Search

Previous vaccination trials have demonstrated that thiol proteins affinity purified from Ostertagia ostertagi excretory-secretory products (O. ostertagi ES-thiol) are protective against homologous challenge. Here we have shown that protection induced by this vaccine was consistent across four independent vaccine-challenge experiments. Protection is associated with reduced cumulative faecal egg counts across the duration of the trials, relative to control animals. To better understand the diversity of antigens in O. ostertagi ES-thiol we used high-resolution shotgun proteomics to identify 490 unique proteins in the vaccine preparation. The most numerous ES-thiol proteins, with 91 proteins identified, belong to the sperm-coating protein/Tpx/antigen 5/pathogenesis-related protein 1 (SCP/TAPS) family. This family includes previously identified O. ostertagi vaccine antigens O. ostertagi ASP-1 and ASP-2. The ES-thiol fraction also has numerous proteinases, representing three distinct classes, including: metallo-; aspartyl- and cysteine proteinases. In terms of number of family members, the M12 astacin-like metalloproteinases, with 33 proteins, are the most abundant proteinase family in O. ostertagi ES-thiol. The O. ostertagi ES-thiol proteome provides a comprehensive database of proteins present in this vaccine preparation and will guide future vaccine antigen discovery projects., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024. Published by Elsevier B.V. All rights reserved.)

The development of effective recombinant vaccines against parasitic nematodes has been challenging and so far mostly unsuccessful. This has also been the case for Ostertagia ostertagi, an economically important abomasal nematode in cattle, applying recombinant versions of the protective native activation-associated secreted proteins (ASP). To gain insight in key elements required to trigger a protective immune response, the protein structure and N-glycosylation of the native ASP and a non-protective Pichia pastoris recombinant ASP were compared. Both antigens had a highly comparable protein structure, but different N-glycan composition. After mimicking the native ASP N-glycosylation via the expression in Nicotiana benthamiana plants, immunisation of calves with these plant-produced recombinants resulted in a significant reduction of 39% in parasite egg output, comparable to the protective efficacy of the native antigen. This study provides a valuable workflow for the development of recombinant vaccines against other parasitic nematodes., (© 2023. The Author(s).)

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., (Published by Elsevier B.V.)

This review addresses the research landscape regarding vaccines against scour worms, particularly Trichostrongylus spp. and Teladorsagia circumcincta. The inability of past research to deliver scour-worm vaccines with reliable and reproducible efficacy has been due in part to gaps in knowledge concerning: (i) host-parasite interactions leading to development of type-2 immunity, (ii) definition of an optimal suite of parasite antigens, and (iii) rational formulation and administration to induce protective immunity against gastrointestinal nematodes (GIN) at the site of infestation. Recent 'omics' developments enable more systematic analyses. GIN genomes are reaching completion, facilitating "reverse vaccinology" approaches that have been used successfully for the Rhipicephalus australis vaccine for cattle tick, while methods for gene silencing and editing in GIN enable identification and validation of potential vaccine antigens. We envisage that any efficacious scour worm vaccine(s) would be adopted similarly to "Barbervax™" within integrated parasite management schemes. Vaccines would therefore effectively parallel the use of resistant animals, and reduce the frequency of drenching and pasture contamination. These aspects of integration, efficacy and operation require updated models and validation in the field. The conclusion of this review outlines an approach to facilitate an integrated research program., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The suitability of a single mid-season targeted selective treatment (TST) for gastrointestinal nematodes control, based on flexible average daily weight gain (ADWG) thresholds, was investigated in 23 groups of first grazing season calves. In each group, animals were weighed three times: before turnout, at mid-season and at housing. Just after the first weighing, each group was divided in two homogenous sub-groups in terms of age, breed and weight, and randomly allocated to one of two sub-groups intented for two different mid-season anthelmintic treatment strategies: (1) a treatment of all calves composing the sub-group (whole-group treatment (WT)) or (2) a targeted selective weight gain-based treatment (TST) of the animals showing an individual pre-treatment ADWG inferior to the mean pre-treatment ADWG of the corresponding WT sub-group. Anthelmintic treatment (levamisole 7.5 mg/kg BW) was performed 3 to 4 months after turnout. At housing, two parasitological parameters (the anti-Ostertagia ostertagi antibody level-Ostertagia optical density ratio (ODR) and the pepsinogen level) and a clinical parameter (the breech soiling score) were assessed at individual level in each group. Then, the high exposed groups to gastrointestinal nematode (GIN) were defined as groups for which untreated animals exhibited a mean Ostertagia ODR ⩾0.7 and among these groups, the ones characterized by high abomasal damage due to Ostertagia for which untreated animals exhibited a mean pepsinogen level ⩾2.5 U Tyr were also identified. Among TST sub-groups, the treatment ADWG thresholds varied from 338 to 941 g/day and the percentage of treated animals from 28% to 75%. Pre- and post-treatment ADWG as well as parasitological and clinical parameters measured at housing were similar between TST and WT sub-groups including the 17 high exposed groups to GIN. Within these 17 groups, the treatment allowed to significantly improve post-treatment ADWG compared with untreated animals. In the six high exposed groups showing mean pepsinogen level ⩾2.5 U Tyr, the average effect of treatment on post-treatment ADWG was the highest and estimated up to 14 kg after a grazing duration of 4 months. In contrast, in six other groups showing mean Ostertagia ODR<0.7 in untreated animals, no effect of treatment was seen suggesting an absence of production losses related to a low level of GIN infection. This study highlighted the suitability of a convenient mid-season TST strategy for first grazing season calves, based on the use of flexible thresholds of ADWG, allowing similar growth compared with a whole-group treatment while keeping a GIN population in refugia.

A simulation study was carried out to assess whether variation in pasture contamination or stocking rate impact upon the optimal design of targeted selective treatment (TST) strategies. Two methods of TST implementation were considered: 1) treatment of a fixed percentage of a herd according to a given phenotypic trait, or 2) treatment of individuals that exceeded a threshold value for a given phenotypic trait. Four phenotypic traits, on which to base treatment were considered: 1) average daily bodyweight gain, 2) faecal egg count, 3) plasma pepsinogen, or 4) random selection. Each implementation method (fixed percentage or threshold treatment) and determinant criteria (phenotypic trait) was assessed in terms of benefit per R (BPR), the ratio of average benefit in weight gain to change in frequency of resistance alleles R (relative to an untreated population). The impact of pasture contamination on optimal TST strategy design was investigated by setting the initial pasture contamination to 100, 200 or 500 O. ostertagi L3/kg DM herbage; stocking rate was investigated at a low (3calves/ha), conventional (5 calves/ha) or high (7 calves/ha) stocking rates. When treating a fixed percentage of the herd, treatments according to plasma pepsinogen or random selection were identified as the most beneficial (i.e. resulted in the greatest BPR) for all levels of initial pasture contamination and all stocking rates. Conversely when treatments were administered according to threshold values ADG was most beneficial, and was identified as the best TST strategy (i.e. resulted in the greatest overall BPR) for all levels of initial pasture contamination and all stocking rates., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Grazing management (GM) interventions, such as reducing the grazing time or mowing pasture before grazing, have been proposed to limit the exposure to gastrointestinal (GI) nematode infections in grazed livestock. However, the farm-level economic effects of these interventions have not yet been assessed. In this paper, the economic effects of three GM interventions in adult dairy cattle were modelled for a set of Flemish farms: later turnout on pasture (GM1), earlier housing near the end of the grazing season (GM2), and reducing the daily grazing time (GM3). Farm accountancy data were linked to Ostertagia ostertagi bulk tank milk ELISA results and GM data for 137 farms. The economic effects of the GM interventions were investigated through a combination of efficiency analysis and a whole-farm simulation model. Modelling of GM1, GM2 and GM3 resulted in a marginal economic effect of € 8.36, € -9.05 and € -53.37 per cow per year, respectively. The results suggest that the dairy farms can improve their economic performance by postponing the turnout date, but that advancing the housing date or reducing daily grazing time mostly leads to a lower net economic farm performance. Overall, the GM interventions resulted in a higher technical efficiency and milk production but these benefits were offset by increased feed costs as a result of higher maintenance and cultivation costs. Because the results differed highly between farms, GM interventions need to be evaluated at the individual level for appropriate decision support., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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.

