Comparison of fecal pooling methods and DNA extraction kits for the detection of Mycobacterium avium subspecies paratuberculosis.

The aim of the study was to develop a sensitive method using quantitative real-time polymerase chain reaction (qPCR) with pooled fecal samples for the screening of Johne's disease (JD). Manufacturer-specified and our new pooling method in combination with five commercial kits for DNA extraction and purification were compared. Different volumes of pooled fecal suspensions were tested, and the results were compared for individual samples and three pool sizes (5, 10, and 50 samples); each of the fecal suspensions, which were prepared from healthy dairy and beef cattle was spiked with 0, 10, 100, or 1000 cultured Mycobacterium avium subspecies paratuberculosis (MAP) organisms or was mixed with fecal suspensions from experimentally infected cattle. The MAP DNA detection proportion with our pooling method in combination with Johne-Spin kit (Fasmac, Japan) was 100% for all models and all pool sizes, except for the low shedder model with a pool size of 50. There was no loss of sensitivity in pools of 10 subjects or less by using the new method. These results suggest that new method is a sensitive, practical, and cost-effective screening test for the detection of MAP-infected cattle and the monitoring of JD-free herds.

Novel mutations of the FASN gene and their effect on fatty acid composition in Japanese Black beef.

Eight novel and four known mutations were detected in the coding sequence of the bovine fatty acid synthase (FASN) gene of an F2 population from Japanese Black and Limousin cattle. Two mutations, g.16024A>G and g.16039T>C, detected in exon 34, which determine amino acid substitutions of threonine (T) to alanine (A) and tryptophan (W) to arginine (R), were clearly separated in the parental breeds. The haplotypes (TW and AR) segregated in F2 individuals and had a significant effect on the fatty acid composition of backfat, intermuscular fat, and intramuscular fat. The TW haplotype was associated with increasing C18:0 and C18:1 content and the ratio of monounsaturated to saturated fatty acids, and decreasing C14:0, C14:1, C16:0, and C16:1 content. The two mutations were screened in two commercial Japanese Black half-sibling populations and similarly determined the contribution to the fatty acid composition of intramuscular fat. The frequency of the TW haplotype was markedly higher in Japanese Black (0.67) than in Holstein (0.17), Angus (0.02), and Hereford (0.07). We conclude that these mutations may contribute to the characteristic fatty acid composition of Japanese Black beef.