GENOTYPING

Heparinised whole blood samples (0.5 to 1.0 ml from each animal) were obtained from 934 of the original 1200 sheep that began the trial. These were remnants of samples utilized for immune assays and were under storage at -20˚C for prolonged periods before they were obtained. The current study was not a component of the original vaccine trial. However, a large between-individual variation in the immune responses to vaccination provided an opportunity for this investigation. By the time blood samples were received in New Zealand (late 2003), some of the sheep were either deceased or culled due to various reasons. Hence, of the original 1200 sheep (600 vaccinates and 600 controls) from the three properties, blood samples from only 934 sheep (detailed in table 4) were available.

Table 4:Source of blood samples from control and Johne’s vaccinated animals

Property Controls Vaccinates Total

P1 161 171 332

P2 138 165 303

P3 131 168 299

Overall 430 504 934

3.4.2 DNA extraction

Before being received from Australia to New Zealand, the heparinised whole blood samples were in storage at -20°C for prolonged periods. This, together with retention of samples for extended periods for bio-security clearance during transit resulted in low DNA yields. Initial attempts to isolate DNA using DNAzol® (Invitrogen Corporation, Carlsbad, California, USA) were unsuccessful. A column-based isolation kit, QIAMP® DNA blood mini kit (Qiagen Private Limited, Clifton Hill, Victoria, Australia), was found to yield more consistent and higher DNA yields. DNA was isolated as per the kit protocol. In brief, leucocytes in whole blood were lysed by incubation in lysis buffer and protease at 56°C for 10 minutes. DNA in lysate was adsorbed onto silica-gel membrane of spin-column and washed twice with supplied wash buffers. Purified DNA was finally eluted using either the supplied elution buffer or plain autoclaved double-distilled water. The kit protocol advocates 200µl of blood as starting material. However, since leucocyte counts were quite low in most of the blood samples, leucocytes separated from around one ml of blood by centrifugation at 1500 rpm for

30 minutes at 4°C were re-suspended in 200µl of phosphate buffered saline and employed as starting material. Quality and quantity of DNA was assessed following electrophoresis through 0.75% agarose gels.

3.4.3 Genetic markers employed

Genotyping of sheep at eight microsatellite loci was undertaken employing nine markers. Four microsatellites were from Ovar-Mhc and two each from SLC11A1 and IFN-γ gene regions. These loci were selected based on reports in the literature regarding their

significance in disease resistance. The primer sequences along with the forward primer 5’ end fluorescent label for the employed markers, together with the location of the microsatellites they amplify are detailed in table 5. Markers OVINRA1 and NRAMP1 amplify the same microsatellite repeat; however, NRAMP1 alleles are 56 base pairs (bp) larger than the respective OVINRA1 alleles. Due to this known difference in allele length, inclusion of both markers provided an internal control for the genotyping method (detailed in section 3.4.5).

3.4.4 Microsatellite DNA amplification

Annealing temperatures were optimised for amplification of target DNA for different markers. Firstly, the target DNA for each marker was amplified in individual reactions and based on proximities in annealing temperatures and primer compatibility, mutiplex

amplifications were tested. It was found that target DNA for the employed nine primer-pairs could be amplified in five PCR reactions. Markers OVINRA1, OVINRA2 and OarKP6 were amplified in one reaction (annealing temperature: 59°C), while markers OLADRB and

NRAMP1 were amplified in a different reaction (63°C). Markers DYMS1 and o(IFN)-γ were amplified together (54°C), while markers SMHCC1 (58°C) and OLADRW (54°C) were amplified individually. Each 20µl-PCR reaction consisted of 5 nmol of each dNTP, 37.5- 45.0 nmol of MgCl2, 10 pmol each of forward and reverse primers for each marker, 1.25 units of Platinum® Taq DNA polymerase (Invitrogen Corporation, Carlsbad, California, USA), 2 µl of 10X PCR buffer and 50 to 100 ng of template DNA. PCR was performed in a

GeneAmp® PCR system 9600 (Applied Biosystems, Foster City, California,USA) with an initial hold at 95°C for 5 min, followed by 30 cycles (94°C for 30 sec; annealing temperature, see table 5, for 30 sec; 72°C for 1 min) and a final extension at 72°C for 7 min. Presence of amplification products and approximate amount of DNA in the PCR products were assessed by running 3.0 µl of each reaction on a 2.5% UltraPure™ agarose 1000 (Invitrogen

Table 5: Details of markers employed in the study Primers Marker* Gene locus* Chromo -some* Position (cM)*† Repeat Sequences Reference Annealing temperature PCR reaction‡ Forward primer 5’ label