Dictyocaulus viviparus, Ostertagia ostertagi (nematode parasites), and Fasciola hepatica (trematode parasite) result in productivity losses on dairy farms and impact on animal health through clinical and sub-clinical disease. Parasite control in livestock systems is largely based on the use of chemoprophylactic agents (anthelmintics), grazing management, or a combination of both. The objective of this study was to document current parasite control measures employed by Irish dairy farmers in a predominantly pasture-based livestock system. A questionnaire survey of 312 geographically representative farmers was completed in 2009 with a follow up survey completed in 2011. Statistical analysis highlighted significant differences in chemoprophylactic usage between 2009 and 2011. In particular, an increase in the use of albendazole for both trematode (19% in 2009 to 36% in 2011) and nematode (30% in 2009 to 58% in 2011) control was observed. This was most likely due to flukicide restrictions introduced in the Republic of Ireland in 2010 for dairy animals. Logistic regression highlighted regional differences in chemoprophylactic use. Farmers in southern parts of Ireland, an area with good quality soil, less rainfall, and a higher density of dairy farms than other regions, were approximately half as likely to dose for F. hepatica and were more likely (OR>2.0) to use albendazole for both nematode and fluke control. Approximately 30% of respondents who used a chemoprophylactic treatment for nematodes, used a product which was 'unsuitable for purpose' (e.g. ivermectin for the treatment of F. hepatica), highlighting the need for increased awareness, continuing research, and regionally targeted education tools regarding optimal parasite control., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Ostertagia ostertagi is considered one of the most economically important bovine parasites. As an alternative to anthelmintic treatment, an experimental host-protective vaccine was previously developed on the basis of ASP proteins derived from adult worms. Intramuscular injection of this vaccine, combined with QuilA as an adjuvant, significantly reduced fecal egg counts by 59%. However, the immunological mechanisms triggered by the vaccine are still unclear. Therefore, in this study, the differences in immune responses at the site of infection, i.e., the abomasal mucosa, between ASP-QuilA-vaccinated animals and QuilA-vaccinated control animals were investigated on a transcriptomic level by using a whole-genome bovine microarray combined with histological analysis. Sixty-nine genes were significantly impacted in animals protected by the vaccine, 48 of which were upregulated. A correlation study between the parasitological parameters and gene transcription levels showed that the transcription levels of two of the upregulated genes, those for granulysin (GNLY) and granzyme B (GZMB), were negatively correlated with cumulative fecal egg counts and total worm counts, respectively. Both genes were also positively correlated with each other and with another upregulated gene, that for the IgE receptor subunit (FCER1A). Surprisingly, these three genes were also correlated significantly with CMA1, which encodes a mast cell marker, and with counts of mast cells and cells previously described as globule leukocytes. Furthermore, immunohistochemical data showed that GNLY was present in the granules of globule leukocytes and that it was secreted in mucus. Overall, the results suggest a potential role for granule exocytosis by globule leukocytes, potentially IgE mediated, in vaccine-induced protection against O. ostertagi.

A randomised clinical trial from the North Island of New Zealand was conducted to assess the effect of repeated anthelmintic treatment on milk production, and to assess factors that affect treatment response. Nine hundred and twenty three multiparous, lactating dairy cattle from three pasture-based, spring-calving dairy herds were enrolled in this trial. Within each herd, cattle were stratified on age and calving date, and were randomly allocated to treatment (n=319) or control (n=604) groups. The treatment group received ≥ 0.05 mg/kg of topical eprinomectin every 28 days for eight treatments during lactation. Pooled-milk from treated cows and bulk-milk samples were obtained at each treatment and analysed with an Ostertagia antibody ELISA, expressed as optical density ratios (ODR). Bi-monthly milk data were collected and expressed as energy-corrected milk (kg/day; ECM). A linear mixed model was used to analyse ECM, with cow as the random effect. The effect of anthelmintic treatment on days from calving, and start-of-mating, to conception were analysed with Cox-proportional hazard models. ODR values ranged from 0.6 to 1.3; there were no differences in ODR between herds (p=0.12), or between pooled-milk from treated cows and bulk-milk (p=0.26). Repeated treatments had no effect on daily ECM yields (p=0.74). However, there was a significant treatment × herd interaction (p=0.03); treatment increased ECM in one herd by 0.781 kg/cow/day (p=0.015), but resulted in a non-significant decrease in the other two herds. A curvilinear interaction existed between days-in-milk and treatment response (p=0.039); the greatest treatment effect occurred during mid-lactation. Previous year milk production (p=0.46) and age (p=0.11) did not influence the effect of treatment on ECM. Treatment had no effect on any reproductive parameter. In conclusion, under New Zealand pastoral conditions, anthelmintic treatment increased milk production in one herd, but had no effect in two other herds. Further work is needed to identify why this variation in gastro-intestinal parasitism occurs., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Ostertagia ostertagi is one of the most important gastrointestinal parasites infecting millions of cattle in temperate climate areas. Because infection leads to significant losses in productivity, farmers are pushed, because of the on-going intensification of the livestock production systems, to frequently administer anthelmintics to minimize the exposure of their animals to this parasite. However, whether such approach is sustainable in the long term, especially in an era of emerging drug resistance and global climatic changes, is still a matter of debate. Immunological control of worm infections through vaccination is often put forward as the most rational and cost-effective alternative for anthelmintics to control helminth infections. However, the development of an immunologically based control strategy requires a thorough knowledge of the host-parasite interactions, the immune responses involved and the biology of the parasite itself. The aim of this review is to consolidate information available in these areas, specifically for O. ostertagi, and identify some critical gaps in our current knowledge., (© 2011 Blackwell Publishing Ltd.)

ConA lectin was used to isolate glycoproteins from detergent extracts of fourth stage Ostertagia ostertagi larvae. This preparation contained proteins additional to those observed in a similar fraction prepared from adult O. ostertagi. Two vaccine trials were conducted with this preparation, and sub-fractions thereof, in groups of 6-8 worm-free calves. All groups were challenged with 50,000 O. ostertagi larvae 1 week after the final immunization, and protection was assessed by comparing the egg and worm counts of the immunized groups with their respective controls. Immunization with the ConA-binding antigen or its sub-fractions induced high titre serum antibody responses. In the first trial, the cumulative egg count of the group immunized with unfractionated antigen was 60% lower than the corresponding control value, and worm counts were 47% lower. In the second trial, the cumulative egg counts of the vaccinated groups ranged from 70% to 85% lower than the corresponding controls, with worm counts up to 64% lower. It was concluded that detergent-soluble, ConA-binding extracts prepared from O. ostertagi fourth stage larvae contained protective immunogens that were as effective as the best antigens published for O. ostertagi to date.

