Biomarkers of gastrointestinal nematodes in beef cattle raised in a tropical area.

Biomarkers are specific molecular, histological, or physiological characteristics of normal or pathogenic biological processes and are promising in the diagnosis of gastrointestinal nematodes (GINs). Although some biomarkers have been validated for infection by Ostertagia sp. in cattle raised in temperate regions, there is a lack of information for tropical regions. The aim of this project was to assess potential biomarkers and validate the most promising. In the first study, 36 bovines (Nelore breed) naturally infected by GINs were distributed into two groups: infected (not treated with anthelmintic) and treated (treated with fenbendazole on days 0, 7, 14, 21, 28, 42, and 56). The variables of interest were live weight, fecal egg count, hemogram, serum biochemical markers, phosphorus, gastrin, and pepsinogen. In the second step, pepsinogen was assessed in cattle of the Nelore breed distributed among three groups: infected (not treated with anthelmintic), MOX (treated with moxidectin), and IVM + BZD (treated with ivermectin + albendazole). In the first study, no difference between groups was found for weight, albumin, hematocrit (corpuscular volume [CV]), erythrocytes, or hemoglobin. Negative correlations were found between pepsinogen and both CV and albumin, and albumin was negatively correlated with the percentage of Haemonchus sp. in the fecal culture. Among the biomarkers, only pepsinogen differentiated treated and infected (beginning with the 28th day of the study). In the second study, a reduction in pepsinogen was found after anthelmintic treatment. Therefore, pepsinogen is a promising biomarker of worms in cattle naturally infected by the genera Haemonchus and Cooperia in tropical areas.

Assessing anthelmintic resistance risk in the post-genomic era: a proof-of-concept study assessing the potential for widespread benzimidazole-resistant gastrointestinal nematodes in North American cattle and bison.

As genomic research continues to improve our understanding of the genetics of anthelmintic drug resistance, the revolution in DNA sequencing technologies will provide increasing opportunities for large-scale surveillance for the emergence of drug resistance. In most countries, parasite control in cattle and bison has mainly depended on pour-on macrocyclic lactone formulations resulting in widespread ivermectin resistance. Consequently, there is an increased interest in using benzimidazole drugs which have been used comparatively little in cattle and bison in recent years. This situation, together with our understanding of benzimidazole resistance genetics, provides a practical opportunity to use deep-amplicon sequencing to assess the risk of drug resistance emergence. In this paper, we use deep-amplicon sequencing to scan for those mutations in the isotype-1 ß-tubulin gene previously associated with benzimidazole resistance in many trichostrongylid nematode species. We found that several of these mutations occur at low frequency in many cattle and bison parasite populations in North America, suggesting increased use of benzimidazole drugs in cattle has the potential to result in widespread emergence of resistance in multiple parasite species. This work illustrates a post-genomic approach to large-scale surveillance of early emergence of anthelmintic resistance in the field.

Impact on beef cattle productivity of infection with anthelmintic-resistant nematodes.

