATCAGTGTTTATTTTTAATCCCATCATGGAAATGATGAGACTGACC 3850 25 TAATTTGGAGGAAATTAAAAATTATGGTTTTCCTCCTTCTAGAACTCGGT 3900 ::@GTTTATAAAGTACTGAACCTATACCAGTGGTTTTCAAACTTGGGTGGTGC 3950 *CTAAaTCACCTGGTGATGTTGGTAACCACACAATGGCTGAGGCCTAGTCC 4000 :GATTTCATCCACCTGGGTGTCAGAGTTCTTCATTAGGTCCTCAGGTTACT 4050 cTCTTACAtACCAAGCATGAGAGCCATTGGCTATAGTAACTCATCTGATA 4100 9 CTTATAGCAAAGACTAAGTaCTAAGCGATAGAAGTGACAGAGACCCTGGG 4150 :GATCATCTAAACAATCTCATACTTAGTGCAGAAAACAGAGATCTGGAGAG 4200 CTTCAGTGACTGGCTTAAAGTCAGATGTATTGCACACACAGA.ATTCACAC 4250 :CCACATTTTTCTGTCACTCCACTTCTGGGTTTTTTTCACTATATAATTTT 4300 .:CAGATCCCTGAAGTGATAAATTTATGGATATGTGATGTGCTCCATCCTGT 4350 TACAGCACAACTGTCAATTaAATTGTTAcAATTTTAGTCTTTATCACTGA 4400 g t AGAAACCAACATTACATATTAAAGTTCATTACTGCTAGTGaAAATAATTT 4450 GTTTTAAAGAATACTTTGATTTTCTTGGGCCTAGACAGCAGAATATCCTA 4500 ATGACTCATATGCTTGAAaTTAATTTTGCTGTTTTCTTTCCCAATAGGTG 4550 g GATGATCTGCAGATCCAGCGCAGCCATCATGAACTCATCAGGTGAT 4600 GAATGACCTGTCGCCAATCTAACCTCAGAGCGGAAGAGAGTCAGA 4650 ATCTCTTTCGAGGCCGGAGAGCATCATGT-TGGTTCTCCTGCCTGCAA 4700 TATTTGAATTTTTCTATCTATTTATTATATTTATATTTTACA 4750 TTATTTATATAGGGATATATATTTAGACTTATCAGTATTTATAATAG 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 the primers used to type the two SNP's.

'GATCCCACAAGAATGGCJ' (SNP iF) 'GATCCCACAAGAATGGc3' (SNP2F)

GAAGACCACTCAACAATCAAAACAATAT

ATC*CAATGACCTT*TTGCCACCACTAAACTTA

AAAGATTGTGGCTATAGTTATCACCCAAGGCC

GAACATTCATACATGTATCTGAATTACATCTCAATCTTGCTCTCTT

ATC TAACTATGATCAG

TCTTCTACTACGCCTGGTGC=C

GGTTCGTCTTGCAGCCTTTAAAAAAAACTAGA

TATTTTGTAAGTATGATCTTTTAATGAGACTTTCTTGTGATTGACTTGATGTTTGATGTT

GACTTGGAATTCTATTTGTGATGGGCTCTCATCTCTAGTCTTCAGTCATTTTGAGAAGAC

3 'GAGTAGAGATCAGAAGTCAGTAA5' (SNP1R) 3 'GAGTAGAGATCAGAAGTCAGTAA5' (SNP2R) 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 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 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.

20 For SNP analysis, competition between oligonucleotides differing only at the point mutation is used. The two allele specific oligonucleotides are labelled with different S. 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 S(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 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 of generations (4-5 max) extreme individuals from these lines were then crossed and a small number of Fl sires were then mated to unrelated and unselected ewes to create large half-sib populations. The resulting outcross progeny were evaluated for host 0 resistance to internal parasites. Then the Fl sires and their outcross progeny, which should be segregating for presumptive alleles affecting host resistance, were genotyped 25 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.

30 To identify the likely polymorphic gene involved, an alternative approach was used.

S 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 S: interest were then identified by the use of database searches and comparative S: mapping using the scientific literature. In this case one of the candidate genes, .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 selection lines were collected and genotyped with the published microsatellite marker.

If the marker was close to 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 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 hence will have an effect on resistance to parasitic nematodes.

