Behavioural response to gastrointestinal parasites of yearling dairy calves at pasture.

AIMS: To investigate the association between gastrointestinal parasites (GIP) and animal behaviour in dairy calves under New Zealand pastoral conditions, using animal-mounted, accelerometer-based sensors. METHODS: Thirty-six, 5-6-month-old, Friesian-Jersey, heifer calves fitted with animal activity sensors to track behaviour were randomly allocated to one of two treatment groups. Half the animals were challenged with an oral dose of 20,000 larvae of Ostertagia ostertagi and Cooperia oncophera once a week for 3 weeks and half were unchallenged. Five weeks after the last dose, seven infected and nine uninfected animals were treated with an oral anthelmintic (AHC) and data collected for a further week. Accelerometer data were classified into minutes per day eating, ruminating, in moderate-high activity or in low activity. Live weight and faecal egg counts (FEC) were recorded weekly over the study period. All animals co-grazed a newly sown pasture not previously grazed by ruminants and were moved every week to fresh grazing. Treatment status was blinded to those managing the animals which were otherwise treated identically. RESULTS: Complete behavioural records were available from 30/36 calves, (13 challenged and 17 unchallenged). Before treatment with AHC, FEC increased in infected and un-treated calves over the study, while uninfected animals maintained a near zero FEC. There was no difference in live weight gain between the two groups over the study period. Bayesian, multinomial regression predicted differences in animal behaviour between infected and uninfected animals that were not treated with AHC over the 7 weeks following initial infection. Parasitised calves not treated with AHC were less active and spent up to 6 (95% highest density interval (HDI) = 1-11) minutes/day less in low level activity and up to 15 (95% HDI = 7-20) minutes/day less in moderate to high level activity. They ruminated up to 9 (95% HDI = 2-15) minutes/day more and ate up to 10 (95% HDI = 2-19) minutes/day more than control calves that were not treated with AHC. The effect of AHC on time spent in each behaviour differed between infected and uninfected calves and increased the coefficient of dispersion of the behavioural data. CONCLUSIONS AND CLINICAL RELEVANCE: Small differences in animal behaviour can be measured in calves with GIP. However, to use this to target treatment, further validation studies are required to confirm the accuracy of behavioural classification and understand the complex drivers of animal behaviour in a dynamic and variable pasture-parasite-host environment.

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.

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.

Efficacy of a fixed-dose combination injectable (0.2 mg/kg doramectin + 6.0 mg/kg levamisole hydrochloride) in Australian cattle against naturally acquired gastrointestinal nematode infections.

Australian producers have long used macrocyclic lactones (MLs) to successfully control cattle gastrointestinal nematodes (GINs) and consequently improve production parameters. However, the trajectory of ML resistance development in cattle GINs is following that of small ruminant nematode populations, highlighting a need for novel treatment options to provide efficacy in the current environment and interrupt the long-term establishment of ML-resistant GIN populations in Australian cattle. Here, we describe three field studies conducted in Australia to evaluate the efficacy of a single administration of a novel fixed-dose combination injectable (FDCI) endectocide against naturally acquired infections of cattle GINs. The FDCI is administered subcutaneously to deliver 0.2 mg/kg doramectin and 6 mg/kg levamisole hydrochloride (HCl). Study sites consisted of three farms in New South Wales (n = 2) and Victoria (n = 1). At each site, cattle were randomly allocated into one of three treatment groups: (1) untreated control (saline), (2) FDCI (0.2 mg/kg doramectin, 6 mg/kg levamisole HCl) or (3) positive control (0.2 mg/kg ivermectin). All treatments were administered on Day 0. Fecal samples were collected prior to treatment on Days -1 (Study 3) or 0 (Studies 1 and 2) and again on Day 14 (post-treatment) to evaluate efficacy via fecal egg count (FEC) and for coproculture. Adequacy of infection was confirmed at all three study sites, with Day 14 geometric mean (GM) FECs for saline-treated cattle ranging from 32.5 eggs per gram (EPG) to 623.7 EPG. FECs for FDCI-treated cattle were significantly reduced compared to saline-treated cattle (p ≤ 0.0001) on Day 14, with GM-based efficacy ≥ 99.7% at all three study sites. In contrast, ivermectin was 97.4% effective against cattle GINs in Study 1 but was only 47.2% and 39.8% effective at study site 2 and 3, respectively. Genus-specific efficacies suggest the presence of ivermectin-resistant Cooperia spp. (Study 1), Haemonchus spp. (Study 2) and Ostertagia spp. (Study 3) populations in the naturally infected cattle used in these studies. The post-treatment FEC and genus-specific efficacy estimations indicate the doramectin + levamisole HCl FDCI was highly efficacious against cattle GINs even in the face of ivermectin LOE at study sites 2 and 3. The efficacy of the new FDCI against both ML-susceptible and ML-resistant economically important cattle GINs in Australia affirms it is a valuable treatment option for producers operating in an environment of ML loss of efficacy.