ABSTRACTAims: The main goal of the current study was to evaluate, on a commercial beef cattle farm, the impact of infection with gastrointestinal nematodes resistant to both ivermectin (IVM) and moxidectin (MXD) on the productivity of calves.Methods: Male Aberdeen Angus calves, aged 9-11 months, with faecal nematode egg counts (FEC) ≥200 epg and body weight ≥190 kg, were allocated to two herds. Herd A (n = 90) grazed a maize-winter forage crop rotation and Herd B (n = 90) grazed a 2-year-old Agropyrum pasture. On Day 0 in each herd, calves were randomly allocated into five groups (n = 18), which were treated with 0.2 mg/kg IVM; 0.2 mg/kg MXD; 3.75 mg/kg ricobendazole (RBZ), both IVM and RBZ, or remained untreated. Faecal samples collected on Days -1 and 19 were used to determine the percentage reduction in FEC, and genera of the nematodes were determined by the identification of the third-stage larvae recovered from faecal cultures. Total weight gain was determined from body weights recorded on Days -1 and 91.Results: Overall mean reduction in FEC was 42% for IVM, 67% for MXD, 97% for RBZ and 99% for IVM + RBZ. The reduction in FEC for Cooperia spp. was ≤78% for IVM and MXD, and for Haemonchus spp. was 0 and 36% for IVM and MXD, respectively, confirming the presence of parasites resistant to both anthelmintics. Only IVM + RBZ treatment resulted in 100% efficacy against Haemonchus spp. The overall estimated mean total weight gain for calves treated with IVM was 15.7 (95% CI = 11.9-19.7) kg and for calves treated with IVM + RBZ was 28.8 (95% CI = 25-32.5) kg (p < 0.001). Mean total weight gain for calves treated with MXD was 23.5 (95% CI = 19.7-27.2) kg.Conclusions and clinical relevance: In calves naturally infected with resistant nematodes, under the production system assessed here, weight gains were lower in calves treated with anthelmintics that were moderately or highly ineffective compared to those treated with highly effective anthelmintics. These results demonstrate to farmers and veterinarians the importance of a sustainable and effective nematode control under field conditions.

A Revised Checklist of Cooperia Nematodes (Trichostrogyloidea), Common Parasites of Wild and Domestic Ruminants.

This review updates the current knowledge on the taxonomy of intestinal nematodes of the genus Cooperia parasitizing in wild and domestic ruminants. The emphasis is put on revision of 19 valid species belonging to the genus. This analysis focuses on main features of the genus Cooperia, including its geographic occurrence and the life cycle details. The most widespread congeners are Cooperia curticei, C. oncophora, C. pectinata, and C. punctata, having nearly worldwide distribution. The fifth species, referred by electronic databases from the European territory as Cooperia asamati Spiridonov, 1985, is unveiled here originally as nomen nudum.

Anthelmintic resistance among gastrointestinal nematodes of cattle on dairy calf to beef farms in Ireland.

BACKGROUND: The control of gastrointestinal nematodes (GIN) of cattle in pasture-based production systems such as Ireland is highly dependent on the availability of efficacious anthelmintics. There is very little information available on the efficacy of the broad-spectrum anthelmintics against GIN of cattle in Ireland and the aim of this study was to determine the prevalence of anthelmintic resistance on dairy calf to beef farms. RESULTS: GIN burden was monitored on thirty-six recruited farms by performing herd level faecal egg counts (FEC) every 2 weeks. Of these, nine farms were lost from the study as calves were treated with an anthelmintic for Dictyocaulus viviparus, two were lost as they treated for GIN, one dropped out of the study and on one the herd FEC did not reach the threshold for carrying out the Faecal Egg Count Reduction Test (FECRT). On the remaining 23 farms, once the herd FEC reached 100 eggs per gram, a FECRT was carried out. Pre and post-treatment larval cultures were also performed to identify the GIN to genus level. The efficacy of fenbendazole, levamisole, ivermectin and moxidectin was evaluated on 15, 11, 16 and 11 farms respectively. Resistance to fenbendazole was identified on 9 farms (60%) with resistance suspected on a further farm. Resistance to levamisole, ivermectin and moxidectin was detected on 2 (18%), 16 (100%) and 8 (73%) farms respectively. The predominant genera detected pre and post-treatment were Cooperia and Ostertagia with both genera detected post-treatment with fenbendazole and ivermectin. Due to the low proportion of Ostertagia spp. pre-treatment, the efficacy of levamisole or moxidectin against this genus could not be reliably established. CONCLUSIONS: Anthelmintic resistance was widespread on the sampled dairy calf to beef farms in Ireland with resistance to benzimidazole, levamisole, ivermectin and moxidectin detected.

Molecular detection of two major gastrointestinal parasite genera in cattle using a novel droplet digital PCR approach.

