The ATP bioluminescence assay: a new application and optimization for viability testing in the parasitic nematode Haemonchus contortus.

The parasitic gastrointestinal nematode Haemonchus contortus causes serious economic losses to agriculture due to infection and disease in small ruminant livestock. The development of new therapies requires appropriate viability testing, with methods nowadays relying on larval motility or development using procedures that involve microscopy. None of the existing biochemical methods, however, are performed in adults, the target stage of the anthelmintic compounds. Here we present a new test for the viability of H. contortus adults and exsheathed third-stage larvae which is based on a bioluminescent assay of ATP content normalized to total protein concentration measured using bicinchoninic acid. All the procedure steps were optimized to achieve maximal sensitivity and robustness. This novel method can be used as a complementary assay for the phenotypic screening of new compounds with potential antinematode activity in exsheathed third-stage larvae and in adult males. Additionally, it might be used for the detection of drug-resistant isolates.

Combination of procedure for ultra rapid extraction (PURE) and loop-mediated isothermal amplification (LAMP) for rapid detection of Mycoplasma bovis in milk.

Polymerase chain reaction (PCR) is typically used for the early detection of mycoplasma in bovine milk; it requires 3 days to obtain results because of the necessary enrichment process. A more rapid, simple, and accurate detection method is required to directly detect the Mycoplasma bovis (M. bovis) gene in milk. In this study, we assess the utility of combining the following two methods to achieve this goal: the loop-mediated isothermal amplification (LAMP), which is more sensitive than PCR, and the procedure for ultra rapid extraction (PURE), which adsorbs and filters components that inhibit DNA amplification/detection. LAMP was examined using DNA extracts obtained by four methods. This showed that PURE had the highest sensitivity and specificity and that the combination of PURE and LAMP was able to detect M. bovis in milk. We then showed that the detection limit of M. bovis was 102 colony-forming units per milliliter of milk using the PURE-LAMP. Finally, the respective sensitivities of the PURE-LAMP and PCR were 57% and 86% for bulk tank milk, 89% and 74% for mature milk, 85% and 92% for colostrum/transitional milk, and 97% and 95% for mastitis milk. The specificity was 100% for all milk samples in both LAMP and PCR. We conclude that PCR was suitable for detecting mycoplasma in bulk tank milk and that the PURE-LAMP could detect mycoplasma within 2 hr and was also effective for mature and mastitis milk.