Previous vaccination trials against the economically important cattle parasite Ostertagia ostertagi have indicated the protective capacity of activation-associated secreted proteins (ASPs). The further development of these antigens into a commercial vaccine will require their recombinant expression. The aim of the current study was to clone and express Oo-asp1 in a baculovirus expression system and to evaluate the protective capacity of the recombinant protein against an O. ostertagi challenge infection in cattle. The full coding sequence of Oo-asp1 was cloned in a baculovirus expression vector in frame with a carboxy-terminal Histidine tag and recombinant virus was used to infect an insect cell culture. Western blot analysis with anti-His and anti-Oo-ASP1 antibodies showed the production of recombinant Oo-ASP1. The cell pellet containing the recombinant was subsequently used to immunize seven calves three times intramuscularly with QuilA as adjuvant. Control animals were solely injected with the QuilA adjuvant. The challenge infection with O. ostertagi consisted of 30,000 L3 larvae per animal given over 30 days (1000 larvae/day, 5 days/week) and started the same day as the final immunization. Immunization with the recombinant Oo-ASP1 did not result in any level of protection against the challenge infection. There was no reduction in faecal egg output or in worm burdens. Moreover, Western blot analyses and ELISA indicated that, although the animals raised an antibody response against the recombinant Oo-ASP1, there was hardly a response against the native Oo-ASP1, suggesting that the baculovirus expressed recombinant was wrongly folded or lacked essential secondary modifications. Further analysis of the structure of the native ASPs and their glycosylations is being done.

Previous vaccination trials against Ostertagia ostertagi in cattle have demonstrated the protective capacity of a protein fraction termed ES-thiol, which is enriched for activation-associated secreted proteins (ASPs) and cysteine proteases. In this study, ES-thiol was subfractionated through Q-Sepharose anion exchange chromatography to determine whether the ASPs and/or the cysteine proteases are responsible for the induced protection. Calves (seven/group) were immunized three times intramuscularly with 100 microg of ES-thiol or equivalent amounts of an ASP-enriched fraction, a cysteine protease-enriched fraction or a rest fraction, with QuilA adjuvant. A negative control group only received QuilA. After the final immunization the animals were challenged with a trickle infection of 25,000 infectious L3 larvae (1000 L3/day; 5 days/week). During a 2-month period the geometric mean cumulative faecal egg count (FEC) of the ES-thiol group was reduced by 62% compared to the QuilA control group (P<0.05). Groups injected with the ASP-enriched, the cysteine protease-enriched and the rest fraction demonstrated a reduction in cumulative FEC of 74, 80 and 70%, respectively (P<0.01). Although no significant reductions in worm burdens were observed, adult male and female worms were significantly smaller in all vaccinated groups (P<0.05), except for male worms from the ES-thiol group. These results suggest the protective capacity of ASPs and the presence of other protective antigens in the ES-thiol fraction.

Drug resistance in sheep gastrointestinal trichostrongyles is a cosmopolitan major constraint to small ruminant production. Despite reports that anthelmintic drug resistance has become common, there are limited information on the presence of drug resistance in Italy. The purpose of this study was to determine the effect of four anthelmintics to control infection in sheep in central Italy. Fifty sheep with fecal egg counts (FEC) > or =150 eggs per gram were selected on each of three farms (n = 150 total sheep) which were randomly allocated to one of five groups. Groups were treated with febantel, levamisole, ivermectin, or moxidectin while the fifth group acted as the control group. A FEC reduction test (FECRT) was conducted on each animal and the mean FEC of each treatment group was compared to that of the control group within farm. Resistance was declared when percentage reduction (R) <95% and the lower 95% confidence interval was <90%. Levamisole (mean R = 89%) resistance was found on all farms and ivermectin (mean R = 93%) resistance was found on two of the three farms. Posttreatment larval cultures showed the presence of Teladorsagia (Ostertagia) circumcincta and Trichostrongylus spp. larvae. Febantel (mean R = 96%) and moxidectin (mean R = 100%) remained effective. This study suggests that drug resistance in sheep gastrointestinal trichostrongyles is present in central Italy and a potential problem which would justify a broader nationwide geographical investigation.

In this study, we isolated and analyzed a small heat shock protein (HSP) of Ostertagia ostertagi (Oo-HSP18). Oo-hsp18 is encoded by a single-copy gene and the full-length cDNA represents an 18-kDa protein. The expression of Oo-hsp18 is highly stage specific and restricted to the adult stage. The protein is synthesized in a tissue-specific manner and localized in the body muscle layer. The levels of Oo-hsp18 mRNAs are sharply induced by heat shock but not by other stressors such as levamisole and H2O2. A vaccination trial with recombinant Oo-HSP18 failed to protect calves against a challenge infection.

Protease inhibitors are thought to protect intestinal parasitic nematodes from their hostile proteolytic environment. In a previous study, screening of Ostertagia ostertagi cDNA libraries with local antibody probes of the abomasal lymph nodes and mucus revealed a (28 kDa) aspartyl protease inhibitor (API), which was exclusively recognised by antibodies from immune calves. Here we report the molecular characterization of Oo-API (sequence analysis, developmental expression and localization) and a vaccine trial in cattle with the native and recombinant baculo-expressed antigen. The full-length open reading frame of api encodes a protein of 28 kDa. The sequence showed 82% significant homology to an Aspin homologue from Trichostrongylus colubriformis (AA034715). The cDNA encoding the full-length sequence was cloned in a bacterial pET expression vector and the pVec 35 baculovirus vector. Polyclonal rabbit serum against the Escherichia coli-expressed protein was used to develop Western Blots of extracts and ES and to localize the antigen on L3, L4 and adult worm sections. The protein was expressed in all life stages, which was confirmed by real-time polymerase chain reaction (RT-PCR), and was mainly localized in the cuticle of L3, the intestinal cells of L4, and the gut and sphincter of adult worms. Polyclonal serum was also used to affinity purify the native protein. Vaccination of calves with native Oo-API and baculovirus-expressed Oo-rbAPI in combination with QuilA resulted in no protection against Ostertagia challenge infections.

The protective capacity of an adult stage Ostertagia ostertagi globin antigen was tested in four vaccination experiments in cattle. In a preliminary experiment, calves were vaccinated three times intraperitoneally with 250 microg globin in Freund's adjuvant and challenged with a trickled infection of 25,000 infective larvae. In three subsequent field studies, calves were vaccinated twice or three times intramuscularly with 80-100 microg globin in Quil A and challenged with a natural gastrointestinal nematode infection on pasture. Higher globin-specific antibody levels were detected in the vaccinated calves than in the control animals in all vaccine trials. In the preliminary experiment, geometric mean cumulative egg counts in the globin group were reduced by 52% and total worm burdens were reduced by 28%, compared to the controls. In the first field trial cumulative faecal egg counts were reduced by 63% in the vaccinated calves. However, the reduction in faecal egg output in these two experiments was not statistically significant and no reduction in faecal egg counts was observed in the vaccinated animals in the two last field trials. In conclusion, vaccination of calves with O. ostertagi globin resulted in highly variable protection levels after challenge infection.