Cooperia sp. and Ostertagia sp. are two cosmopolitan parasitic nematodes often found in mixed gastrointestinal infections in cattle across temperate regions. In light of the recent increase in the emergence of anthelmintic resistance in these and other nematodes derived from cattle around the globe, and their negative impact on animal health and productivity, novel molecular assays need to be put forth in order to facilitate the monitoring of parasite burden in infected herds, using pasture and/or fecal samples. Here, we describe a novel droplet digital PCR platform-based concept for precise identification and quantification of the two most abundant and important parasite genera in grazing western European cattle. By exploiting a single nucleotide difference in the two parasites' ITS2 sequence regions, we have developed two specific hydrolysis probes labeled with FAM™ or HEX™ fluorophores, which can not only distinguish between the DNA sequences of the two, but also quantify them in mixed DNA samples. A third, newly developed universal probe was also tested along the genus-specific probes to provide a robust and accurate reference. It was evident that the universal probe displayed congruent results to those obtained by the genus-specific probes when used with DNA from both parasites in a single sample. All in all, the results of our assay suggest that this novel protocol could be used to distinguish and quantify cattle parasites belonging to the two most important genera (i.e., Cooperia and Ostertagia) in a single mixed DNA sample.

Morphological variability, molecular phylogeny, and biological characteristics of the nematophagous fungus Duddingtonia flagrans.

This study aims to investigate the molecular phylogenetic analysis, morphological variability, nematode-capturing ability, and other biological properties of Chinese Duddingtonia flagrans isolates. We isolated 13 isolates of D. flagrans and found features that have never been reported before, such as two to three septa incluing club-shaped conidia. Meanwhile, we conducted molecular phylogenetic analysis of the seven isolates and tested the radical growth of the isolates under different pH values, temperatures, and media. The capturing ability against infective larvae (L3) of Cooperia spp. in yak was detected in vitro. Finally, one isolate was selected for scanning electron microscopy (SEM) to investigate the trap formation process. The fungal sequence was obtained and submitted to GenBank (Accession no. KY288614.1, KU881774.1, KP257593.1, KY419119.1, MF488979.1, MF488980.1, and MF488981.1), and the tested isolates were identified as D. flagrans. Except for three isolates, the radial growth of the other isolates on 2% corn meal agar and 2% water agar exhibited faster growth than on other media. The fungus could not grow at 10 and 40°C but grew within 11 to 30°C. Moreover, it did not grow at pH 1-3 and 13-14, but instead at pH 4-12. In the in vitro experimental, L3s were reduced by 94.36%, 88.15%, and 91.04% for SDH035, DH055, and F088, respectively. SEM results showed that at 8 hr post addition of nematodes, some of the latter were captured. In the later stages of the interaction of the fungus with nematodes, a large number of chlamydospores were produced, especially on the predation trap. Results of the present study provided information about the molecular phylogenetic analysis, morphological variability, nematode-capturing ability, and other biological properties of Chinese Arthrobotrys flagrans isolates before administering them for biocontrol.

Nematode parasites in young cattle: what role for unexpected species?

AIMS To investigate the timing of infection of beef calves with sheep nematode species on three sheep and beef farms, and to determine the prevalence of cross-infection in calves before weaning across a larger number of farms. METHODS Farms in the Far North, Gisborne and Tararua districts, in the North Island of New Zealand, were enrolled in 2014. Fresh faecal samples were collected from approximately 10 calves on each farm between birth and up to 5 months after weaning. In 2016, faecal samples were collected from calves before weaning from 22 farms across the upper North Island. For both trials faecal samples were assessed for faecal nematode egg counts and cultured to determine parasite genus. For samples from the three farms, larvae were identified to species using a multiplex PCR assay. RESULTS On the three farms, the median percentage of sheep nematode species detected in faecal cultures was 25 (min 3, max 77)%. The main sheep species detected were Cooperia curticei and Haemonchus spp. (putatively contortus). In faecal samples collected before weaning from 22 farms, Haemonchus spp. were present in 19/22 samples, and the median prevalence was 15 (min 0, max 73)% of the total larvae cultured. CONCLUSIONS AND CLINICAL RELEVANCE The implications of sheep nematode species being present in calves should be considered by farmers and veterinarians when undertaking anthelmintic efficacy testing, as they may contribute to false conclusions regarding anthelmintic efficacy. Pre-weaning calves may also be a possible source of contamination and/or refugia for Haemonchus spp. on farms and should be considered when developing parasite control plans for sheep.

