Influence of Temperature and Humidity on Sample Preservation prior to the Molecular Identification of Major Gastrointestinal Strongylids (GIS) in Small Ruminants

Abstract

The exponential development of resistance to anthelmintics is increasingly threatening the small ruminant production sector. Current diagnostic methods for gastrointestinal strongylid infections depend on the collection of fresh faeces, maintained at a consistent refrigeration temperature of 4°C, with rapid transport and laboratory processing to prevent egg hatching. Implementing molecular diagnostic techniques offers a solution to these strict requirements, simplifying both the diagnostic process and associated logistics. To assess the influence of temperature and humidity on coprological samples prior to genomic DNA extraction, this study sought to develop a simple and rapid method using faecal samples from sheep that had been dewormed for over three months. For this experiment, half of the samples were kept at room temperature and the other half refrigerated at 4°C for 24 hours in order to investigate the influence of temperature prior to DNA genomic extraction. Samples were then pressed onto filter paper (3 mm in thickness), and, to study the impact of the natural humidity of samples on DNA extraction, half of each previous groups was placed in a oven at 25°C for 16 hours (dry samples) prior to extraction while the remaining groups were, immediately, extracted (fresh samples). The DNeasy Blood & Tissue kit (Qiagen) was used for genomic DNA extraction, successfully extracting genomic DNA from all tested samples. It was possible to correlate the extracted quantities with the level of humidity present in the samples. Amplification of a segment of the rRNA ITS1 nematode gene (Internal Transcriber Spacer) was only achieved in samples that underwent the drying process in the oven, regardless of temperature. Although still in the early stages, the results presented here indicate that the storage temperature of the samples is not decisive for identifying gastrointestinal strongylids, but rather the humidity level is critical. The methodology is quick to execute and highly adaptable, allowing it to be performed in the field, thus facilitating the transport, storage, and routine laboratory processing and analysis of samples. Consequently, we will continue to optimise the technique under study and investigate why only oven-dried samples achieved ITS1 gene amplification. This work was funded by SUMO: Sustainability of Montado PRR-C05-i03-I-000066; FCT – Foundation for Science and Technology, I.P. (Portugal) through projects UIDB/00276/2020; LA/P/0059/2020 - AL4AnimalS.