The objective of this study was to investigate the consequences of short-term grazing on bioactive forages on (i) the viability and fecundity of established adult Teladorsagia circumcincta population and (ii) the establishment and development of incoming T. circumcincta infective larvae. Forty-eight, parasite naive, 3-month old, grazing lambs were artificially infected with 8000 infective larvae of T. circumcincta on day 1 of the experiment. On day 21 p.i., lambs were allocated to one of three bioactive forage grazing treatments; chicory (Cichorium intybus), sulla (Hedysarum coronarium), lotus (Lotus pedunculatus), and the control grass/clover (Lolium perenne/Trifolium repens) forage. On day 28 of the experiment a second dose of 8000 T. circumcincta infective larvae was administered to the lambs to investigate the effects of forages on the ability of infective larvae to establish within the host. All animals were slaughtered for worm recovery on day 35, while liveweight gain, feacal egg counts (FEC) and total worm egg output were monitored regularly throughout the experiment. Although FEC or total egg output were similar among the groups, adult worm burdens at slaughter were significantly affected (P<0.05) by forage treatment during the 2 week grazing period. Lambs grazing chicory had the lowest adult worm burdens and significantly lower numbers of male worms compared to those grazing on grass/clover (P<0.01), while the lambs grazing on sulla or lotus had similar adult populations to grass/clover fed animals. The results from the worm recoveries of the second dose (immature worm burdens) were affected by physiologically and/or immunologically mediated mechanisms, which reduced larval establishment in all treatments. Nevertheless, immature worm burdens at slaughter were similar between chicory, sulla and grass/clover group, while the immature worm recoveries from the lotus group were significantly higher (P<0.05) compared to those from lambs grazing grass/clover. Overall, the results of the present study support the view that chicory can be a promising candidate species in pasture management practices to control T. circumcincta burdens.

In this study, we describe the molecular analysis of zinc-metalloproteases from the abomasal nematode Ostertagia ostertagi which were exclusively recognized by local antibodies of immune cattle. Full-length or partial coding sequences of 4 different zinc-metalloprotease cDNAs of Ostertagia (met-1, -2, -3 and -4) were amplified using gene-specific primers using the 3'- and 5'-Rapid Amplification of cDNA Ends (RACE) technique. Sequence analysis identified the cDNAs as encoding zinc-metalloproteases, which showed between 62% and 70% homology to a metalloprotease 1 precursor of Ancylostoma caninum. The full-length cDNA of met-1 consists of an open reading frame (ORF) of 586 amino acids which contains 5 potential N-glycosylation sites and a predicted zinc-binding domain (HEBXHXBGFXHEXXRXDRD). The complete coding sequence of met-3 contains an ORF of 508 aa and the same conserved zinc-binding domain. These domains are signature sequences of the astacin family of the superfamily of metzincin metalloproteases. The presence of a threonine amino acid after the third histidine in MET-1 and MET-3, however, may place them in a new family or subfamily. Real-time PCR analysis of L3, exsheathed L3, L4 and adult cDNA identified transcription of the 4 metalloproteases in different life-stages. The protein MET-1 was expressed in insect cells using the baculovirus expression system but the immunization of calves with this molecule did not lead to protection against challenge infection.

As an alternative to antihelminthic drugs, we are exploiting vaccination to control infections with the abomasal nematode Ostertagia ostertagi in cattle. Our focus for vaccine targets is excretory-secretory (ES) products of this parasite. One of the most abundant antigens in larval and adult Ostertagia ES products is a protein homologous to nematode polyprotein allergens. We found that the Ostertagia polyprotein allergen (OPA) is encoded by a single-copy gene. OPA comprises three or more repeated units, and only the 15-kDa subunits are found in ES products. The native antigen is localized in the intestinal cells of third-stage larvae and in the hypodermis and cuticle of fourth-stage larvae and adult parasites. Vaccination of cattle with native OPA (nOPA) in combination with QuilA resulted in protection against Ostertagia challenge infections. The geometric mean cumulative fecal egg counts in the nOPA-vaccinated animals were reduced by 60% compared to the counts in the control group during the 2-month course of the experiment. Both male and female adult worms in nOPA-vaccinated animals were significantly shorter than the worms in the control animals. In the abomasal mucus of vaccinated animals the nOPA-specific immunoglobulin G1 (IgG1) and IgG2 levels were significantly elevated compared to the levels in the control animals. Reductions in the Ostertagia egg output and the length of the adult parasites were significantly correlated with IgG1 levels. IgG2 titers were only negatively associated with adult worm length. Protected animals showed no accumulation of effector cells (mast cells, globular leukocytes, and eosinophils) in the mucosa. In contrast to the native antigen, recombinant OPA expressed in Escherichia coli did not stimulate any protection.

Intramuscular immunization of calves with an excretory-secretory antigen fraction enriched for cysteine proteinase activity (ES-thiol) and QuilA as adjuvant induces a protective immune response against the abomasal nematode Ostertagia ostertagi. The objectives of the present study were to confirm the protective capacity of ES-thiol in combination with QuilA, to test Al(OH)(3) as adjuvant for vaccination against O. ostertagi and to look for correlations between protection and immunological effector responses. Calves(seven animals/group) were vaccinated three times intramuscularly with 100 micro g antigen and/or adjuvant (ES-thiol with QuilA, ES-thiol with Al(OH)(3), QuilA alone and Al(OH)(3) alone) and subsequently challenged with a trickled oral infection of 25 000 infective larvae in total over 25 days. Faecal egg counts in the ES-thiol QuilA group were reduced by 56% during the two-month period of the trial compared to the QuilA control group (P < 0.002). Calves immunized with ES-thiol QuilA had significantly smaller adult worms (P < 0.002) and less eggs/female worm (P < 0.05) compared to the QuilA control group. No differences in egg output, worm counts or parameters of worm fitness were observed in the ES-thiol Al(OH)(3) group compared to the Al(OH)(3) control group. Although the protective immune mechanism against O. ostertagi remains unknown, protection in the ES-thiol QuilA group was associated with high levels of parasite-specific antibodies in the abomasal mucosa.

Sixty-four Romney ewe lambs were allocated to 12 groups on the basis of liveweight. Four groups (n=5) were administered oral ivermectin (IVM), 4 (n=6) oral moxidectin (MOX) and the remaining 4 (n=5) controlled-release capsules containing IVM (IVM-CRCs). Nine and 10 days later, the groups within each treatment type were challenged with infective-stage larvae (L3) of 1 of 4 different isolates of Teladorsagia circumcincta (two doses each of 5000 L3). The first of these (S) was known to be anthelmintic-susceptible; the second (OR) was a multiple anthelmintic-resistant strain recovered from the field following therapeutic failure of both ivermectin and moxidectin and subsequently maintained in the laboratory without further anthelmintic selection; the third (R) was derived from OR but had been passaged for five generations indoors with each generation being screened with all three broad-spectrum anthelmintic classes; and the fourth (RxS) was an F1 cross between the R and S isolates. As anticipated, because of its limited residual activity, IVM had no significant effect on the establishment, 9 and 10 days post-treatment, of any of the parasite isolates. In contrast MOX, which has greater residual activity, was highly effective at preventing the establishment of the S isolate but showed no significant effect against the OR, R or RxS isolates. The IVM-CRC was also highly effective at preventing the establishment of the S isolate and furthermore it significantly reduced establishment of both the OR and RxS isolates, although it had no significant effect against the R isolate. The results suggest that with respect to the establishment of T. circumcincta L3s following anthelmintic treatment, macrocyclic lactone (ML) resistance is effectively a dominant trait in the presence of MOX, while it behaves as a partially dominant/recessive trait under treatment with IVM-CRCs. The potential implications of this finding in relation to selection for ML resistance in T. circumcincta are discussed.