Ivermectin treatment failure on four Irish dairy farms.

We report on the use of the faecal egg count reduction test to evaluate the performance of ivermectin in treating gastrointestinal nematode infections in first grazing season (FGS) calves on four dairy farms in Co. Kilkenny, Ireland. On each farm, FGS calves were injected subcutaneously with ivermectin in accordance with their live weight (day 0). Calves were individually faecal sampled on both day 0 and day 14. Faecal egg counts were determined using the Mini-FLOTAC technique. Composite faecal cultures for each farm were performed on each sampling occasion. The faecal egg count reductions (mode) ranged from 17.3-80.2% with the lower 95% confidence limit ranging from 3.1-72.3% on the four farms, respectively. Ivermectin-resistant nematodes were detected on all farms, with evidence of Ostertagia resistance on one farm. This study highlights the urgent need for Irish producers to reappraise their parasite control practices.

Anthelmintic effect of 2H-chromen-2-one isolated from Gliricidia sepium against Cooperia punctata.

Gliricidia sepium is a tropical legume with known anthelmintic-like properties. The aim of this study was to: (1) perform a bio-guided fractionation of an acetonic extract of G. sepium leaves using the egg hatch assay (EHA); (2) elucidate the anthelmintic (AH)-like phytochemical using nuclear magnetic resonance (NMR); and (3) assess the ultrastructural damage of the Cooperia punctata treated eggs. The anthelmintic activity of G. sepium was traced from an acetonic extract using the EHA. Phytochemicals were isolated through silica gel columns and elucidated through spectroscopic measurements (1H and 13C). Final fraction was evaluated with EHA at decreasing concentrations of: 1.100; 0.500, 0.250, 0.125, 0.060, 0.001 and 0.00001 mg mL-1. Egg hatching inhibition was calculated using the formula: 100*(1-HT/HC). The maximal half of effective concentration (EC50) was calculated with GraphPad. Bio-guided isolation procedures lead to the elucidation of 2H-chromen-2-one, which inhibited both hatching and embryo development of C. punctata (EC50 of 0.024 ± 0.082 mg mL-1) (P < 0.05). Scanning and Transmission Electron Microscopy (SEM and TEM) revealed electrodensity alterations and fractures in the eggshell layers. After toxicity evaluations and in vivo assessment, 2H-chromen-2-one can be suggested as a novel AH-phytochemical for reducing larval density in pastures and worm burdens inside the host.

A comparison of two methods for quantifying parasitic nematode fecundity.

Accurate measures of nematode fecundity can provide important information for investigating parasite life history evolution, transmission potential, and effects on host health. Understanding differences among fecundity assessment protocols and standardizing methods, where possible, will enable comparisons across different studies and host and parasite species and systems. Using the trichostrongyle nematode Cooperia fuelleborni isolated from wild African buffalo (Syncerus caffer), we compared egg recovery and enumeration between two methods for measuring the fecundity of female worms. The first method, in utero egg count, involves visual enumeration of the eggs via microscopic inspection of the uterine system. The second method, ex utero egg count, involves dissolving the same specimens from above in a sodium chloride solution to release the eggs from the female's uterus, then enumeration under an inverted microscope. On average, the ex utero method resulted in 34% more eggs than the in utero method. However, results indicate that the two methods used to quantify parasitic nematode fecundity are highly correlated. Thus, while both methods are viable options for estimating relative nematode fecundity, we recommend caution in undertaking comparative studies that utilize egg count data collected using different methods.