Review and Evaluation of Ostertagia ostertagi Antibody ELISA for Application on Serum Samples in First Season Grazing Calves.

The O. ostertagi-Ab ELISA assay is widely used as a diagnostic tool for monitoring gastrointestinal (GI) nematodes using milk samples from adult dairy cows. This assay is potentially also useful to analyse serum samples from first-season grazing (FSG) calves, providing a more cost-effective and robust diagnostic technique than the current serum pepsinogen assay. However, a comprehensive evaluation of its use in serum samples from FSG calves has not yet been conducted. In this study, we first reviewed the available scientific literature in which the O. ostertagi-Ab ELISA was applied to serum samples from FSG calves. Then, a field study was conducted to compare results from the O. ostertagi-Ab ELISA assay with a serum pepsinogen assay on a set of 230 serum samples from 11 commercial dairy herds (seven in Belgium and four in Sweden). The literature review showed an increase in mean antibody levels, expressed as optical density ratio (ODR) values, from <0.4 (early grazing season) to values of 0.7-1.1 (late grazing season). Three out of five studies found a negative correlation between O. ostertagi antibody levels measured during the late grazing season and weight gain, while the other two studies found no correlation between the two variables. Our field studies showed a good correlation between O. ostertagi antibody levels and the results from the pepsinogen assay. Both indicators were negatively related to average daily weight gain in the Belgian herds, but not in the Swedish herds. Overall, the results suggest that the O. ostertagi-Ab ELISA test can be a useful tool in FSG calves and could replace the use of the serum pepsinogen assay at the end of the grazing season for general monitoring purposes.

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.

Practical guide to the diagnostics of ruminant gastrointestinal nematodes, liver fluke and lungworm infection: interpretation and usability of results.

The diagnostics of ruminant parasites remains one of the cornerstones for parasite control best practices. Field veterinarians have several techniques at their disposal (fecal egg count, coproculture, FAMACHA®, plasma pepsinogen, ELISA-Ostertagia, ELISA-Fasciola, Baermann and ELISA-Lungworm) for the identification and/or quantification of gastrointestinal nematodes, lungworms and liver fluke infecting small ruminants and cattle. Each of these diagnostic tools has its own strengths and weaknesses and is more appropriate for a specific production operation and/or age of the animal (young and adults). This review focuses on the usability and interpretation of the results of these diagnostic tools. The most advanced technical information on sampling, storage, advantages and limitations of each tool for different types of production operations and animal categories is provided.

Life history matters: Differential effects of abomasal parasites on caribou fitness.

Parasites can impact wildlife populations through their effects on host fitness and survival. The life history strategies of a parasite species can dictate the mechanisms and timing through which it influences the host. However, unravelling this species-specific effect is difficult as parasites generally occur as part of a broader community of co-infecting parasites. Here, we use a unique study system to explore how life histories of different abomasal nematode species may influence host fitness. We examined abomasal nematodes in two adjacent, but isolated, West Greenland caribou (Rangifer tarandus groenlandicus) populations. One herd of caribou were naturally infected with Ostertagia gruehneri, a common and dominant summer nematode of Rangifer sspp., and the other with Marshallagia marshalli (abundant; winter) and Teladorsagia boreoarcticus (less abundant; summer), allowing us to determine if these nematode species have differing effects on host fitness. Using a Partial Least Squares Path Modelling approach, we found that in the caribou infected with O. gruehneri, higher infection intensity was associated with lower body condition, and that animals with lower body condition were less likely to be pregnant. In caribou infected with M. marshalli and T. boreoarcticus, we found that only M. marshalli infection intensity was negatively related to body condition and pregnancy, but that caribou with a calf at heel were more likely to have higher infection intensities of both nematode species. The differing effects of abomasal nematode species on caribou health outcomes in these herds may be due to parasite species-specific seasonal patterns which influence both transmission dynamics and when the parasites have the greatest impact on host condition. These results highlight the importance of considering parasite life history when testing associations between parasitic infection and host fitness.

