Parasitic helminth infections have a considerable impact on global human health as well as animal welfare and production. Although co-infection with multiple parasite species within a host is common, there is a dearth of tools with which to study the composition of these complex parasite communities. Helminth species vary in their pathogenicity, epidemiology and drug sensitivity and the interactions that occur between co-infecting species and their hosts are poorly understood. We describe the first application of deep amplicon sequencing to study parasitic nematode communities as well as introduce the concept of the gastro-intestinal “nemabiome”. The approach is analogous to 16S rDNA deep sequencing used to explore microbial communities, but utilizes the nematode ITS-2 rDNA locus instead. Gastro-intestinal parasites of cattle were used to develop the concept, as this host has many well-defined gastro-intestinal nematode species that commonly occur as complex co-infections. Further, the availability of pure mono-parasite populations from experimentally infected cattle allowed us to prepare mock parasite communities to determine, and correct for, species representation biases in the sequence data. We demonstrate that, once these biases have been corrected, accurate relative quantitation of gastro-intestinal parasitic nematode communities in cattle fecal samples can be achieved. We have validated the accuracy of the method applied to field-samples by comparing the results of detailed morphological examination of L3 larvae populations with those of the sequencing assay. The results illustrate the insights that can be gained into the species composition of parasite communities, using grazing cattle in the mid-west USA as an example. However, both the technical approach and the concept of the ‘nemabiome’ have a wide range of potential applications in human and veterinary medicine. These include investigations of host-parasite and parasite-parasite interactions during co-infection, parasite epidemiology, parasite ecology and the response of parasite populations to both drug treatments and control programs.
Next-generation deep amplicon sequencing, or metabarcoding, has revolutionized the study of microbial communities in humans, animals and the environment. However, such approaches have yet to be applied to parasitic helminth communities. We recently described the first example of such a method - nemabiome sequencing - based on deep-amplicon sequencing of internal transcribed spacer 2 (ITS-2) rDNA, and validated its ability to quantitatively assess the species composition of cattle gastro-intestinal nematode (GIN) communities. Here, we present the first application of this approach to explore GIN species diversity and the impact of anthelmintic drug treatments. First, we investigated GIN species diversity in cow-calf beef cattle herds in several different regions, using coproculture derived L3s. A screen of 50 Canadian beef herds revealed parasite species diversity to be low overall. The majority of parasite communities were comprised of just two species; Ostertagia ostertagi and Cooperia oncophora. Cooperia punctata was present at much lower levels overall, but nevertheless comprised a substantive part of the parasite community of several herds in eastern Canada. In contrast, nemabiome sequencing revealed higher GIN species diversity in beef calves sampled from central/south-eastern USA and Sao Paulo State, Brazil. In these regions C. punctata predominated in most herds with Haemonchus placei predominating in a few cases. Ostertagia ostertagi and C. oncophora were relatively minor species in these regions in contrast to the Canadian herds. We also examined the impact of routine macrocyclic lactone pour-on treatments on GIN communities in the Canadian beef herds. Low treatment effectiveness was observed in many cases, and nemabiome sequencing revealed an overall increase in the proportion of Cooperia spp. relative to O. ostertagi post-treatment. This work demonstrates the power of nemabiome metabarcoding to provide a detailed picture of GIN parasite community structure in large sample sets and illustrates its potential use in research, diagnostics and surveillance.
Abundances of macrobenthic species were monitored twice yearly (March and September) at 6 locations in Tees Bay, UK, between 1973 and, and once yearly at 4 stations in the outer Tees estuary and 7 stations in the inner estuary between 1980 and 1999. In the Bay, multivariate analysis revealed a serial pattern of community change over years for all areas, but with a major shift in community composition between 1986 and 1988. Inter-annual variability in community composition was significantly greater after 1987 than before 1987 in all areas. Overall, inter-annual variability was greater in areas near the estuary mouth than in areas farther away, although the direction of community change and the timing of the discontinuity were the same in all areas. The serial nature of community change with time was also weaker in the areas close to the estuary mouth. Although there was no clear pattern of change in the number of species present over the sampling period, a dramatic increase in Shannon diversity (H ') occurred after 1987, due to an increase in evenness that resulted from the reduction of a few previously dominant species, notably the small polychaete Spiophanes bombyx. Although biodiversity measures describing the taxonomic breadth of the species assemblages also showed a marked step change in 1987, this was one of reduced diversity, with average taxonomic distinctness (∆ + ) decreasing and the variation in taxonomic distinctness (Λ + ) increasing. These abrupt, detrimental changes coincided with a well-documented change in a variety of components of the North Sea ecosystem during the same period. Traditional species diversity measures, such as H ', therefore gave a false impression of improving environmental quality over this period: given that the average taxonomic spread was reduced, certain taxa were under-represented with respect to others, and community composition as measured by a multivariate stability index (MSI) became less stable. H ' also failed to distinguish putatively impacted areas close to the estuary mouth compared with those more distant, despite clear differences in ∆ + , Λ + , and in community stability (MSI). Overall patterns of biodiversity and community composition in the Bay have thus been affected temporally by regional changes in the North Sea ecosystem, and spatially by the effects of the estuarine outflow. In the estuary itself, multivariate analysis also revealed a serial pattern of community change, with a major shift in composition in 1994 in both the outer and inner estuary which coincided with the construction of a barrage in the estuary. The numbers of both individuals and species began to increase at this time in the outer estuary. H ' showed no obvious changes over the period, but in the outer estuary a step change in ∆ + and Λ + occurred at the same time as that in the Bay. However, the direction of change was the reverse of that in the Bay, suggesting an improvement in environmental quality or a shift to more saline conditions.
Traditionally, the overall diluting capacity of an estuary is characterized using a coefficient of longitudinal dispersion, K xe , which is given by the ratio of the dispersive flux of a dissolved substance to its tidally averaged longitudinal gradient. A steady-state model, which assumes a balance between the dispersive and non-dispersive fluxes and an exponential increase in estuary cross-sectional area towards the sea, has been used to derive expressions for the axial salinity distribution and the dispersive flux of salt. The model was set up assuming either a constant dispersion coefficient along the estuary or one that increased with distance towards the sea. By comparing salinity predictions with data from five UK estuaries, estimates were made of the maximum dispersive salt flux and the corresponding maximum salinity gradient for each system. The results indicated that there was an approximately linear relation between the fluxes and gradients, and the slope of a line plotted through the origin provided an estimate of a common K xe for all five estuaries. The magnitude of K xe was found to be about 90 m 2 s À1 with a standard deviation of approximately AE32 m 2 s À1 . It is concluded that a representative value of 100 m 2 s À1 for K xe is a reasonable first choice when setting up a cross-sectionally averaged estuary model. The results also showed that larger systems, such as the Thames, had lower salinity gradients and lower dispersive salt fluxes, whilst smaller estuaries displayed the opposite characteristics. The model was used to predict the variation in the non-advective flux of salt along an estuary. The distribution was found to be similar to the corresponding flux distribution estimated from observations at the seaward end of the Tees estuary, despite appreciable spatial variations in the individual flux components. Allowing for a small decrease in freshwater flow, the model indicated that there was a decrease in the maximum dispersive flux between neap and spring tides. It is argued that such a reduction in flux can result in a seaward shift in the salinity distribution to a region of greater cross-section, where the freshwater transport per unit area again balances the reduced upstream dispersive flux, as found in the neap to spring response in the Tees estuary.
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