Anthelmintic effects of forage chicory (Cichorium intybus) against gastrointestinal nematode parasites in experimentally infected cattle.

Two experiments studied the effects of dietary chicory against gastrointestinal nematodes in cattle. In Experiment (Exp.) 1, stabled calves were fed chicory silage (CHI1; n = 9) or ryegrass/clover hay (CTL1; n = 6) with balanced protein/energy intakes between groups. After 16 days, all calves received 10 000 Ostertagia ostertagi and 66 000 Cooperia oncophora third-stage larvae (L3) [day (D) 0 post-infection (p.i.)]. In Exp. 2, calves were assigned to pure chicory (CHI2; n=10) or ryegrass/clover (CTL2; n = 10) pastures. After 7 days, animals received 20 000 O. ostertagi L3/calf (D0 p.i.) and were moved regularly preventing pasture-borne infections. Due to poor regrowth of the chicory pasture, CHI2 was supplemented with chicory silage. At D40 p.i. (Exp. 1) and D35 p.i. (Exp. 2) calves were slaughtered for worm recovery. In Exp.1, fecal egg counts (FEC) were similar between groups. However, O. ostertagi counts were significantly reduced in CHI1 by 60% (geometric mean; P < 0·01), whereas C. oncophora burdens were unaffected (P = 0·12). In Exp. 2, FEC were markedly lowered in CHI2 from D22 p.i onwards (P < 0·01). Ostertagia ostertagi adult burdens were significantly reduced in CHI2 by 66% (P < 0·001). Sesquiterpene lactones were identified only in chicory (fresh/silage). Chicory shows promise as an anti-Ostertagia feed for cattle and further studies should investigate its on-farm use.

Impact of chemical structure of flavanol monomers and condensed tannins on in vitro anthelmintic activity against bovine nematodes.

Plants containing condensed tannins (CT) may have potential to control gastrointestinal nematodes (GIN) of cattle. The aim was to investigate the anthelmintic activities of four flavan-3-ols, two galloyl derivatives and 14 purified CT fractions, and to define which structural features of CT determine the anti-parasitic effects against the main cattle nematodes. We used in vitro tests targeting L1 larvae (feeding inhibition assay) and adults (motility assay) of Ostertagia ostertagi and Cooperia oncophora. In the larval feeding inhibition assay, O. ostertagi L1 were significantly more susceptible to all CT fractions than C. oncophora L1. The mean degree of polymerization of CT (i.e. average size) was the most important structural parameter: large CT reduced larval feeding more than small CT. The flavan-3-ols of prodelphinidin (PD)-type tannins had a stronger negative influence on parasite activity than the stereochemistry, i.e. cis- vs trans-configurations, or the presence of a gallate group. In contrast, for C. oncophora high reductions in the motility of larvae and adult worms were strongly related with a higher percentage of PDs within the CT fractions while there was no effect of size. Overall, the size and the percentage of PDs within CT seemed to be the most important parameters that influence anti-parasitic activity.

Simultaneous resistance to multiple anthelmintic classes in nematode parasites of cattle in New Zealand.

