Effects of fecal collection and storage factors on strongylid egg counts in horses

Fecal analyses are becoming increasingly important for equine establishments as a means of parasite surveillance and detection of anthelmintic resistance. Although several studies have evaluated various egg counting techniques, little is known about the quantitative effects of pre-analytic factors such as collection and storage of fecal samples. This study evaluated the effects of storage temperature, storage time and airtight versus open-air storage on fecal egg counts. The experimental protocols were replicated in two study locations: Copenhagen, Denmark and Athens, Georgia, USA. In both locations, the experiment was repeated three times, and five repeated egg counts were performed at each time point of analysis. In experiment A, feces were collected rectally and stored airtight at freezer (-10 to -18 °C), refrigerator (4 °C), room (18-24 °C), or incubator (37-38 °C) temperatures. Egg counts were performed after 0, 6, 12, 24, 48, and 120 h of storage. In experiment B, feces were collected rectally and stored airtight or in the open air in the horse barn for up to 24 h. Egg counts were performed after 0, 3, 6, 12, and 24 h of storage. In experiment A at both locations, samples kept in the refrigerator showed no decline in egg counts, whereas storage in the freezer and incubator led to significantly declining egg numbers during the study. In contrast, storage at room temperature yielded marked differences between the two study locations: egg counts remained stable in the U.S. study, whereas the Danish study revealed a significant decline after 24 h. In experiment B, the Danish study showed no differences between airtight and open-air storage and no changes over time, while the U.S. study found a significant decline for open-air storage after 12 h. This difference was attributed to the different barn temperatures in the two studies. To our knowledge, this is the first study to evaluate the pre-analytic factors affecting egg counts in horses using an experimental protocol replicated in two contrasting geographic and climatic locations. Our results demonstrate that refrigeration is the best method for storage of fecal samples intended for egg count analysis, but that accurate results can be derived from fecal samples collected from the ground within 12 h of passage.