To determine the time required for accurate viability assess

To determine the time required for accurate viability assessment, approximately 1000 A. caninum eggs were transferred into 1.5mL centrifuge tubes, suspended in 1mL Phosphate Buffer Saline (PBS), and heat-inactivated on a heating block for 30min at 80°C. Tubes were centrifuged at 800× for 3min, and the supernatant discarded. The heat-inactivated eggs were kept at a room temperature (24±1°C) for 1, 6, 12 and 24h to allow eggshells to become permeable. The eggs were stained with aqueous solution (0.1%) of eosin Y, methyl red and methylene blue and enumerated using a microscope. Stained eggs were considered as non-viable and unstained were considered as viable (Fig. 1). Each experiment was conducted in triplicates. In addition, approximately 1000 eggs not subjected to heat-inactivation were incubated at room temperature for 1h, stained with above three stains, and assessed by microscopy to determine the numbers of stained non-viable and unstained viable eggs in the stock solution. An analysis of variance (ANOVA) was performed to determine whether the numbers of stained non-viable eggs obtained using different stains were statistically significant at the level of P<0.05. The potential for false viability results using all three staining methods as opposed to the incubation time were calculated by subtracting stained non-viable eggs from total numbers of eggs.
Results and discussion The stock solution contained 17–23% stained non-viable eggs as per microscopic observation. For heat-inactivated eggs, 32–53% became stained non-viable after 1h of incubation at room temperature (Table 1). The numbers of stained non-viable hookworm eggs increased to 88–94% after 6h of incubation. Our results suggest that the vital stain method could potentially over estimate up to 11% viable eggs after 6h of incubation (Fig. 2). This is particularly important considering the infectious dose of hookworms is low (1–10 larvae). All 1000 heat-inactivated eggs became stained non-viable after 12h of incubation at room temperature. Similar results were also observed for 24h incubation experiment. Our results also suggest that accurate assessment of the viability of hookworm eggs from environmental samples can be achieved if they Brefeldin A are incubated at room temperature for up to 12h prior to microscopic observation. We also evaluated whether molecular weight of a particular stain can influence the viability assessment of hookworm eggs. Among the stains used in this study, methylene blue was found to be more effective in penetrating eggshells of inactivated hookworm eggs after 1 and 6h of incubation compared to methyl red and eosin Y. However, the difference was not statistically significant (P>0.05) (Table 1). Similar result was also reported by de Victorica and Galvan (2003). In conclusion, the accurate viability assessment of hookworm eggs depends on incubation time, which is determined to be up to 12h. The molecular weight of different stains may not play any significant role in determining the accuracy of viability assessment.
Acknowledgment The authors appreciate the financial support received from the Water Corporation, WA (Grant number XX-09-1390) and the Commonwealth Scientific and Industry Research Organization (CSIRO), Land and Water Flagship (Grant number EOP 77576). The authors thank Lyn Knott, Laboratory Manager Faculty of Veterinary Science, University of Queensland, Australia for providing A. caninum positive fecal samples from dogs.
Introduction The protozoans Giardia and Cryptosporidum are recognized as major causes of diarrhea and nutritional disorders in institutional and community settings (Savioli et al., 2006). Among the waterborne pathogens, Giardia and Cryptosporidium are the most common causes of major diarrheal outbreaks globally (Karanis et al., 2007). A FAO/WHO Expert Commitee ranked Cryptosporidium and Giardia as the 5th and 11th most important global food-borne zoonoses, respectively, using a multicriteria ranking approach (WHO/FAO, 2014). The role of water and food, particularly fresh produce as a source for these protozoan agents is now well-recognized as documented in traceback-outbreak investigations, which is carried out through the detection of oocysts and cysts in vegetables and water samples, e.g. when irrigated in fields (Amoros et al., 2010; Armon et al., 2002; Robertson and Gjerde, 2001; Smith et al., 2006; Smith and Nichols, 2010; Vuong et al., 2007). Forage and fresh produce becomes contaminated when improperly treated with reclaimed low quality water, for example, wastewater is used to irrigate agricultural land, leading to human exposure and disease. Alternatively, direct contamination of fresh produce through handling by farm workers may also occur. The contamination of fruit and vegetables with oocysts and cysts is an important source of human infection because these products are frequently eaten raw or lightly cooked (Cook et al., 2007).