Resistance to the benzimidazole and macrocyclic lactone anthelmintics is widespread in Cooperia spp. on cattle farms in New Zealand. Since this was first documented in 2006 little has changed in cattle farming systems except for the widespread use of levamisole to control Cooperia spp. in young cattle (i.e., parasite control has maintained an almost total reliance on use of anthelmintics). Here we report the emergence of simultaneous resistance to the benzimidazole, macrocyclic lactone and levamisole anthelmintics in Cooperia spp. and in Ostertagia spp. Anthelmintic efficacy against nematode parasites of cattle was investigated on four commercial farms following reports of poor animal growth rates and welfare, and positive faecal egg counts, despite routine treatment with combination anthelmintics, which included levamisole. Faecal egg count reduction tests involved 15 animals per treatment group, individual egg counts (paired samples) conducted pre- and post-treatment, with eggs counted to ≤ 15 eggs per g faeces and larval cultures for morphological identification. Actives tested varied between farms but always included levamisole alone and several combination products containing levamisole. Of the 20 tests conducted (i.e., 5 products on each of 4 farms) only 3 exceeded 90% efficacy against Cooperia spp. even though 8 of the products tested were combinations containing levamisole and at least one other broad-spectrum anthelmintic. Levamisole used alone achieved efficacies between 44% and 71% against Cooperia spp. across the four trials. The only product to exceed 95% efficacy against Cooperia spp. was a combination of monepantel + abamectin which was 100% effective against all parasites. Resistance to oxfendazole in Ostertagia spp. was indicated on 3 farms, while on one farm efficacy of all the tested products was ≤75% against this parasite. All the farms involved in this study were farming intensive cattle operations with an almost total reliance on anthelmintics to control parasitism. The results clearly demonstrate the emergence of simultaneous resistance to oxfendazole, levamisole and the macrocyclic lactone anthelmintics. Despite years of advice and recommendations to change farming practices away from intensive monocultures, many farmers have continued with the practice, and some are now faced with the very real possibility of being unable to control cattle parasites on their farms.

Corrigendum: A revision of the trichostrongylid nematode Cooperia Ransom, 1907, from deer game: recent integrative research confirms the existence of the ancient host-specific species Cooperia ventricosa (Rudolphi, 1809).

[This corrects the article DOI: 10.3389/fvets.2024.1346417.].

A revision of the trichostrongylid nematode Cooperia Ransom, 1907, from deer game: recent integrative research confirms the existence of the ancient host-specific species Cooperia ventricosa (Rudolphi, 1809).

The trichostrongylid roundworms of the genus Cooperia, which are important in veterinary medicine, currently comprise 19 valid species that parasitize the small intestine of both free-living and domestic ruminants. Only four Cooperia spp. have been reported in Europe, namely C. oncophora, C. punctata, C. curticei and C. pectinata. In 2018-2022, 25 red deer (Cervus elaphus) and 30 sika deer (Cervus nippon) of both sexes and various ages from several remote locations in the Czech Republic were parasitologically examined. Intestinal nematodes of the genus Cooperia were found only in two northern regions. Using the globally recognized key book on trichostrongylid nematodes, they were preliminarily identified as C. pectinata. However, a molecular analysis of cox2 and ITS rDNA gene sequences revealed that Cooperia sp. parasitizing Czech deer is a separate taxon that is more closely related to C. oncophora than to C. pectinata. A subsequent morphological analysis and literature survey confirmed the independence of deer Cooperia sp., which is similar but not identical to bovid C. pectinata. Previous long-term correct identifications of bovid C. pectinata and misidentifications of deer Cooperia species were caused by a fundamental error in the key book mentioned above. Interestingly, the ancient trichostrongylid nematode Strongylus ventricosus from the type host red deer (Cervus elaphus) shot near Greifswald (Germany) was described by Rudolphi in 1809. Rudolphi's type material (one male and four females) was deposited in the Museum für Naturkunde (Berlin). Later, the ancient species S. ventricosus was taken as a synonym for various Cooperia spp. Our current re-examination of the type male indicated that there is a relatively good agreement with our new material from Czech deer regarding the most important characteristics of S. ventricosus (i.e., the shape and size of the male spicules); however, Rudolphi's type material is in rather poor condition. The suggested resurrection of the deer Cooperia sp. in this study as Cooperia ventricosa (Rudolphi, 1809) requires verification by collecting and analyzing new nematode material from the type locality near Greifswald.

