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The aim of the study was the analysis of the nucleotide-binding oligomerization domain containing 2 (NOD2, formerly CARD15) as a candidate gene for Mycobacterium avium ssp. paratuberculosis infection in cattle. Eleven SNPs in the NOD2 gene were identified, and finally, four SNPs were included in a case–control study using 324 German Holstein cows tested for paratuberculosis using fecal culture and ELISA. The SNP (GenBank) AY518738S04:g.521G>A in exon 4 showed a significant association between the fecal culture status of the animals and NOD2 allele variants. The other three SNPs showed no associations in German Holstein cows.