Novel epidemiological model of gastrointestinal nematode infection to assess grazing cattle resilience by integrating host growth, parasite, grass and environmental dynamics.

Gastrointestinal nematode (GIN) infections are ubiquitous and often cause morbidity and reduced performance in livestock. Emerging anthelmintic resistance and increasing change in climate patterns require evaluation of alternatives to traditional treatment and management practices. Mathematical models of parasite transmission between hosts and the environment have contributed towards the design of appropriate control strategies in ruminants, but have yet to account for relationships between climate, infection pressure, immunity, resources, and growth. Here, we develop a new epidemiological model of GIN transmission in a herd of grazing cattle, including host tolerance (body weight and feed intake), parasite burden and acquisition of immunity, together with weather-dependent development of parasite free-living stages, and the influence of grass availability on parasite transmission. Dynamic host, parasite and environmental factors drive a variable rate of transmission. Using literature sources, the model was parametrised for Ostertagia ostertagi, the prevailing pathogenic GIN in grazing cattle populations in temperate climates. Model outputs were validated on published empirical studies from first season grazing cattle in northern Europe. These results show satisfactory qualitative and quantitative performance of the model; they also indicate the model may approximate the dynamics of grazing systems under co-infection by O. ostertagi and Cooperia oncophora, a second GIN species common in cattle. In addition, model behaviour was explored under illustrative anthelmintic treatment strategies, considering impacts on parasitological and performance variables. The model has potential for extension to explore altered infection dynamics as a result of management and climate change, and to optimise treatment strategies accordingly. As the first known mechanistic model to combine parasitic and free-living stages of GIN with host feed-intake and growth, it is well suited to predict complex system responses under non-stationary conditions. We discuss the implications, limitations and extensions of the model, and its potential to assist in the development of sustainable parasite control strategies.

Tracking gastrointestinal nematode risk on cattle farms through pasture contamination mapping.

Gastrointestinal nematode (GIN) parasites in grazing cattle are a major cause of production loss and their control is increasingly difficult due to anthelmintic resistance and climate change. Rotational grazing can support control and decrease reliance on chemical intervention, but is often complex due to the need to track grazing periods and infection levels, and the effect of weather on larval availability. In this paper, a simulation model was developed to predict the availability of infective larvae of the bovine GIN, Ostertagia ostertagi, at the level of individual pastures. The model was applied within a complex rotational grazing system and successfully reproduced observed variation in larval density between fields and over time. Four groups of cattle in their second grazing season (n = 44) were followed throughout the temperate grazing season with regular assessment of GIN faecal egg counts, which were dominated by O. ostertagi, animal weight and recording of field rotations. Each group of cattle was rotationally grazed on six group-specific fields throughout the 2019 grazing season. Maps and calendars were produced to illustrate the change in pasture infectivity (density of L3 on herbage) across the 24 separate grazing fields. Simulations predicted differences in pasture contamination levels in relation to the timing of grazing and the return period. A proportion of L3 was predicted to persist on herbage over winter, declining to similar intensities across fields before the start of the following grazing season, irrespective of contamination levels in the previous year. Model predictions showed good agreement with pasture larval counts. The model also simulated differences in seasonal pasture infectivity under rotational grazing in systems that differed in temperature and rainfall profiles. Further application could support individual farm decisions on evasive grazing and refugia management, and improved regional evaluation of optimal grazing strategies for parasite control. The integration of weather and livestock movement is inherent to the model, and facilitates consideration of climate change adaptation through improved disease control.

Development of Bovine Gastric Organoids as a Novel In Vitro Model to Study Host-Parasite Interactions in Gastrointestinal Nematode Infections.

