Determination of Ancylostoma caninum ova viability using metabolic profiling

Abstract

Differentiation between viable and non-viable hookworm ova in environmental samples is necessary in order to implement strategies to mitigate re-infections in endemic regions. In this study, an untargeted metabolic profiling method was developed that utilised gas chromatography-mass spectrometry (GC-MS) in order to investigate hookworm ova viability. Ancylostoma caninum was used to investigate the metabolites within viable and non-viable ova. Univariate and multivariate statistical analyses of the data resulted in the identification of 53 significant metabolites across all hookworm ova samples. The major compounds observed in viable and non-viable hookworm ova were tetradecanoic acid, commonly known as myristic acid [fold change (FC) = 0.4], and dodecanoic acid, commonly known as lauric acid (FC = 0.388). Additionally, the viable ova had self-protecting metabolites such as prostaglandins, a typical feature absent in non-viable ova. The results of this study demonstrate that metabolic profiling using GC-MS methods can be used to determine the viability of canine hookworm ova. Further studies are needed to assess the applicability of metabolic profiling using GC-MS to detect viable hookworm ova in the mixed (viable and non-viable) populations from environmental samples and identify the metabolites specific to human hookworm species.

Hookworm-Derived Metabolites Suppress Pathology in a Mouse Model of Colitis and Inhibit Secretion of Key Inflammatory Cytokines in Primary Human Leukocytes

“…Our findings concur with previous reports (39) on the presence of SCFAs in the ES products of A. caninum, in which volatile fatty acids such as acetic, propionic, isobutyric, and branched-chain C 5 acids were detected (39). More recently, saturated fatty acids were reported from the eggs of A. caninum (42), but the authors did not report the presence of SCFAs. The importance of SCFAs in human metabolism and disease has become evident recently, particularly in terms of perturbations in the microbiota (43,44).…”

Hookworm-derived small molecule extracts suppress pathology in a mouse model of colitis and inhibit secretion of key inflammatory cytokines in primary human leukocytes

“…Our findings concur with previous findings (62) on the presence of SCFA in the ES products of A. caninum , where volatile SCFA such as acetic, propionic, isobutyric and branched chain C 5 acids were detected (62). More recently, saturated fatty acids were reported from the ova of A. caninum (65), but the authors did not report the presence of SCFA.…”

Detection of viable hookworm ova from wastewater and sludge

“…Quantitative detection and differentiation between viable and non-viable hookworm ova in point source samples such as environmental soil, wastewater, sludge and contaminated foods are necessary in order to implement strategies to eliminate re-infections in endemic regions (McCarthy et al, 2012;Gyawali et al, 2016b). Despite the advancements of PCR/qPCR methods, the viability of hookworm ova has been determined by either culture-based or vital stain methods.…”

“…Differentiation between viable and non-viable hookworm ova in environmental samples such as wastewater and sludge is necessary in order to identify hookworm contamination and implement strategies to mitigate re-infections in endemic regions (Gyawali et al, 2016b). …”

“…In addition, treated wastewater or sludge can have a high level of background debris which may provide a similar profile to helminths ova during detection/quantification and compromise the sensitivity of the method (Barbosa et al, 2008). (Bowman et al, 2003;Nocker et al, 2007b;McCarthy et al, 2012) Vital stain  Cheap and easy process  Assessment can be done in few hours  Less chemical and equipment  Can be done in small scale laboratory  Difficulty on viability assessment  Sensitivity depends on detection threshold of a microscope  Stain might not effective on recently inactivate ova  Lack specificity (Weber et al, 1991;Nelson and Darby, 2001;Cabaret et al, 2002;Victorica and Galvan, 2003;Verweij et al, 2007;McCarthy et al, 2012;Gyawali et al, 2016b) Flow cytometry  Quick and easy process  Higher sensitivity than microscopy  Automated process  Quantification can be done  Lack of specificity  Difficulty on viability assessment of ova  Difficulty distinguishing background debris from ova (Hammes et al, 2008;Barbosa et al, 2008;Wang et al, 2010) Molecular (PCR/qPCR)  Quick and easy process  Higher sensitivity and specificity  Viability could be possible  Automated process  a Multiple species can be identified from single sample  b Quantification is possible  Require advance laboratory and equipment  Genomic information is essential  Require right genomic target for viability  Possibility of providing false positive result by extracting DNA from inactivated ova  Possibility of false negative result via inhibitors present in the samples  a Multiple sets of primers require which can reduce the sensitivity  b Triplicate sample required (Pecson et al, 2006;Verweij et al, 2007;Traub et al, 2007;Traub et al, 2008;Janwan et al, 2011;Jonker et al, 2012, Ngui et al, 2012b a = Multiplex PCR, b =Quantitative PCR…”