A three-year grazing experiment (1998-2000) was conducted with first-season grazing cattle (FSGC) on improved pastures in central-eastern Sweden. Comparison was made between five groups with 10 calves in each group where four of these were set stocked and either (1) untreated, (2) ivermectin bolus treated, (3) subjected to biological control with the nematophagous fungus Duddingtonia flagrans, or (4) treated with a copper wire particle bolus. The fifth treatment was an evasive grazing strategy, whereby untreated calves were turned out onto pasture used by older cattle the previous year and then these calves were moved to silage aftermath in mid-July. To introduce low-levels of parasite infection to the experiment, each animal received a 'priming dose' of approximately 5,000 Ostertagia ostertagi and 5,000 Cooperia oncophora infective third stage larvae immediately prior to the start of the first grazing year of the trial. Results showed that efficient and sustainable parasite control of FSGC was possible to achieve without the use of anthelmintics by using turnout pastures that the previous year had been grazed by older cattle, in combination with a mid-July move to aftermath leys. Biological control also proved beneficial but the efficacy was impaired if high faecal egg counts coincided with rapid dung pat degradation due to heavy rainfall. No indication of parasite control was observed with the copper wire particle bolus. It was also demonstrated that the impact of gastrointestinal (GI) parasitism varied between years and that the level of overwintering contamination is important but likewise, is unpredictable. Although faecal egg counts in 1999 were low, due both to a delayed turnout and drought for the major part of the grazing season, deposited eggs successfully developed to infective larvae and overwintered in large numbers. The population of overwintered infective larvae at the time of turnout in early May played an important role in the course of infection in 2000 and resulted in an average 65 kg advantage of the ivermectin treated calves compared with the untreated calves.Thus, this three-year grazing experiment has emphasised the importance of subclinical gastrointestinal nematode infections in FSGC in Sweden. In addition, the study has shown that adequate parasite control may be achievable without the use of anthelmintics.

Vaccination is being considered as the most feasible alternative for anthelmintic drugs to control gastrointestinal nematode infections in cattle. However, despite the identification of several candidate protective antigens, no vaccines against gastrointestinal nematode parasites are currently available. The main problems that hamper the development of nematode vaccines in ruminants are that vaccination with recombinant nematode proteins produced in bacterial or eukaryotic expression systems did not induce a protective immune response and no suitable antigen delivery system is available for presentation of protective worm antigens to the bovine mucosal immune system. The present review will focus on recent advances and remaining obstacles in vaccine development against gastrointestinal nematodes in cattle, in particular against the abomasal parasite Ostertagia ostertagi.

A clinical trial was carried out in two provinces of Canada to investigate the effect of treatment with eprinomectin at calving on production parameters in adult dairy cattle in 1999-2000. One of the objectives of this study was to evaluate the impact of treatment on reproductive performance as measured by: calving-to-conception interval, calving-to-first service interval and number of services per conception. The ability of an indirect ELISA using a crude adult Ostertagia ostertagi antigen to predict response to treatment also was evaluated. All lactating cows in 20 dairy herds were allocated randomly to receive either eprinomectin pour-on or placebo at calving. Information on reproductive parameters was obtained from computerised cow records. Survival models were used to evaluate the effect of treatment on the two intervals and a Poisson model was used to evaluate the number of services to conception. A total of 549 cows were included in these analyses. A marginally significant treatment effect on calving-to-conception interval was observed (hazard ratio=1.24, P=0.06) but not on calving-to-first service interval. A significant reduction in the number of breedings to conception for treated animals also was observed with a longer effect in cows with short interval to first service. Milk samples from a subset of 109 late-lactation cows were tested for antibodies against O. ostertagi. The ELISA optical-density ratio (ODR) values obtained between 120 days before calving and drying off were categorised as high ODR (>or=0.5) and low ODR (<0.5). Among untreated animals, the hazard of conception was lower (hazard ratio=0.38, 95% CI=[0.19,0.75]) for high-ODR cows compared to low ODR cows suggesting that higher parasite burdens had an adverse effect on reproductive performance. Treated high-ODR cows had a hazard of conception equivalent to the hazard for all cows in the low-ODR group (indicating that treatment prevented the negative effect associated with these higher parasite burdens).

Cysteine proteinase enriched fractions obtained by thiol-sepharose chromatography of Ostertagia ostertagi membrane-bound protein extract (S3-thiol) or total adult excretory-secretory (ES-thiol) products were tested in a vaccination experiment to evaluate their protective efficacy against O. ostertagi in cattle. Calves were vaccinated three times and subsequently challenged with a trickled infection of 25,000 infective larvae in total over 25 days (1000 L3/day, 5 days/week). Geometric mean cumulative egg counts in the ES-thiol group were reduced by 60% during the 2-month period between the first challenge infection and necropsy, compared to the control group (P < 0.002). No reduction in egg output was observed in the S3-thiol group. At necropsy, calves immunized with ES-thiol had a significantly higher percentage of inhibited L4 larvae (9.8%) and had in total 18% less worms than the control calves, but this reduction was not statistically significant. Both the female and male adult worms were significantly smaller in the ES-thiol group than in the control group. Although no significant difference was observed in the number of eggs per female worm between the groups, there was a trend to less eggs per female worm in the ES-thiol group. Number of worms, size of adult worms and number of eggs per female worm were not significantly different between the S3-thiol group and the control group. Systemic immunization with QuilA as adjuvant induced a significant rise in Ostertagia-specific antibody levels in the abomasal mucosa. Ostertagia-specific local antibody levels showed a significant negative correlation with the size of the adult worms, the number of eggs per female worm and the cumulative faecal egg counts. However, these correlations were quite weak and did not appear to be isotype-specific.

Biological options for nematode parasite control are being sought, as the long-term efficacy of conventional anthelmintics comes increasingly under threat from drug-resistant parasites. Three biological methods with the potential to reduce pasture contamination by parasitic nematode larvae were examined: (a) killing of larvae developing in dung by nematophagous fungi; (b) removal of dung through earthworm ingestion; (c) burial of dung in soil as might occur through the action of dung beetles. Field trials with the test bio-control agents were carried out in autumn and spring by adding dung from sheep infected with Ostertagia (Teladorsagia) circumcincta to pots of ryegrass/white clover. The factorial treatment structure included five fungal treatments (individual applications of Duddingtonia flagrans, Monacrosporium gephyropagum and Harposporium helicoides, a combination of all the three fungi together and an untreated control), two dung burial treatments (dung buried or deposited on the soil surface) and two earthworm treatments (earthworms present or absent). D. flagrans and H. helicoides, individually or in combination, reduced recovery of infective stage larvae in experiment 1, while only H. helicoides reduced recovery in experiment 2. In both the experiments, dung burial increased the total number of larvae recovered, while the number of infective larvae were reduced by the action of earthworms. Increased recovery following burial, along with the fact that larvae moved rapidly from soil onto herbage, suggests that soil may provide a protective reservoir for infective larvae infesting herbage.

Two different deworming strategies based on the use of moxidectin, a broad spectrum endectocide with persistency against Ostertagia ostertagi, were evaluated in grazing dairy heifers. Eighty-four heifers unexposed to parasitic infection were randomly allocated to 3 different groups: untreated (Group 1), treated at 3 and 10 weeks after turnout with 0.5 mg/kg/body weight (BW) of topical moxidectin (Group 2), or treated at turnout and 10 weeks later with 0.5 mg/kg/BW of topical moxidectin (Group 3). The heifers were turned out on June 6 (Day 0) to a naturally contaminated pasture divided into 3 sections by an electrical fence. Each group of heifers was put on a different section of pasture. The trial ended when they were housed on October 29 (Day 143). The results of this experiment confirmed the beneficial effect of 2 strategic treatments with moxidectin pour-on under field conditions in Quebec. The parasitological parameters showed a statistical difference in terms of mean fecal egg counts between treated and control groups (ANOVA, P < or = 0.006). After turnout, pasture larval counts were consistently higher for Group 1 compared with counts for Group 2 or 3. All heifer groups gained weight during the trial period, but the weight gain profile of both treated groups was significantly higher than that of the untreated controls (ANOVA, P < 0.03). During the trial period, Group 2 and Group 3 gained 77.7 kg BW and 73.2 kg BW, respectively, while Group 1 gained only 57.9 kg BW, despite the fact that heifers of Group 1 received a greater quantity of supplementary feed while on pasture.