Gastro-intestinal nematode (GIN) parasites are a major cause of production losses in grazing cattle, primarily through reduced growth rates in young animals. Control of these parasites relies heavily on anthelmintic drugs; however, with growing reports of resistance to currently available anthelmintics, alternative methods of control are required. A major hurdle in this work has been the lack of physiologically relevant in vitro infection models that has made studying precise interactions between the host and the GINs difficult. Such mechanistic insights into the infection process will be valuable for the development of novel targets for drugs, vaccines, or other interventions. Here we created bovine gastric epithelial organoids from abomasal gastric tissue and studied their application as in vitro models for understanding host invasion by GIN parasites. Transcriptomic analysis of gastric organoids across multiple passages and the corresponding abomasal tissue showed conserved expression of tissue-specific genes across samples, demonstrating that the organoids are representative of bovine gastric tissue from which they were derived. We also show that self-renewing and self-organising three-dimensional organoids can also be serially passaged, cryopreserved, and resuscitated. Using Ostertagia ostertagi, the most pathogenic gastric parasite in cattle in temperate regions, we show that cattle gastric organoids are biologically relevant models for studying GIN invasion in the bovine abomasum. Within 24 h of exposure, exsheathed larvae rapidly and repeatedly infiltrated the lumen of the organoids. Prior to invasion by the parasites, the abomasal organoids rapidly expanded, developing a 'ballooning' phenotype. Ballooning of the organoids could also be induced in response to exposure to parasite excretory/secretory products. In summary, we demonstrate the power of using abomasal organoids as a physiologically relevant in vitro model system to study interactions of O. ostertagi and other GIN with bovine gastrointestinal epithelium.

A model for the development of the free-living stages of Ostertagia leptospicularis, used in conjunction with on-farm egg count data, to estimate sources of pasture contamination on New Zealand red deer (Cervus elaphus) farms.

Farmed red deer face challenges from nematode parasites, primarily the pulmonary species Dictyocaulus eckerti and the complex of Ostertagiinae nematodes in the abomasum. Previous investigations on New Zealand deer farms identified limited seasonality in faecal egg and larval output in all stock classes, however, this does not indicate the key times of year those eggs develop into infective-stage larvae, and subsequently contribute to infection risk. A simple temperature-driven model was developed for the free-living stages of a representative deer-specific Ostertagiinae species; Ostertagia leptospicularis. The model was then validated using development data generated from seasonal plot trials. Using faecal egg counts and animal numbers from the previous monitoring study, and local weather data from each of the sampling farms, the relative contribution of each stock class to infective third-stage larvae population on pasture was estimated for each month of the year (2018-2019). The mixed-age adult females were the primary source of infective larvae on most of the sampled farms, principally due to their numerical dominance (i.e., venison breeding farms). The frequent anthelmintic use in the youngest stock class (< 12 months old) on some farms limited their output of eggs and hence their contribution to pasture infestation with infective larvae. However, the trends of larval development on pasture differed between the farms (due to local climate), and between the sampling years (2018-2019). Therefore, calendar-based application of anthelmintics to older stock is not recommended, as contribution to pasture contamination is multifactorial. The study provides a better understanding of deer parasite epidemiology on which to base improved management practices.

Gastrointestinal nematode and lungworm infections in organic dairy calves reared with nurse cows during their first grazing season in western France.

The rearing system of dairy calves with nurse cows has been developing since 2010 in organic farms in western France. This system allows cow-calf contact until a weaning age close to the natural weaning for cattle and is characterized by an early turnout for calves at around one month of age with their nurse cows and a first grazing season with mixed grazing of calves and adults at a ratio of 2-4 calves per nurse cow. The objectives of this study were to assess the gastrointestinal (GIN) and lungworm infections in such reared calves and their variability during the first grazing season. Faecal egg count (FEC), pepsinogen (PEP) concentration and Ostertagia ELISA optical density ratio (ODR) were determined in calves (n = 497) at housing in 33 groups from 24 farms in 2018, and in calves (n = 405) and nurse cows (n = 199) throughout the 2019 grazing season in 41 groups from 20 farms. For lungworm infection, information was obtained during 2019 through the recording of coughing episodes along the grazing season and the Dictyocaulus ELISA ODR determination at housing both in calves and nurses. Results indicated that the level of GIN infection was overall low for calves during the first grazing season with PEP and Ostertagia ODR group-average values ranging from 0.97 to 1.6 U Tyr and 0.23 to 0.71 ODR respectively. No anthelmintic treatment being given in any group of calves. Ostertagia ODR values increased with the duration of the grazing season (>240 d) and with the ratio calves/nurse (>2). GIN parameters for nurses remained fairly stable during the grazing season with mean FEC, PEP and Ostertagia ODR group-average values of 13 epg, 2.28 U Tyr and 0.81 ODR, respectively. Antibodies against lungworms were detected in 3-62 % of calves depending on the duration of grazing, but only 6% of calves showed a coughing episode. The dilution effect due to the mixed grazing of resistant (nurse cows) and susceptible (calves) animals associated with predominant milk diet of calves during the first months of grazing in combination with protective grazing management allow calves to be turned out at an early age without using anthelmintic treatments. Further studies are needed to assess the GIN infection dynamics during the second grazing season in weaned heifers.

