BMC Genomics 2006, 7:42 http://www.biomedcentral.com/1471-2164/7/42 ney sheep during primary infection of naïve animals with T. colubriformis [4]. Therefore, while UBD is expressed more highly in naïve susceptible animals, upon infection with nematodes the resistant animals may induce UBD expression to a higher level than the susceptible animals in a TNFα-dependent manner. The previous study also identified smooth muscle function and Major Histocom- patibility Complex II expression as important mediators of parasite resistance [25]. The current study did not iden- tify smooth muscle or MHC II genes as differentially expressed between the lines in the absence of challenge and so shows that these genes are induced in response to challenge. Resistance to gastrointestinal nematodes may therefore be mediated, in part, by the ability to induce expression of these genes and this response is generated upon exposure to infection. Conclusion Despite divergent selection over many years relatively few genes were differentially expressed between the selection lines in the absence of nematode challenge. Many more genes were found to be differentially expressed between the lines in response to natural parasite challenge [25] however, the genes identified in the present study may contribute to an animals innate resistance or susceptibil- ity. The genes more highly expressed in resistant animals had a variety of functions, but some were involved in maintaining a healthy immune system, while some were pro-apoptotic genes. A number of genes more highly expressed in the susceptible animals were related to cellu- lar response to stress and infection indicating that the sus- ceptible animals may have a compromised gastrointestinal tract, even in the absence of nematode infection, and this may contribute to their innate suscep- tibility. It is notable that the human orthologs of the dif- ferentially expressed genes are located on a number of different human chromosomes (Tables 1 and 2). It is not known whether the polymorphisms that give rise to the differences in expression reported here are due to cis or trans mutations. Although it is possible that a mutation in one or a small number of genes may give rise to the expression differences, the diversity of the actions and pathways of the genes differentially expressed is consist- ent with genetic resistance to nematodes being due to many genes with small effects rather than a mutation in a single locus. The recent release of the bovine genome [70], a closely related species, will greatly aid mapping and rese- quencing of sheep genes responsible for parasite resist- ance. Methods Selection lines and tissue collection Resistant and susceptible lines of Perendale sheep have been selected based on faecal egg count (FEC) since 1986. These lines now differ in faecal egg count by 4.9 fold [24]. Pregnant ewes were adjusted to concentrate feed, treated with anthelmintic, brought indoors and subsequently lambed indoors. The lambs were raised indoors to insure their immune system remained naïve with respect to gas- trointestinal nematodes. During this period the ewes and lambs had access to commercially formulated sheep nuts and chopped Lucerne hay ad libitum. All animals were faecal sampled periodically to ensure no parasitic nema- todes were present. Four female lambs per selection line were chosen and at an average age of 84 days (standard deviation, 6.8 days) these animals were sacrificed and duodenum tissue promptly collected. The tissue was fro- zen in liquid nitrogen and stored at -80°C. No adult nem- atodes could be detected in the abomasums or intestines of the animals confirming their naïve status. The average live weight of the lambs pre-slaughter was 23.4 kg (stand- ard deviation, 3.8 kg). No significant differences were observed between the weights of the animals in the two lines at any stage. All procedures were approved by the AgResearch Invermay Animal Ethics Committee, formally constituted under the New Zealand Animal Welfare Act. RNA preparation Total RNA was isolated from the duodenum of each ani- mal using TRIzol (Invitrogen) and was cleaned using an RNeasy kit (Qiagen). RNA integrity was confirmed by denaturing agarose gel electrophoresis and RNA was quantitated using a NanoDrop® ND-1000 Spectrophoto- moter (NanoDrop Technologies). First-strand cDNA was made from 25 µg of total RNA by anchored oligo(dT)20- primed reverse transcription incorporating amino-modi- fied dNTPs, and was subsequently labelled indirectly by fluorescent coupling of Cy™3 and Cy™5 mono-reactive dyes (Amersham) using the SuperScript™ Indirect cDNA Labeling System (Invitrogen) according to the manufac- turer's instructions. Array preparation Ovine cDNA libraries were prepared from 27 tissues by MWG Biotech (Germany). These libraries were single-pass sequenced from the 5' end generating expressed sequence tags (ESTs). The inserts, representing ovine expressed sequences, were then amplified in 96 well plates using the universal primers SP6 and T7 in 50 µl reactions (1.5 mM MgCl2, 2 mM each dNTP, 45 pmol primers and 2.5 units AB Red Hot Taq polymerase). The reactions were dena- tured at 94°C for 3 minutes then cycled 36 times at 94°C for 45 sec, 55°C for 45 sec and 72°C for 60 sec. Finally products were extended at 72°C for 5 min. The PCR prod- ucts were verified by agarose gel electrophoresis, precipi- tated and resuspended in printing solution as described previously [25]. At the University of Otago Genomics Facility an ovine microarray consisting of 19,968 spots was printed onto poly-L-lysine coated glass slides using an ESI array robot with 32 split pinheads depositing 0.6 nl Page 8 of 12 (page number not for citation purposes)