Haemonchus contortus is known as a prolific parasite, producing high numbers of eggs. It could therefore be used as a cheap source of larvae for immunisation of lambs. The value of immunisation would be improved if the immunity produced gave protection against not only homologous but also heterologous infections. Because antibody cross-reactivity between Haemonchus and Ostertagia has been well established, we wanted to know whether drug-abbreviated infections of H. contortus would stimulate production of antibodies that would cross-react with Trichostrongylus colubriformis. The results obtained from these trials indicated that H. contortus drug-abbreviated infections produced significant immunity against not only Haemonchus but also O. circumcincta. Immunisation increased the level of immunoglobulin E (IgE) and IgE-specific antibodies against T. colubriformis, but the differences between experimental and control animals were not statistically significant. Significantly higher levels of IgG-specific antibodies against T. colubriformis were observed.

Five groups of ten 7-month-old Romney lambs were challenged twice-weekly (2 x 3,000) with either Ostertagia circumcincta (Groups 1-3) or Trichostrongylus colubrifornzis (Groups 4 and 5) infective-stage larvae (L3) following administration of controlled release capsules (CRC) containing either albendazole (ABZ-CRC) (Groups 2 and 5) or ivermectin (IVM-CRC) (Group 3). Larval challenge comprised a mixture of equal numbers of drug-susceptible and -resistant L3 (OR/TR:OS/TS) during the 14-week period of drug release. Positive faecal egg counts (FEC) were recorded in each group during this period. Eggs recovered from faeces were used in an egg hatch assay (EHA) utilizing different concentrations of ABZ. O. circumcincta eggs from Groups 2 and 3 had significantly higher LC50 values than Group 1; eggs of T. colubriformis from the ABZ-CRC treated Group 5 had a higher LC50 than control Group 4. Following the exhaustion of drug release, larval challenge was changed to consist of only susceptible parasites and EHA performed wveekly to determine whether LC50 declined, which would indicate dilution or replacement of drench survivors. In those animals challenged with O. circumcincta, LC50 values were still significantly higher in Groups 2 and 3 at week 19 than in the previously untreated Group 1. For T. colubriformis, LC50 values declined steadily following exhaustion of drug release and were not significantly different between Groups 4 and 5 by week 20. This decline in LC50, and corresponding rise in FEC, was largely the result of 3 animals which dominated with high FEC. The remaining 6 animals showed little decline in LC50 or rise in FEC. The results demonstrate the ability of persistent drugs to screen for resistant parasites and establish that the period of selective advantage for drug-resistant parasites of both species is significantly longer than the period of drug release from CRCs.

The protective capacity of vaccination with Haemonchus placei whole gut homogenate against challenge with the non-blood-feeding nematode Ostertagia ostertagi was evaluated in calves. Ten helminth-free calves were randomly assigned to two groups. Group 1 received 100microg H. placei intestinal homogenate in the adjuvant 5% dextran sulfate/PBS, while Group 2 received the adjuvant alone. Injections were administered subcutaneously on Days 0 and 28. All calves were challenged with approximately 26,100 O. ostertagi larvae on Day 42. Serum antibody response and counts of nematode eggs per gram of feces (EPG) were determined throughout the study. Calves were necropsied at 5.5 weeks post-challenge for recovery of nematodes. Although significant increases were detected in both serum IgG(1) and IgG(2) of Group 1 calves (p<0.05), there was no significant difference in the total number of O. ostertagi recovered from the two groups (p>0.05). Lengths of adult nematodes were not significantly different between groups nor were the numbers of eggs present in adult females recovered from each group significantly different (p>0.05). There were also no significant differences between groups regarding fecal egg counts (p>0.05). Results suggest either: (1) the antigens targeted by the induced antibodies were not present in O. ostertagi; (2) the antigens targeted by the induced antibodies were present, but not essential to O. ostertagi survival; or (3) the antigen was present and essential, but amount of antibody ingested was insufficient to cause damage to the nematode.

Background and Objective: A hypersensitive inflammatory response, associated with the ingestion of trichostrongylid larvae, is a major cause of diarrhoea and soiling of the breech with faeces in adult Merino sheep ('hypersensitivity scours'). This response is characterised by an infiltration of eosinophils and altered lymphocyte populations in the gut of affected sheep. The objective of this study was to investigate the numbers of Ostertagia circumcincta and Trichostrongylus vitrinus infective larvae that were needed to induce diarrhoea in adult Merino sheep grazing improved pastures., Method: Sheep from three farms were categorised as either being susceptible, or not susceptible, to hypersensitivity scours, then transported to an experimental farm and grazed on pastures with low numbers of trichostrongylid infective larvae. They were ranked by farm of origin and previous worm egg count, then systematically allocated to one of five dose-rate groups and infected with 0, 2, 4, 10 or 20 x 10(3) trichostrongylid infective larvae per week for 13 weeks., Results: In sheep selected as being susceptible to hypersensitivity scours, the lowest dose rate of infective larvae (2000 per week) was as effective in inducing diarrhoea as the highest dose rate (20,000 per week). In contrast, even the highest larval dose did not induce diarrhoea in sheep selected as not susceptible to hypersensitivity scours. Sheep selected with an increased susceptibility to hypersensitivity scours also had a significantly higher amount of moisture in their faeces at all times except during summer., Conclusions: The apparent lack of a threshold dose of trichostrongylid infective larvae needed to induce hypersensitivity scours suggests that even improved worm control programs, which substantially decrease the ingestion of larvae, may not be able to reduce the prevalence of hypersensitivity scours or the severity of breech soiling. Controlled-release anthelmintic capsules will control breech soiling but are often not a cost-effective strategy. Thus, genetic selection of less susceptible sheep is probably the best long-term option for the control of hypersensitivity scours, and the assessment of faecal moisture may be a convenient marker of susceptibility to this syndrome.

Two of the most important questions for immunologists studying nematode infections are what effect does the host response have on the parasite and which components of the host response are responsible for these effects. The number of nematodes and the mean length of adult female Ostertagia circumcincta was measured in over 500 6-7 month old lambs of the Scottish Blackface breed. Quantitative genetic analyses indicated that there was substantial genetic variation among lambs in faecal egg counts and in worm length but the analyses provided no evidence for genetic variation in worm numbers. Separate analyses have shown a strong relationship between decreased worm length and decreased worm fecundity. Therefore, genetic resistance in lambs appears to be due to control of worm growth and not to control of worm numbers. The only immune response that is consistently associated with reduced worm length is the IgA response to fourth-stage larvae. The association is remarkably strong. After allowing for the influence of worm number on worm length (density-dependence) essentially all of the variation among deliberately infected lambs in worm length can be accounted for, in a statistical sense, by variation in the strength and specificity of the local IgA response. Therefore, the IgA mediated suppression of worm growth and fecundity appears to be the major mechanism of resistance to O. circumcincta in lambs.

The nematocidal effectiveness of moxidectin, administered topically at the rate of 500 mcg/kg BW, was determined for lactating dairy cows. Naturally infected animals were given either topical vehicle or moxidectin (Cydectin Pour-On Fort Dodge Animal Health) at the rate of 1 ml/10 kg BW (10 animals per treatment group), and sacrificed 14-18 days post-treatment for nematode enumeration. 100% efficacies were recorded for Ostertagia lyrata males, Cooperia punctata males and Oesophagostomum radiatum L4, with treatment group differences in geometric means significant (P < 0.05) for all. Populations of Trichostrongylus L4 and adult O. radiatum were also reduced by 100%, but low prevalence rates in the control animals precluded meaningful statistical inference. Nematode populations for which efficacies ranged from 96.7 to 99.6% (based on geometric means) and for which treatment group differences were significant (P < 0.05) included Ostertagia spp. adult females, inhibited L4 and developing L4, O. ostertagi adult males, Trichostrongylus axei adults and Cooperia spp. adult females. For all nematodes combined, moxidectin was 98.9% efficacious. In addition to exhibiting excellent nematocidal effectiveness, topical moxidectin was demonstrated to be safe, with animal health and milk production unaffected during the study.