Gastrointestinal nematodes and Fasciola hepatica in Norwegian cattle herds: a questionnaire to investigate farmers' perceptions and control strategies.

BACKGROUND: Pasture management influences the prevalence and impact of the pasture parasites (PP) in cattle herds, which cause production-limiting disease worldwide. Evaluating farmer management strategies is vital when considering sustainable PP control practices. The aim of this questionnaire-based study was to describe the pasture management and control strategies regarding PP in Norwegian beef cattle (BC) and dairy cattle (DC) production systems with a focus on gastrointestinal nematodes (GIN) and Fasciola hepatica. RESULTS: A total of 745 responses from BC (return rate 20.5%) and 1347 responses from DC farmers (30.7%) were included. The mean total pasture time for DC was 4.2 months for first-season grazers and 4.3 months for second-season grazers and cows, while the corresponding finding in BC was 5.4 months. Home pasture was used for most of the pasture period, particularly for first-season grazer dairy heifers (81%), which were also commonly grazed on the same pasture every year (79%). For most farmers it was necessary for grazing areas to be used for cattle for more than one season (77% of BC farmers and 89% of DC farmers). However, changing the pasture during the season was common in both DC (67%) and BC (70%) herds. The majority of DC farmers (60%) stated that they did not consider that they had a problem with PP. Of the remaining 40%, few respondents could specify whether their herds had a problem due to infection by GIN (11%) or liver flukes (12%). Treatment for GIN was performed by 52% of DC and 34% of BC farmers. Diagnostic faecal samples were collected upon suspicion of parasitic disease by 5% of DC and 16% of BC farmers. Veterinarians were stated as a central source of information about parasite management and treatment. CONCLUSIONS: Potential risks for exposure to PP were identified, such as use of the same pasture every year for first-season grazers and frequent use of home pasture. The perception of problems related to PP appeared low. Regular anthelmintic treatment without concurrent use of diagnostic faecal samples seems to be common practice.

Seroprevalence of Major Pasture-Borne Parasitoses (Gastrointestinal Nematodes, Liver Flukes and Lungworms) in German Dairy Cattle Herds, Association with Management Factors and Impact on Production Parameters.

Pasture-borne parasites adversely affect bovine health and productivity worldwide. In Europe, gastrointestinal nematodes, especially Ostertagia ostertagi, the liver fluke Fasciola hepatica and the lungworm Dictyocaulus viviparus represent the most important parasites of dairy cattle. The present study assessed exposure towards these parasites among 646 cattle herds in three parts of Germany during 2017-2019 via antibody detection in bulk tank milk (BTM). Overall, O. ostertagi levels indicative of production losses were detected in 41.2% (266/646; 95% confidence interval (CI): 37.4-45.1%) of BTM samples, while F. hepatica seroprevalence amounted to 14.9% (96/646; 95% CI: 12.2-17.9%). Only 2.3% (15/646; 95% CI: 1.4-3.9%) of samples were D. viviparus antibody-positive. Significantly lower O. ostertagi as well as F. hepatica seroprevalence was detected in dual-purpose breeds compared to high-performance breeds from the same region. Management factors related to parasite exposure included access to fresh grass and hay, silage quality and anthelmintic treatment. Furthermore, F. hepatica and O. ostertagi seropositivity was significantly associated with suboptimal herd-level body condition. Interestingly, the relationship between seropositivity and productivity differed between breed types. Negative impacts on milk yield were detected only in high-performance breeds, while O. ostertagi seropositivity was associated with a lower milk fat content in dual-purpose herds.