MATERIALS AND METHODS Breeding the Wallaceville selection lines and establishing the outcross 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 and ewe lambs. At present, the lambs in the high line (susceptible animals) have an 8 fold higher FEC than the low line (resistant animals) during the autumn. On a logarithmic scale this translates to a 2.6 ap divergence between lines. As the selection lines involved a relatively small number of animals outcross rather than backcross pedigrees

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: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).

oo Table 1: Half-sib outcross sheep families used to detect QTL for disease resistance S. 3 Sire identification Number of progeny 0 Sire 1 92/0066 225 Sire 2 92/0153 175 35 Sire 3 92/0154 348 SSire 4 92/0155 111 Sire 5 93/0124 101 *Q 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 the end of each challenge and results averaged. Mean strongyle and Nematodirus infection levels (epg) of 1091 (FEC1) 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 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 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 has a CO containment categorisation from the University of Otago Safety Committee.

DNA samples were collected from all the parents of the Fl rams, the rams themselves and all their progeny. In addition DNA from randomly selected individuals from the S parental selection lines was also obtained.

2 Genotyping.

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

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I.I: 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 S* 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.

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 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 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 (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 (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 25 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.

S. Genotype differences between lines The analysis technique and the assumptions used have been described Dodds et al 35 (1997).

17 RESULTS AND DISCUSSION Outcross pedigrees.

The 22 most susceptible and 22 most resistant animals from the 5 outcross pedigrees (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 heterozygous 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(FEC1 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 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 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 (0.33p, difference in FEC) and 920155 (0.66(7p, 29%) for log(mean FEC1+50). Corresponding 25 figures were derived for log(meanFEC2 +50) for sire 920066 (0.438cp, 16%) and 930124 (0.534,p, 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.

9 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 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 susceptible selection lines using PEDDRIFT. The results are presented in Table 2. The result shown for the IFNG microsatellite is clearly significant 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 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) Significancel 9.e AA AB BB IFNG microsatellite Susceptible 1 15 34 P 0.021 Resistant 17 25 8 S* 1Significance 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.

4 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 GTIT sequence whose presence or absence is the basis of the microsatellite marker is bracketed.

CAAGGAGTGAAAGGAAACTCTAACTACAAGACCAAAATGCCACAAAACCATAGTTATTAA

TACCAACTAACTAGCATCTCTGTCTATCTGTCACCATCTCATC'ITAAAAAACTTGTGAAAAT

ACGTAATCTTGATGAGACTTCAATTAGGTATAAATACCAGCACCAAAAGGAGACACAGCAC

ATTCTICTGATCATCTGCAGATCAACAATTAGAAAAGAAAGATCAGCTACCTCCTIGGGAC

CTGATCATAACGC(g/a)GGAGCTAC(c/t)GATTTCAACTACTCCGGCCTAACTCTCTCCTAA ACGATGAAATACACAAGCTCCTTCTTAGCTTTACTGCTCTGTTGCTT-TGGGTT7T

CTGG

TTTCTATGGCCAGGGCCCA TAAGAAATAG CAAAGGAGTATGTAAGTA

TGATCTTTTAATGAGACTTTCTTGTGATTGACTTGATGTTTGATGTTGACTTGGAATT

20 CTATTTGTGATGGGCTCTCATCTCTAGTCTTAGTCATTTTGAGAAGACTTGGTGTTA *9

TTGTGACTGTTGGCTAGCTGTGTTTGTTTGTTTGTTTGTTT(GTTT)GACTAAACGATCT

CTGCTCAGTTT 3' The sequence was derived from three sheep from the parasite susceptible line and S 25 three sheep from the parasite resistant line. Parts of this sequence have been published previously (McInnes et al. 1990 Genbank Accession X52640; Schmidt,P et al. 1997 Genbank Accession Z92887) but this is the first time that the complete untranslated sequence has been identified. Two SNP's were identified in the 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.

The typing method of Xhu and Clarke (1996) was used to type the SNP2 Polymorphism. In the diagram of the sequence below ail the primers used to type SNP2 are shown above or below the DNA sequence they are derived from.

5 'GATCCCACAAGATGGC3' (SNP2F)

'GGATCCCACAAGAATGGCATGGGTGGGCATAATGGGTCT

GTTCCTAAGCCAGGTAAGACCACAAGCAATCAA

AACCATAGTTATTAATACCAACTAACTAGCATCTCTGTCTATCTGTCACCATCTCATCTTAA,

ACTTGTGAAAATACGTATCTTGATGAGACTTCAATTAGGTATAATACCAGCAC

CAAAAGGAGA

CACAGCACATTCTTCTGATCATCTGCAGATCCTTAGAAGAAGATCAGCTACCTCCTTG

GGACCTGATCATAACGC GGAGCTAC

GATTTCAACTACTCCGGCCTAACT

(SNP2C) (SNP2T) CTCTCCTAAACGATGAATACACAAGCTCCTTCTTAGCTTTACTGCTCTGTGfPGCTTTTGGG.