DYMS1 DYA 20 17.9 (CA)n F: 5’AACAACATCAAACAGTAAGAG3’

R: 5’CATAGTAACAGATCTTCCTACA3’

Buitkamp et al. (1996)

54ºC _Three PETTM

(Red)

OLADRW DRB1 20 56.8 (GT)n(GA)m F: 5’TCTCTGCAGCACATTTCCTGG3’

R: 5’CGTACCCAGAGTGAGTGAAGTATC3’ Gruszczynska (1999) 54ºC Five VIC ® (Green)

OLADRB DRB2 20 57.7 (AC)n F: 5’CTGCCAATGCAGAGACACAAGA3’

R: 5’GTCTGTCTCCTGTCTTGTCATC3’ Gruszczynska et al.(2002b) 63ºC Two NED™ (Yellow) SMHCC1 MHC1 20 60.6 (CA)n F: 5’ATCTGGTGGGCTACAGTCCATG3’ R: 5’GCAATGCTTTCTAAATTCTGAGGAA3’ Groth and Wetherall (1994) 58ºC Four FAM™ (Blue)

NRAMP1 SLC11A1 2 248.5 (TG)n F: 5’GATGAGTGGGCACAGTGGCCT3’

R: 5’TTCAAGTGTCTTATTTACACCCATTG3’ Matthews and Crawford (1998) 63ºC Two FAM™ (Blue)

OVINRA1 SLC11A1 2 248.5 (TG)n F: 5’GCCACGGGTGGGATGAGT3’

R: 5’TGAGCTAGGAGATAGCAGG3’

Pitel et al. (1996) 59ºC

One VIC

® (Green)

OVINRA2 SLC11A1 2 248.5 (GT)n F: 5’GGGACACTGAGCAGGACA3’

R: 5’CCATAGGGAGAGTCTTAGGT3'

Pitel et al. (1996) 59ºC

One FAM™

(Blue)

o(IFN)-γ IFNG 3 192.1 TGT(GT3)5 or 6 F: 5'TTGTGACTGTTAGCTAGATGTGTT3'

R: 5'ATACACATATTATGCCCATCTTTT3' Schmidt et al. (1996) 54ºC Three NED™ (Yellow)

OarKP6 IFNG 3 192.1 (AC)n F: 5'GCCCTGTGTCTCGTGTAACTCAC3'

R: 5'CCACAGGGTTGCAAAGAATCA3' Paterson and Crawford (2000) 59ºC One NED™ (Yellow) * Marker, locus and chromosome details as per the Australian sheep gene mapping website, available online at http://rubens.its.unimelb.edu.au/~jillm/jill.htm (last accessed 15/01/2007) †

Positions of marker loci on respective chromosomes are sex-averaged values; ‡ Nine markers were amplified in five different PCR reactions

3.4.5 Determining PCR product lengths and scoring genotypes

Fluorescent labeling at the 5’ end of forward primers for the nine markers with four different dyes facilitated automated length determination of the PCR products employing an ABI3730 Genetic Analyzer (Applied Biosystems, Foster City, California,USA). For each individual, PCR products from the five different PCR reactions were diluted and pooled based on their DNA concentrations and 1.0 µl of the pooled product used for the capillary run. Resulting chromatogram data from the genetic analyzer were analyzed using ABI Prism®

GeneMapper™ software version 3.7 (Applied Biosystems, Foster City, California,USA), to obtain the individual genotypes for all the markers, based on the amplified product lengths. Genotypes from 771 and 865 sheep out of the 934 DNA sampleswere recovered for the NRAMP1 and OVINRA1 marker loci, respectively. Genotypes were available for both the loci in 741 individuals and in each of those individuals, NRAMP1 alleles were longer exactly by 56 bp than corresponding OVINRA1 alleles. Hence, in 30 individuals having exclusive amplification for NRAMP1, genotypes were assigned to OVINRA1 locus based on

corresponding NRAMP1 genotypes, increasing the number of available OVINRA1 genotypes to 895. Since these two markers amplify the same microsatellite region, only OVINRA1 genotypes were employed for further analyses.

Prior to the actual genotyping study, exactness of genotype scoring in the pooled PCR products was validated as follows. Individual amplifications were carried out for the nine markers, employing ten randomly chosen DNA samples and the lengths of resulting products determined individually in the genetic analyzer. From the same 10 DNA templates,

amplification of the nine marker regions was also carried out in five PCR reactions as detailed in table 5 and the resulting products pooled and analyzed as a single run in the analyser for length determination. Chromatogram data from the two methods resulted in identical genotypes at all the nine marker loci for each of the ten individuals.

3.5 STATISTICAL ANALYSES