Repeated, drug-truncated infections with Ostertagia ostertagi elicit strong humoral and cell-mediated immune responses and confer partial protection in cattle.

Bovine ostertagiasis causes significant production losses to the cattle industry. Protective immunity induced by natural infection is slow to develop and anthelmintic resistance is rapidly developing. There is a need to advance alternatives for control of gastrointestinal nematode parasites. The present study investigated the effects of repeated, drug-truncated infections (rDTI) on development of protective immunity and attenuation of a challenge infection by O. ostertagi. Helminth-free calves were randomly assigned to either a rDTI or a control group (n = 5). The rDTI group received daily oral infections of 5000 Ostertagia L3 for 5 consecutive days, then were drug-treated on 14 and 15 days post infection (dpi), to attenuate O. ostertagi at the late fourth larval (L4) through young adult stages. DTI was repeated 3 weeks after the drug treatment. A total of 5 DTIs were administered to the DTI-treated animals. Non-DTI-treated, control animals received tap water as infection control. All animals were drug-treated at the same time. Animals were challenge-infected 4 weeks following the final round of rDTI. The results show that eggs per gram of feces (EPG) in the rDTI group were significantly reduced (P < 0.05) from 21 to 39 dpi, with an overall reduction in cumulative EPG. The control group exhibited reduced (P = 0.0564) average weight gains when compared to those of the rDTI group during weeks 4-5 post infection, a period coinciding with peak EPG output of control animals. Antigen-specific IgG, IgE and IgA responses were detected after the 2nd DTI, and stronger antibody recall responses were elicited by challenge infection. High levels of antigen-specific peripheral blood mononuclear cell (PBMC)/T cell proliferation to whole worm and excretory-secretory (ES) antigens were detected in rDTI-treated animals. These data indicate that partial protective immunity against ostertagiasis, involving cell-mediated and humoral responses, can be attained by rDTI which allowed for maximal antigen exposure from staggered parasitic developmental stages. The data suggest that rDTI can be used as a model to study host-parasite interactions and identify parasite antigens responsible for eliciting host protective immune responses.

Ostertagia ostertagi Mediates Early Host Immune Responses via Macrophage and Toll-Like Receptor Pathways.

Ostertagia ostertagi is an abomasal parasite with significant economic impact on the cattle industry. Early host immune responses are poorly understood. Here, we examined time course expression of Toll-like receptors (TLRs) in peripheral blood mononuclear cells (PBMC) during infection where PBMC macrophages (Mϕ) generated both pro- and anti-inflammatory responses when incubated with excretory/secretory products (ESP) from fourth-stage larvae (OoESP-L4) or adult worms (OoESP-Ad). First, changes in cell morphology clearly showed that both OoESP-L4 and OoESP-Ad activated PBMC-Mϕ in vitro, resulting in suppressed CD40 and increased CD80 expression. Expression of mRNAs for TLR1, -4, -5, and -7 peaked 7 days postinfection (dpi) (early L4), decreased by 19 dpi (postemergent L4 and adults) and then increased at 27 dpi (late adults). The proinflammatory cytokine tumor necrosis factor alpha (TNF-α) (transcript and protein) increased in the presence of OoESP-Ad, and the anti-inflammatory cytokine interleukin 10 (IL-10) (protein) decreased in the presence of OoESP-L4 or OoESP-Ad; however, IL-10 mRNA was upregulated, and IL-6 (protein) was downregulated by OoESP-L4. When PBMC-Mϕ were treated with ligands for TLR4 or TLR5 in combination with OoESP-Ad, the transcripts for TNF-α, IL-1, IL-6, and IL-10 were significantly downregulated relative to treatment with TLR4 and TLR5 ligands only. However, the effects of TLR2 ligand and OoESP-Ad were additive, but only at the lower concentration. We propose that O. ostertagi L4 and adult worms utilize competing strategies via TLRs and Mϕ to confuse the immune system, which allows the worm to evade the host innate responses.