The efficacy of an isolate of the nematophagous fungus Duddingtonia flagrans against gastrointestinal nematodes of cattle was examined at 2 dose levels on 2 permanent pastures, with high and low stocking rates, respectively. Thirty calves, experimentally infected with Ostertagia ostertagi, were divided into 3 comparable groups and allocated to 3 similar paddocks in each of the 2 trials. Two of the 3 groups received fungal material once per day during the initial 2 months, either at high dose (10(6) fungal spores/kg body weight) or low dose (5 x 10(5) or 2.5 x 10(5) fungal spores/kg body weight). The third group remained as an untreated control group. Faecal, blood, and herbage samples were collected and animals were weighed every month from May to September. The pasture grazed at a high stocking rate had a large number of overwintering infective larvae, while the pasture grazed at a low stocking rate had a low overwintering herbage larval infectivity. The results showed that, at a high stocking rate, the recovery of infective larvae on pasture was diminished and calves were prevented from clinical ostertagiosis by using the D. flagrans Troll A-isolate. At low stocking rate, the parasite burden seemed not to be very heavy, and a conclusive effect of the fungi at the dose-level used could not be detected.

One of three groups of sheep was challenged twice-weekly with infective-stage larvae (L3) of the sheep parasite O. circumcincta, another with the cattle parasite O. ostertagi while the third received no larval challenge. Positive faecal egg counts (FEC) and a rise in plasma pepsinogen levels were observed only in those animals given O. circumcincta. Anti-O. circumcincta L3 IgG titres were rapidly elevated during parasite challenge with either O. circumcincta or O. ostertagi. Throughout the experiment, no rise in anti-adult IgG titres or eosinophil numbers was observed in peripheral blood in any group. On evidence of self-cure of the trickle-infection, determined by a reduction in FEC, all groups were drenched and challenged with 15,000 O. circumcincta L3. No effect of previous challenge on parasite establishment or FEC was observed, although egg viability was significantly reduced in both groups given prior challenge. Significant differences in adult female worm length were observed between groups. Those recovered from animals previously challenged with O. circumcincta were shorter than from those given O. ostertagi which were in turn shorter than those from previously unchallenged animals. In utero egg counts were significantly lower in worms from animals previously challenged with O. circumcincta than in those from unchallenged control animals. The results indicate that a level of immunity to O. circumcincta can be conferred by exposure to O. ostertagi.

Various parameter estimates were assessed at housing in calves that had been exposed to gastrointestinal nematodes during a first grazing season. The analysis involved 41 groups of first grazing season (FGS) calves on 15 different farms in Belgium and comprised groups that had received chemoprophylactic treatment and untreated controls. Serum pepsinogen levels gave the clearest division between chemoprophylactic-treated calf groups (all were <2.6 U tyr), and untreated calf groups in which sub-clinical (range: 2.0-4.1 U tyr) and clinical infections (range 3.7-6.3 U tyr) occurred. There was also a tight relationship between individual pepsinogen values and adult Ostertagia burdens obtained at slaughter. In chemoprophylactic-treated groups there was a significant negative relationship between mean serum pepsinogen levels at housing and the proportion of the grazing season covered by different chemoprophylactic systems. Although only limited data on crude adult Ostertagia antigen ELISA were available, a good relationship between optical densities and estimated exposure was also found. The parasitological parameters, faecal egg counts and pasture Ostertagia larval counts at housing, and weight gain per day, gave less clear divisions among the three categories (chemoprophylaxis, sub-clinical and clinical). Distinguishing how much exposure a calf group has experienced during a first grazing season could help in designing more appropriate control measures for the FGS calves in the next year, assuring good protection and at the same time allowing sufficient exposure for the development of acquired immunity to Ostertagia, and for this serum pepsinogen is recommended.

The objective of this study was to investigate the influence of different types of chemoprophylaxis in first season grazing calves on their resistance against a natural reinfection with Ostertagia ostertagi and Cooperia oncophora in the second grazing season. Thirty helminth-naive crossbred calves were randomly divided in three groups of 10 animals. The animals of group B received an ivermectin sustained release bolus on day 0. The calves of group D were treated on days 0 and 56 with a subcutaneous injection of doramectin (0.2 mg kg(-1) BW). Group C was the untreated control group ('immune' controls). Although exposure to gastrointestinal nematodes in the first grazing season was only limited, the chemoprophylactic treatments in groups B and D resulted in three distinctly different infection levels (group C > group D > group B). At the start of the second grazing season, six helminth-naive steers (group N, 'susceptible' controls) were turned out together with the second season animals. After 3 weeks of grazing, the 'susceptible' controls were slaughtered, together with four animals from each other group. Parasitological and immunological parameters indicated that resistance to reinfection with Ostertagia was reduced in the chemoprophylactic treated animals, and was negatively related to the degree of suppression of host-parasite contact in the first grazing season (group C > group D > group B > group N). None of the groups had developed a complete resistance against Cooperia yet. A negative relationship was observed between reduction of first grazing season exposure, and weight gains early in the second grazing season. The remaining animals stayed on pasture until the beginning of November. At the end of the second grazing season, levels of acquired resistance against Ostertagia infection were similar in all groups, and all animals had become immune against Cooperia. No effect of first year chemoprophylaxis on total weight gains could be demonstrated. Because of discrepancy between pasture larval counts and tracer worm counts, it was not possible to draw firm conclusions on the effect of chemoprophylaxis on pasture infestation levels in the second year.

The build up of gastrointestinal nematode infections was followed in two grazing experiments. Both experiments included four groups of six calves, a permanently housed non-infected control group and three groups which were grazed from May to October. One of these was moved to aftermath in the beginning of July, the second in the beginning of July and August and the third in the beginning of July, August and September. The build up of gastrointestinal nematode infections was followed by performing faecal egg counts, differentiation of faecal larval cultures, pasture larval counts, serum pepsinogen values, serum antibodies against Cooperia oncophora, weight gain and worm counts. In the second experiment four of the principal trial animals of each group were treated with oxfendazole and subsequently challenged with 100,000 larvae of Ostertagia ostertagi to examine development of immunity against O. ostertagi. The faecal egg counts and the worm counts of the sentinels necropsied in July indicated low initial infections in both experiments. Infection levels in experiment 1 remained low in each group until the beginning of September. However, during the last month, moderate to high infections were acquired by the groups which were moved once or twice. In contrast, low to moderate infections were maintained in the group moved three times. In the second experiment moderate C. oncophora burdens were already observed in the sentinels grazed until the beginning of August. Tracers grazing in August-September with the group moved once acquired high O. ostertagi and C. oncophora infections, whereas those grazed with both other groups acquired moderate infections. In October high infections with both species occurred in the groups moved once and twice, whereas low to moderate infections were observed in the group moved three times. The challenge infection demonstrated a reduction of establishment of O. ostertagi of approximately 70% in all three groups on pasture. The results demonstrate that moving calves at monthly intervals to clean pasture can be an effective method for the control of parasitic gastroenteritis. In addition, the data indicate that it is essential that the last move does not occur more than 1 month before the end of the grazing season.