Toltrazuril + fenbendazole for cattle: Pharmacokinetics and efficacy against Eimeria spp. and gastrointestinal nematodes.

The present work evaluated the pharmacokinetics and efficacy of the association of 15cmg/kg toltrazuril +5cmg/kg fenbendazole against Eimeria spp. and gastrointestinal nematodes (GINs) in calves of different regions of Brazil (Center-West, Southeast, and South). A total of seven experiments were carried out, five of which determined formulation efficacy against Eimeria spp., considering the following aspects: therapeutic, preventive, metaphylactic, and residual efficacy. Therapeutic efficacy experiments for GINs were carried out by parasitological necropsy. The toltrazuril + fenbendazole association demonstrated ≥95% efficacy against Eimeria spp. for 21 days post-treatment (DPT). When used preventively and metaphylatically, the same association demonstrated ≥97% efficacy against E. zuernii, E. ellipsoidalis, E. cylindrica, E. bovis, E. wyomingensis and E. auburnensis. Toltrazuril + fenbendazole administered seven days before challenge was 100% effective against all these Eimeria species. Results of therapeutic, preventive, metaphylactic and residual efficacies can be related to the pharmacokinetic results, especially considering toltrazuril sulfone, which was detected in animal plasma for a longer period than the parent compound. Toltrazuril + fenbendazole achieved 100% anthelminthic efficacy against the GINs Haemonchus placei (L4), Cooperia pectinata and Oesophagostomum radiatum; 99.94% against adult H. placei; and 99.98% against C. puntacta. The association of toltrazuril + fenbendazole, associated with other measures, is an important and suitable tool for the control and treatment of Eimeria spp. and GINs in young cattle.

The identification of small molecule inhibitors with anthelmintic activities that target conserved proteins among ruminant gastrointestinal nematodes.

Gastrointestinal nematode (GIN) infections are a major concern for the ruminant industry worldwide and result in significant production losses. Naturally occurring polyparasitism and increasing drug resistance that potentiate disease outcomes are observed among the most prevalent GINs of veterinary importance. Within the five major taxonomic clades, clade Va represents a group of GINs that predominantly affect the abomasum or small intestine of ruminants. However, the development of effective broad-spectrum anthelmintics against ruminant clade Va GINs has been challenged by a lack of comprehensive druggable genome resources. Here, we first assembled draft genomes for three clade Va species (Cooperia oncophora, Trichostrongylus colubriformis, and Ostertagia ostertagi) and compared them with closely related ruminant GINs. Genome-wide phylogenetic reconstruction showed a relationship among ruminant GINs structured by taxonomic classification. Orthogroup (OG) inference and functional enrichment analyses identified 220 clade Va-specific and Va-conserved OGs, enriched for functions related to cell cycle and cellular senescence. Further transcriptomic analysis identified 61 taxonomically and functionally conserved clade Va OGs that may function as drug targets for new broad-spectrum anthelmintics. Chemogenomic screening identified 11 compounds targeting homologs of these OGs, thus having potential anthelmintic activity. In in vitro phenotypic assays, three kinase inhibitors (digitoxigenin, K-252a, and staurosporine) exhibited broad-spectrum anthelmintic activities against clade Va GINs by obstructing the motility of exsheathed L3 (xL3) or molting of xL3 to L4. These results demonstrate valuable applications of the new ruminant GIN genomes in gaining better insights into their life cycles and offer a contemporary approach to discovering the next generation of anthelmintics.IMPORTANCEGastrointestinal nematode (GIN) infections in ruminants are caused by parasites that inhibit normal function in the digestive tract of cattle, sheep, and goats, thereby causing morbidity and mortality. Coinfection and increasing drug resistance to current therapeutic agents will continue to worsen disease outcomes and impose significant production losses on domestic livestock producers worldwide. In combination with ongoing therapeutic efforts, advancing the discovery of new drugs with novel modes of action is critical for better controlling GIN infections. The significance of this study is in assembling and characterizing new GIN genomes of Cooperia oncophora, Ostertagia ostertagi, and Trichostrongylus colubriformis for facilitating a multi-omics approach to identify novel, biologically conserved drug targets for five major GINs of veterinary importance. With this information, we were then able to demonstrate the potential of commercially available compounds as new anthelmintics.