Effect of Mixed Invasions of Hypoderma bovis and Ostertagia ostertagi in Cattle on Milk Yield and Contents in Polish Dairy Farms.

Wide distribution of ecto- and endoparasites in cattle is a serious problem in the sustainability of a farm, due to the negative impact on animals' health and productivity. The aim of this study was to determine the presence of antibodies against Ostertagia and Hypoderma in udder milk samples and the comparison of milk yield and content of the basic components of milk in ELISA-positive and ELISA-negative cows. Milk samples were collected from 148 lactating cows from 3 herds. Antibody detection was performed using specific ELISAs for Ostertagia ostertagi and Hypoderma bovis. Milk yield and content of protein, fat, and dry matter were studied in samples from each individual cow 11 times per year at 4 week intervals. The extensiveness of dual parasitic invasions in individual herds, estimated on the basis of udder milk testing with the ELISA test, varied and amounted to 3.22%, 11.36%, and 4.76% in the three studied herds, respectively. No antibodies were found in 61.2%, 22.7%, and 57.1% of the milk samples from the cows in each herd. The milk yield of ELISA-positive cows was significantly lower in comparison to the efficiency of ELISA-negative cows and amounted to 294 kg and even to 3672 kg of milk per year, per cow. No significant differences were found between the fat and protein contents of milk between ELISA-positive and -negative cows for both parasites.

Morphometric and Molecular Analyses of Ostertagia leptospicularis Assadov, 1953 from Ruminants: Species Diversity or Host Influence?

Ostertagia leptospicularis Assadov, 1953 was formally described in roe deer Capreolus capreolus and has been reported in a wide range of ruminants, including other Cervidae, as well as Bovidae. Nematode specimens derived from various host species exhibit morphological similarity; however, some differences can be observed. It is unclear if this is due to the differential reaction of one nematode species in different host species (i.e., host-induced changes) or because of distinct nematode species in these hosts (i.e., species complex). This paper focuses on specimens resembling O. leptospicularis f. leptospicularis and its closely related species (Ostertagia ostertagi f. ostertagi) collected from various hosts. Morphometric and molecular techniques were applied to assess host-induced changes in nematode morphology and to clarify its systematic classification. There was an overall effect of host species on measurements of nematodes resembling O. leptospicularis (both males and females), but the distinctiveness of the specimens from cattle Bos taurus were highlighted. The results obtained may suggest that the specimens of O. leptospicularis from cattle in Germany and cervids in central Europe belong to different strains. Furthermore, nematodes from the cervid strain appear to circulate within particular host species, which can be seen in the stated morphological variations.

Molecular and phenotypic characterisation of fenbendazole resistance in a field-derived isolate of Ostertagia ostertagi.

The prevalence of anthelmintic resistance in the bovine nematode Cooperia oncophora has been well documented globally but lack of efficacy against the more pathogenic nematode species Ostertagia ostertagi is less common. The sensitivity of an O. ostertagi isolate to the benzimidazole class of anthelmintic was investigated using classical parasitological techniques following apparent clinical failure of controlled release fenbendazole capsule administration in first season grazers at pasture. A controlled efficacy test (CET) was conducted in conjunction with sequencing of the ß-tubulin isotype 1 gene of larvae pre- and post-fenbendazole administration. Twelve helminth-naïve calves were infected experimentally with 20,000 third stage larvae; six received oral fenbendazole (7.5 mg/kg bodyweight) 28 days post infection. Total abomasal nematode burdens were compared between treatment and control groups to determine efficacy. Fenbendazole resistance in O. ostertagi was confirmed with a total treatment failure in reducing worm burden: efficacy of 0%. Sequence analysis of the ß-tubulin isotype-1 gene from forty-five infective larvae from both control and treated groups was performed. The three commonest single nucleotide polymorphisms (SNPs) associated with benzimidazole resistance, namely F167Y, E198A and F200Y, were examined. The predominant resistance-associated SNPs were F200Y (78 % control and 79 % treated groups) and F167Y (remaining genotypes) and emphasises the importance of these SNPs in clinical disease in this isolate. The development of diagnostic molecular tools based on a characterised field-derived isolate of benzimidazole-resistant Ostertagia will enable future prevalence surveys to be undertaken to assess the possible risk posed by resistance in this economically important species.