Detection of APAF1 mutation in Holstein cows and mummified foetuses in Japanese dairy herds.

Some of the highest genetic merit sires have been shown to harbour recessive mutations affecting fertility, which may spread rapidly in the population through AI. These disorders may result in abortion and decline in pregnancy per insemination in cows. This study was carried out on 240 Holstein-Friesian cows and 15 mummified foetuses. Blood and tissue samples were collected from the cows and mummified foetuses, respectively, for DNA extraction. Allele-specific PCR was designed for the detection of the cows and foetuses carrying the nonsense mutation (C/T) in apoptosis peptide activating factor 1 gene (APAF1). The mutant allele frequency of the APAF1 in carrier cows and mummified foetuses was calculated. Milk samples were taken from the carrier and non-carrier cows for progesterone assay. The allele-specific PCR reaction efficiently distinguished the C/T mutation in APAF1. Of 240 cows, seven cows (2.9%) were diagnosed to carry one copy of the mutant allele of APAF1. However, the carrier frequency was 33.3% in mummified foetuses (five of 15). The mutant allele frequency was 0.02 and 0.17 in the cows and mummified foetuses, respectively. Concentrations of progesterone did not differ between cows with APAF1 mutation and non-carrier cows during 45 days post-insemination. This study provided allele-specific PCR for the detection of APAF1 mutation in cows. Moreover, it reports the carrier and mutant allele frequencies of APAF1 in dairy cows and mummified foetuses in Japan.

ELISA for bovine interleukin-1 receptor antagonist and its application to mastitic sera and whey.

A sandwich ELISA for the bovine IL-1 receptor antagonist (bIL-1ra) was developed using recombinant (r) bIL-1ra produced by Escherichia coli, anti-rbIL-1ra rabbit IgG, its biotinylated one and avidin-peroxidase. This ELISA system enabled detection of rbIL-1ra at a concentration of more than 2 ng/ml. This ELISA was applied to quantitation of bIL-1ra in sera and whey of mastitic and healthy cows. The results indicate that although IL-1ra levels in healthy and mastitic sera and whey were comparable, serum IL-1ra/IL-1beta ratio of euthanized cows was significantly lower than that of the recovered.

Interferon-gamma and tumor necrosis factor-alpha levels in sera and whey of cattle with naturally occurring coliform mastitis.

Concentrations of interferon-gamma (lFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) were determined in serum and whey samples from cattle with naturally occurring coliform mastitis for two weeks after onset using bovine INF-gamma and TNF-alpha-specific ELISA. In serum and whey samples from healthy cows. IFN-gamma was almost undetectable and TNF-alpha was detected at low levels. At the onset of illness, INF-gamma in sera and whey and TNF-alpha in whey from the mastitic cows were significantly higher than their respective values in healthy cows. Concentrations of IFN-gamma and TNF-alpha in whey from mastitic cattle decreased significantly as the cows recovered.

Concentrations of IL-6 in serum and whey from healthy and mastitic cows.

Inflammatory cytokines, such as interleukin (IL)-6, have been shown to reflect clinical signs in certain conditions in diseased animals. In this study, we quantified the IL-6 concentrations in the serum and milk whey from 94 dairy cows with acute clinical mastitis and 55 healthy lactating cows. The IL-6 concentrations in serum from mastitic cows were significantly higher on the first day of illness compared to those of normal cows. Higher concentrations of IL-6 were also detected in the whey from mastitic cows, whereas low concentrations of IL-6 were detected in both serum and whey samples from normal cows. IL-6 concentrations in the serum taken at the onset of illness from cows that later required euthanasia were significantly higher than those in samples from cows that later recovered. These results suggest that serum IL-6 concentrations may be of prognostic value in identifying cows with severe mastitis.