Benzimidazole Resistance in Cattle: The First Report of the Presence of F200Y Mutation in Cooperia in Ecuador.

Anthelmintic resistance among GINs in cattle is a worldwide issue. Identifying the early signs of anthelmintic resistance (AR) is necessary to sustainably manage bovine parasitic infections. This study aimed to evaluate the resistance status of bovine parasitic nematodes against FBZ on a farm with a known history of broad-spectrum anthelmintic usage in Ecuador. FBZ efficacy was analyzed using a fecal egg count reduction test (FECR test) and ß-tubulin 1 mutation identification in Cooperia spp., the dominant nematode parasite identified before and after treatment. According to the FECR test, the nematode population was susceptible to FBZ. After amplifying and cloning the ß-tubulin 1 of Cooperia spp., an F200Y mutation was found in 43% of the pooled larva coproculture after treatment. This study reports, for the first time, the presence of F200Y resistance-conferring mutation in Cooperia spp. in Ecuador. Although the nematode population was phenotypically susceptible to FBZ, the presence of F200Y suggests the existence of resistance in the early stages. Our findings highlight the need to implement alternative control strategies for parasitic infections besides broad-spectrum anthelmintics.

Integration of ITS-2 rDNA nemabiome metabarcoding with Fecal Egg Count Reduction Testing (FECRT) reveals ivermectin resistance in multiple gastrointestinal nematode species, including hypobiotic Ostertagia ostertagi, in western Canadian beef cattle.

A large-scale Fecal Egg Count Reduction Test (FECRT) was integrated with ITS-2 rDNA nemabiome metabarcoding to investigate anthelmintic resistance in gastrointestinal nematode (GIN) parasites in western Canadian beef cattle. The study was designed to detect anthelmintic resistance with the low fecal egg counts that typically occur in cattle in northern temperate regions. Two hundred and thirty-four auction market-derived, fall-weaned steer calves coming off pasture were randomized into three groups in feedlot pens: an untreated control group, an injectable ivermectin treatment group, and an injectable ivermectin/oral fenbendazole combination treatment group. Each group was divided into six replicate pens with 13 calves per pen. Individual fecal samples were taken pre-treatment, day 14 post-treatment, and at monthly intervals for six months for strongyle egg counting and metabarcoding. Ivermectin treatment resulted in an 82.4% mean strongyle-type fecal egg count reduction (95% CI 67.8-90.4) at 14 days post-treatment, while the combination treatment was 100% effective, confirming the existence of ivermectin-resistant GIN. Nemabiome metabarcoding of third-stage larvae from coprocultures revealed an increase in the relative abundance of Cooperia oncophora, Cooperia punctata, and Haemonchus placei at 14 days post-ivermectin treatment indicating ivermectin resistance in adult worms. In contrast, Ostertagia ostertagi third-stage larvae were almost completely absent from day 14 coprocultures, indicating that adult worms of this species were not ivermectin resistant. However, there was a recrudescence of O. ostertagi third stage larvae in coprocultures at three to six months post-ivermectin treatment, which indicated ivermectin resistance in hypobiotic larvae. The calves were recruited from the auction market and, therefore, derived from multiple sources in western Canada, suggesting that ivermectin-resistant parasites, including hypobiotic O. ostertagi larvae, are likely widespread in western Canadian beef herds. This work demonstrates the value of integrating ITS-2 rDNA metabarcoding with the FECRT to enhance anthelmintic resistance detection and provide GIN species- and stage-specific information.