NOD2 - a candidate gene for susceptibility to paratuberculosis in sheep Gabbianelli F.1, De Grossi L.2 , Valentini A. 1, Sezzi E.2, De Sanctis B.2, Gelli A.2, Pariset L.1 1 Dipartimento per l’innovazione nei sistemi biologici, agroalimentari e forestali, Università della Tuscia, Viterbo, Italy 2 Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, Viterbo, Italy   Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana name fragment (bp) fragment amplified sequenced   1 986 exon1 yes partial intron 1-2 2 5001 no intron 1-2 exon2 intron 2-3 exon3 partial intron 3-4 3 1002 partial intron 2-3 4 999 5 1003 exon4 6 4998 partial intron 4-5 7 1001 partial exon 4 8 891 9  5003 exon5 intron 5-6 exon 6 intron 6-7 exon 7 10 920 partial intron 7-8 11 5003 exon 8 intron 8-9 exon 9 intron 9-10 exon 10 partial intron 10-11 12 1000 partial intron 9-10 partial exon 10 13 996 14 15 994 16 997 17 2215  exon 11 partial intron 11-12 partial exon 12 18 19 20 973 21 Paratuberculosis or Johne's disease (JD) is a chronic intestine infection of ruminants caused by a slow to develop, contagious, and resistant to antibiotics bacterium, Mycobacterium avium paratuberculosis (MAP). This disease is not easily amenable by classical control methods such as treatment, vaccination and current control strategies. Experimental animal models, widespread of MAP and low prevalence suggest that there could be genetic factors responsible for susceptibility or resistance to the causative agent (Mycobacterium avium paratuberculosis). In livestock, a number of candidate genes have been studied, selected on their association to susceptibility in other mycobacterial diseases, their known role in disease pathogenesis or links to susceptibility of humans to Crohn's disease. A previous study performed 95 samples and identified polymorphisms within NOD2 exon 4 and intron 5-6; although no significant association between SNPs and the disease was found the p-value was borderline (p 0,0560). We therefore decide to sequence entire NOD2 gene, whose sequence in sheep is still unpublished, performing a research for SNPs potentially associated with sheep paratuberculosis. This study was aimed at exploring correlations between genetic polymorphisms and susceptibility to paratuberculosis in Sarda sheep breed by amplifying the full sequence of the ovine NOD2 gene, still unpublished in sheep Table 1 - Primers pairs and fragment amplified and sequenced Materials and Methods bp SNP 7248 C 7286 A 7503 7676 R 7681 7832 7982 G 13893 13900 13911 T 13912 13919 13926 13933 13934 13938 13943 13953 13957 13962 13963 13972 13987 13998 14004 15268 DEL/G 15715 17181 17344 17655 17714 18657 29358 29374 29586 31967 T/C Blood and faeces of 95 individuals were collected in a flock positive to Paratuberculosis. We first performed an Elisa test on blood, identifying 51 positives and 44 negatives. To verify the results, faeces samples were then analyzed by PCR. Genomic DNA was extracted from blood of the same individuals using NucleoSpin Tissue kit (Macherey&Nagel) checked for quality on agarose gel and quantified using a fluorimeter DTX Multimode Detector 880 (Beckman Coulter) with Picogreen method according with manufacturer’s instructions (Quant-iT, Invitrogen). Primers were designed using Primer3 software (http://frodo.wi.mit.edu/) and tested with RepeatMasker (http://www.repeatmasker.org/) on bovine NOD2 gene (ENSBTAG00000020936) in order to amplify the entire ovine sequence. Samples were amplified and sent to Macrogen (www. Macrogen.com) for sequencing. Results In a previous study we identified 3 SNPs in the NOD2 gene, candidate for susceptibility to paratubercolosis in ruminants and to Chron disease in humans. None of the SNPs resulted significantly associated with the disease (p≤0.05) but two borderline values (p≤0.056 and p≤0.083) were obtained for two of the SNPs. This could suggest that other SNPs within the same gene could be associated with paratuberculosis in sheep. 17 primers pairs (tab.1) were successfully amplified and sequenced in a panel of 10 affected and 10 control samples. The sequences were BLASTed to verify the correspondence with bovine sequence. About 12.000 bp on a total of 31.000 bp according to the bovine NOD2 sequence were successfully sequenced and submitted to Genbank (KC795825). We found 36 polymorphic sites (tab.2) that will be tested for association with the disease. Table 2 – Polymorphic sites