The persistent efficacy of some anthelmintics brings advantages in nematode control in domestic animals. However, scientific assessment of persistent efficacy is relatively new, and a feature of published data has been variability in the reported endpoint for this activity. Trial design and method of calculating efficacy have a large bearing on the results obtained. Three types of studies used to evaluate the persistent efficacy of anthelmintics are briefly discussed and compared. In the first type of study, animals are treated followed by a single infection at 7, 14 or more days after treatment. The reduction in worm counts compared to an untreated control group gives a good indication of the persistent efficacy of the product at each time point. One control group can be used for several time points. In the second type of study, the animals are treated and then infected daily from day 1 until 7 days, 14 days or longer after treatment. The animals are slaughtered approximately 3 weeks after the last infection. This approach may better mimic a natural infection but the results obtained are an average reduction over the whole infection period. At the end of the evaluated period, the actual protection may be considerably lower than the average. From this test, it is difficult to define when the protection decreases or disappears. In this test, a control group is required for each period. In the third type of study, a modification of the second, the animals are treated and infected as before but animals are slaughtered soon (2-5 days) after the last infection. Based on the reduction of, for example, the different Ostertagia stages a more specific determination of the persistent efficacy 0-3 days (L3), 3-7 (EL4), 7-14 (LL4 + EL5) and more than 14 days (LL5 + adults) before slaughter can be obtained. Only two groups of animals are required to cover a 3 week period and the average efficacies can be reduced to about one week.

Models of the population biology and transmission of helminth parasites of domestic ruminants do not generally include any explicit reference to production because that would require the identification of a systematic relationship between parasitism and production loss. This is difficult because (1) the relationship between parasitism and production loss (in so far as we can identify one) is non-linear, (2) it is by no means clear which index of parasitism we should use in defining the nature of the relationship and (3) time delays in the system and the multicausal nature of production losses make it difficult to tease out those components of the loss that are attributable to parasitism.

Anthelmintics and endectocides used for the treatment and prophylaxis of Ostertagia sp. in ruminants include benzimidazoles, levamisole, morantel and the avermectins and milbemycins. Most of these agents are excreted to some extent in the faeces of treated animals and it has been demonstrated that members of the avermectin/milbemycin group may have deleterious effects on non-target organisms utilising the faeces. The environmental impact of antiparasitic chemotherapy depends on the deleterious effect which the agent or its metabolites have on organisms in the locus of the excreta, the amount of active agent excreted, the temporal nature of the excretion and the stability of the ecotoxic residues. These have to be considered in the context of the overall proportion of excreted faeces from a herd which is contaminated and thus the availability of non-contaminated faeces which may act as refugia for dung utilising organisms. The contribution which weathering, faunal inhabitants, trampling by cattle and disturbance by birds have on the rate of dung degradation must also be considered. The greatest ecotoxicological risk is associated with sustained release delivery devices, delivering endectocides with potent activity against dipteran flies and coleopteran beetles. The relatively large proportion of most cattle herds excreting faeces with no endectocidal contamination is likely to reduce the impact that such treatment or prophylactic strategies have on non-target organisms.

A study was conducted to investigate reinfection with nematodes in calves following treatment with doramectin or ivermectin administered subcutaneously at a dose rate of 200 micrograms kg-1 of body weight under conditions of natural challenge. Thirty calves were allocated to three groups of 10 calves each based on body weight, sex, breed and pre-treatment faecal egg counts (FEC) after grazing together on a common pasture for three months. Treatments were doramectin, ivermectin and no treatment. Calves were returned to the same pasture for 56 days, placed on dry lot from days 56 to 63 and then necropsied over days 64-66. Faecal egg counts were done at days -1 and 0, then bi-weekly from day 14 to 56 and day 63. Mean FEC in control calves continued to rise throughout the grazing period. Trichostrongyle FEC were significantly (P < 0.05) greater in the control group compared to either treated group at each sample time following treatment. At necropsy, a total geometric mean of 19,847 nematodes per calf was recovered from untreated controls, of which eight genera were identified. The predominant nematode genera were Ostertagia (8749), Nematodirus (3702) and Cooperia (1927). In the ivermectin-treated calves, geometric mean worm burden was similar to that of the untreated controls: A total of 20,349 nematodes per calf was present including the genera Nematodirus (8633), Ostertagia (4700) and Cooperia (1740). In contrast, the geometric mean worm burden in doramectin-treated calves was 12,173, including Ostertagia (4310), Cooperia (1141) and Nematodirus (1667). Doramectin was more effective than ivermectin in protecting calves from reinfection over a 56-day post-treatment grazing period under conditions of natural challenge as measured by accumulated mean worm burdens.

Three groups of newly-weaned Romney lambs were given either a standard oral dose of albendazole, a controlled-release capsule containing albendazole, or a standard oral dose of moxidectin. At 10, 20, 30 and 40 days after treatment, sub-groups of lambs were given 10,000 infective-stage larvae of either a drug-resistant or a drug-susceptible strain of Ostertagia circumcincta. The recommended oral dose of albendazole removed 32 per cent of the resistant strain and over 99.9 per cent of the susceptible O. circumcincta. The recommended oral dose of moxidectin removed 91 per cent of the resistant strain and over 99.9 per cent of the susceptible parasites. None of the lambs treated with controlled-release capsules was challenged at 20 or 30 days after treatment. Twenty-one days after challenge, samples of faeces were taken to determine the presence of nematode eggs and cultured to establish the proportion of eggs developing to infective-stage larvae (L3). Abomasa were recovered after slaughter and worm burdens determined. In the lambs given controlled-release capsules only the resistant parasites were able to establish, and there were significantly fewer than in the lambs treated orally with albendazole. The proportion of the eggs from resistant parasites which developed to L3 was not reduced by the presence of the capsules. Oral moxidectin provided no protection against the establishment of the resistant strain and viable L3 were recovered after challenge with resistant parasites 10 days after treatment; however, the establishment of susceptible O. circumcincta was reduced by more than 99 per cent. The establishment of the susceptible parasites in the lambs treated with moxidectin increased with time and was not significantly lower than in the other groups by 30 days after treatment.

Four groups of 8 parasite-naive Dorset-crossbred lambs, 3-4 months old, were turned out on infected pasture on 2 May and allocated to 4 separate paddocks. From May to September, 2 groups received Duddingtonia flagrans (10(6) chlamydospores per kg body weight per lamb per day) mixed in 100 g of barley, while the other 2 groups received barley only. All groups remained set-stocked until slaughter for worm counts on 10 October. In late June, all lambs were treated with fenbendazole due to severe parasitic gastroenteritis in all groups. The faecal egg counts were comparable for the 2 treatments throughout the grazing period. Larval development of Ostertagia/Trichostrongylus spp. in faecal cultures was 1-28% in the fungi-fed groups compared with 60-80% in the untreated groups (P < 0.05). In September, pasture larval counts of Ostertagia/Trichostrongylus were 930 and 4400 L3 kg-1 on paddocks of fungi-fed and untreated groups, respectively. Corresponding figures for Nematodirus spp. were 7200 and 11600 L3 kg-1, respectively. At slaughter, the number of immature Ostertagia spp. was 62% lower in the fungi-fed groups compared with the untreated groups (P < 0.05). Four parasite-free lambs were introduced to each paddock during the period 3-23 October and slaughtered for worm counts after 3 weeks of housing. The total worm burden of tracers on paddocks previously grazed by fungi-fed lambs was reduced 86% (P < 0.05; geometric means) compared with control groups, while significant reductions were also seen in abomasal worm counts (68%; P < 0.05), N. spathiger (98%; P < 0.05) and for N. battus (97%; P < 0.01). It is concluded that dosing sheep with D. flagrans while grazing may limit the build up of pasture contamination in the late grazing season and subsequently limit the intake of larvae in sheep.