1. 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 We therefore decide to sequence entire NOD2 gene, whose sequence in sheep is still unpublished, performing a research for SNPs potentially associated with sheep paratuberculosis.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susceptibility to paratuberculosis in Sarda sheep breed by amplifying the full sequence of the ovine NOD2 gene, still unpublished in sheep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 ( and tested with RepeatMasker ( on bovine NOD2 gene (ENSBTAG ) 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 bp on a total of 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