The maximum settling time examined in

The maximum settling time examined in our study was 90min, but oocyst sedimentation would probably continue if the settling time was extended resulting in increased oocyst reduction. A time estimate required for all oocysts to reach the bottom of the glass jars was calculated as 3.6h in treated water and 74.6h in untreated water. Hence, the oocyst load was reduced 20.7 times faster in wastewater treated with MO Tozadenant extract than by natural sedimentation. Our study was based on treatment with a single dose of MO seed extract but further studies including treatment with different doses, different water types and longer settling periods are needed. In conclusion, this laboratory based-study showed that adding 4mLL of a 5% w/v extract from seed powder of the M. oleifera tree significantly reduced the number of C. parvum oocysts as compared to natural sedimentation of 90 min in wastewater.
Acknowledgements This research was supported by a grant from the PATHOS project funded by the Strategic Research Council of Denmark (ENV 2104-07-0015). The Ph.D. fellowship of Heidi Huus Petersen was jointly financed by the PATHOS project and the Graduate School of Health and Medical Sciences, University of Copenhagen.
Introduction Hookworm infection is responsible for malnutrition, anemia, and impaired cognitive development in humans (Brooker et al., 2008; WHO, 2012). It is estimated that approximately 8.0×106 people worldwide are infected with hookworm (Hotez et al., 2014). Despite implementation of several interventions to reduce hookworm infection in developing countries, it is difficult to control (WHO, 2012; WHO, 2015). Land application of untreated wastewater and sludge is a common practice in developing countries (Do et al., 2007; Gupta et al., 2009). As a result, a high load of viable hookworm eggs can be present in the environment, which may pose a significant human health risk (Karkashan et al., 2014). Several guidelines have been developed by national and international authorities to minimize the risk of hookworm infection associated with wastewater and sludge (US EPA, 2003; NRMMC, 2004; WHO, 2006). These guidelines, however, do not provide a standardized protocol for reliable detection and quantification of viable hookworm eggs from wastewater matrices. It is necessary to detect viable eggs in order to determine accurate health risk if wastewater is used for land application. Conventional egg viability assessment method such as incubation is time consuming, expensive and laborious (Karkashna et al., 2014; Gyawali et al., 2015). Vital stain method provides information on the viability of hookworm eggs more rapidly than the incubation method (Dabrowaska et al., 2014; Karkashan et al., 2014). Vital stain method takes advantage of structural integrity of viable and non-viable eggshells. A viable egg has three layers of intact shells that act as an alternative barrier and prevent stain from entering into the cytoplasm (Matthews, 1986). Once the egg becomes non-viable, the integrity of eggshells is compromised and becomes permeable to stains (Bae and Wuertz, 2009). The eggshells, however, may not be permeable to vital stains immediately after inactivation. In this study, we evaluated time required for accurate viability assessment of hookworm eggs using three commonly used vital stains. These stains include eosin Y (MW=647.8), methyl red (MW=269.3), and methylene blue (MW=319.8). They were chosen to evaluate, whether molecular weight of a particular stain can influence the assessment of viability.
Material and methods Dog fecal samples were collected after obtaining ethics approval from the Animal Ethics Committee, The University of Queensland, Australia (Reference number: AEC/QU/12/2013). Eggs of the dog hookworm [Ancylostoma caninum] were isolated from the fecal samples using magnesium sulfate (specific gravity 1.2) flotation method (Gyawali et al., 2015). The isolated eggs were enumerated by microscopy using a Sedgewick-Rafter Counting chamber (PYSER-SGI, UK).