The use of nemabiome metabarcoding to explore gastro-intestinal nematode species diversity and anthelmintic treatment effectiveness in beef calves.

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.

Exploring the Gastrointestinal "Nemabiome": Deep Amplicon Sequencing to Quantify the Species Composition of Parasitic Nematode Communities.

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.

Current Status of Parasite Control at the Feed Yard.

Fly and louse infestations are readily discerned and remedied in feedlot cattle. Tapeworm and fluke infections are accepted as probable but, given the lack of anthelmintics with realistic efficacy against these infections, these helminths are allowed to persist without treatment. Nematode infections are considered ubiquitous with cattle coming from pasture and are targeted with a macrocyclic lactone, usually in combination with a benzimidazole. Populations of nematodes seem to be effectively controlled by a combination of anthelmintic treatment, animal resistance and resilience, lack of reinfection, and diet.

A sampling of factors relative to the epidemiology of gastrointestinal nematode parasites of cattle in the United States.

Gastrointestinal nematodosis of cattle is a parasitic condition resulting from an immense and seemingly forever-expanding array of factors. Countless determinants influence the incidence and severity of the species-specific infections that occur in cattle, determinants that affect the free-living or environmental stages of the parasites and the parasitic stages. The vast majority of animals have a subclinical or economic level of parasitism undetectable to the eye but quantified more accurately by treatment-induced improved performance (e.g., feed efficiency, nitrogen balance, weight gain, milk production). Unfortunately, the results of treatment (effectiveness and improved animal performance) sometimes can be as varied as the parasitisms that are being treated.