TTTGTTAGCAGCCTTTAAAAAAACTAGATTTGA

GTATGATCTTTTAATGAGACTTTCTTGTGATTGACTTGATGTTTGATGTTGACTTGGATTCTAT

TTGTGATGGGCTCTCATCTCTAGTCTTCAGTATTTTGAGAAGACTTGGTGTTATTGTGACTGTT

3 'GAGTAGAGATCAGAAGTCAGTA5' (SNP2R) As shown in the sequence above the 4 primers used to type the SNP2 polymorphism were: SNP2F 5' GATCCCACAAGAATGGC SNP2R 5' AATGACTGAAGACTAGAGATGAG 25 SNP2C SNP2T 5' GCCGGAGTAG'ITGAAATCA *Using primers SNP2F, SNP2R and SNP2C, a CC animal will give 2 PCR products whereas TTL animals will give only 1.

Using primers SNP2F, SNP2R and SNP2T, a 'VT animal will give 2 PCR products whereas CC animals will give only 1.

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 IFNG Point mutation Selection line Marker Alleles (No. of animals) Significance Susceptible Resistant P 0.001 EXAMPLE 3 USE OF RFLP MARKER a.

a.

a.

a.

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. McInnes, 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 Marker Selection line Marker Alleles (No. of animals) Significance* IFNG Point mutation Susceptible Resistant P =0.04 significance determined using PEDRIFT software (Dodds and McEwan 1997) Other RFLP's have been detected for this gene (Penty and Hill 199 1) using Rsal, Msp I and Pst I restriction enzymes. It is likely that these other RFLP's wilfl also prove to be useful in detecting those animals resistant or susceptible to parasites.

CONCLUSION

The above results demonstrate that a conservative mutation in the gene encoding 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 animnals resistant to nematode parasites, or against animals susceptible to such parasites. The method has particula 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 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 .,.oooparasites and also to kits containing these materials. The kits may contain other o.,•°conventional reagents useful for determining the presence or absence of sequences 25 associated with resistance to nematode parasites.

:'°":Par-ticularly preferred are primers/ sequences for use in detecting mutations in the "°.:°ovine interferon gamma gene, including SNPIF, 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 SNP1F and SNPIR; and ":°.fragments of these containing at least 15 nucleotides provided that for SNPIA, SNPIG.7 .°°:SNP2C and SNP2T the missing nucleotides are from the 5' terminal. Other primers °eo •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.

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.

*0 0*

C

bed U C *s.

o

S

a.

d

REFERENCES

Baker, Watson, et al. (1991). In "Breeding for Disease Resistance" p19 editors G.D. Gray and R.R. Woolaston, Wool Research Development Corporation, Melbourne, Australia Bisset, S. A. Vlassoff, et al. (1996). "Nematode burdens and immunological responses following natural challenge in Romney lambs selectively bred for low or high faecal worm egg count." Veterinary Parasitology 61(3-4): 249-263.

Crawford, 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. 51: 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. Green R. S. and Risdon P. L. 1994. Antibody responses of sheep to challenge with Trichostrongylus colubriformis and the effect of dexamethasone treatment. International Journal for Parasitology 24: 921-928.

Georges, D. Nielsen, et al. (1995). "Mapping quantitative trait loci controlling milk production in dairy cattle by exploiting progeny testing." Genetics 139(2): 907- 25 920.

e Holland Abrahamson, 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 S Lander, E. and L. Kruglyak (1995). "Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results." Nature Genetics 11(3): 241-247.

35 McEwan, J. 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.

Mclnnes,C.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. 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.L.M. Hodgson,et al. (1991). Cloning and sequencing of the ovine gamma interferon gene. Australian Veterinary Journal 68: 82-84 Sambrook, Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning a Laboratory 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 Schmidt, Ludt, C. et al (1996). A dialleleic tetranucleotide repeat (GT) 5 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 21: 586-587.

Claims (8)

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. 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. p

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 20 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: 25 a g at position 188; (ii) a g at position 529; (iii) c t at position 538; (iv) g a at position 905; t c at position 1149; 30 (vi) c t at position 1270; (vii) a t at position 1294; (viii) c a at position 1349; (ix) a g at position 1620; a g at position 1686; (xi) a c at position 1717; (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; (xxxv) a g at position 4441; (xxxvi) a g at position 4519. 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: g t t t at positions 853-856; (ii) t at position 1524; and (iii) g a t g g g g g at positions 2448-2455. C DATED THIS 26 DAY OF MAY 1999 NEW ZEALAND PASTORAL AGRICULTURE RESEARCH INSTITUTE LIMITED Patent Attorneys for the Applicant:- F.B.RICE CO C o*