World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.): Third edition of the guideline for evaluating efficacy of anthelmintics in ruminants (bovine, ovine, caprine).

This guideline is aimed at those who are involved in the assessment of anthelmintic efficacy in ruminant livestock species (bovine, ovine and caprine). The intent is to provide a framework that can be adopted worldwide for the testing of anthelmintics in ruminants, such that studies carried out in different countries can be compared and thereby unnecessary duplication can be reduced. Recommendations are made for the selection, housing and feeding of study animals, the type of studies required, the method used to conduct those studies, the assessment of results and the standards for defining anthelmintic efficacy.

Dictyocaulus viviparus bulk tank milk seropositivity is correlated with meteorological variables.

Control of infections with Dictyocaulus viviparus is difficult due to its volatile epidemiology. In the absence of predictive models, 'vigilance and treatment' is today's mainstay for control. In order to evaluate the potential of predictive model development to support a more preventative approach, this longitudinal study aimed at understanding the influence of weather factors on D. viviparus bulk tank milk antibody ELISA results. Bulk tank milk samples were analysed with a Major Sperm Protein-based ELISA (expressed as an optical density ratio) twice monthly on 717 Flemish dairy farms during the grazing season (April-October) in 2018. Meteorological data of the sampled farms were obtained at 1 km spatial scale using the ALARO-SURFEX climate model. A mixed effects model showed that the bulk tank milk optical density ratio was significantly associated with the month of sampling, evapotranspiration, temperature and its quadratic term, the number of hot days and the number of rainy days in the 7-8 weeks prior to sampling. There were significant farm effects involved. The model's accuracy to predict bulk tank milk optical density ratio infection status was 80%, while optical density ratios were generally overestimated by 38%. Inclusion of the previous (2-week-old) optical density ratio values increased accuracy to 86% and reduced the mean square error. We conclude that meteorological parameters have a predictive value for bulk tank milk optical density ratio results, while further research should evaluate model improvements through the addition of herd management factors as well as confirm the predictive power through external validation in additional farms and years.

Anthelmintic resistance in ruminants: challenges and solutions.

Anthelmintic resistance (AR) is a growing concern for effective parasite control in farmed ruminants globally. Combatting AR will require intensified and integrated research efforts in the development of innovative diagnostic tests to detect helminth infections and AR, sustainable anthelmintic treatment strategies and the development of complementary control approaches such as vaccination and plant-based control. It will also require a better understanding of socio-economic drivers of anthelmintic treatment decisions, in order to support a behavioural shift and develop targeted communication strategies that promote the uptake of evidence-based sustainable solutions. Here, we review the state-of-the-art in these different fields of research activity related to AR in helminths of livestock ruminants in Europe and beyond. We conclude that in the advent of new challenges and solutions emerging from continuing spread of AR and intensified research efforts, respectively, there is a strong need for transnational multi-actor initiatives. These should involve all key stakeholders to develop indicators of infection and sustainable control, set targets and promote good practices to achieve them.

Myostatin mutation causing double muscling could affect increased psoroptic mange sensitivity in dual purpose Belgian Blue cattle.

Belgian Blue cattle are known for their high degree of muscling and good carcass qualities. This high degree of muscling is mainly caused by a mutation in the myostatin gene (MSTN). Although the MSTN mutation is considered as fixed in the Belgian Blue breed, segregation is occurring in a sub-population bred for dual purpose. In the latter population, we observed an association between the mutation in MSTN and susceptibility to psoroptic mange, a skin disease caused by Psoroptes ovis mites that heavily plagues Belgian Blue cattle. In total, 291 animals were sampled and screened for their susceptibility for mange lesions and their MSTN genotype. Via linear mixed modelling, we observed that homozygous mutant animals had a significant increase in the size of mange lesions (+2.51% lesion extent) compared to homozygous wild type. These findings were confirmed with zero-inflated modelling, an animal model and odds analysis. Risk ratios for developing severe mange lesions were 5.9 times as high for homozygous mutant animals. All analyses confirmed an association between the MSTN genotype and psoroptic mange lesion size.

Evaluation of a Cooperia oncophora double-domain ASP-based vaccine against Cooperia spp. infections in cattle and sheep.

A double-domain activation-associated secreted protein (dd-Co-ASP) isolated from the bovine small intestinal parasite Cooperia oncophora was previously shown to be an effective vaccine candidate to protect calves against a homologous challenge infection. The aim of this study was to investigate whether the dd-Co-ASP protein, purified from a Belgian C. oncophora isolate, would offer protection against a C. oncophora isolate from the southern hemisphere as well as other Cooperia species such as C. punctata in cattle and C. curticei in sheep. Two vaccination studies were performed, i.e. one in cattle and one in sheep, in which the protective effects of dd-Co-ASP, supplemented with Quil A as an adjuvant, were compared with an adjuvant control. Whereas our results showed a 75 % reduction in Cooperia spp. cumulative faecal egg counts, the results obtained in sheep demonstrated that dd-Co-ASP was ineffective in raising a protective immune response against a C. curticei challenge infection. Even though sequence analysis of the dd-Co-ASP gene revealed restricted sequence heterogeneity in the double domain ASP within and between bovine Cooperia species, the results of the vaccine study suggest that there is sufficient conservation at the protein level to yield cross-protection, holding promise for the development of a general Cooperia vaccine for use in cattle.

Risk factors for lungworm-associated milk yield losses in grazing dairy cattle.

Dictyocaulus viviparus, the causative agent of bovine parasitic bronchitis, is an important parasite of dairy cattle. Infections can lead to substantial economic losses, due to mortality, reduced weight gain and milk production and treatment costs. There have been relatively few studies investigating herd management risk factors for infections with D. viviparus and lungworm-associated production losses. The aims of this study were (1) to assess the impact of (sub)clinical lungworm infections on productivity in dairy cows and, (2) to identify or confirm risk factors, related to herd management, for infections in grazing dairy cattle. Using a recombinant Major Sperm Protein (MSP)-based ELISA, the presence of D. viviparus antibodies in bulk tank milk (BTM) samples was evaluated on 717 and 634 farms at two-week intervals during two grazing seasons (2018 and 2019). Associations between milk antibody levels and production data (mean milk yield in kg/cow/day, percentage of fat and protein) were assessed, as well as associations with putative risk factors in the herd management, gathered through a questionnaire survey. In both years, there was a substantial, but non-significant, difference in the annual mean milk yield on farms with at least one BTM sample above the cut-off of 0.41 ODR, compared with the mean milk yield on farms that stayed under this threshold on each sampling day (-0.17 and -0.70 kg milk/cow/day in 2018 and 2019, respectively). In 2019, this association was stronger, and significant, when the cut-off was exceeded in at least two consecutive BTM samples (-1.74 kg milk/cow/day). BTM results were also significantly negatively associated with the closest milk production data during the two-weekly BTM sampling intervals in 2019. A single or two consecutive positive tests were used in the risk factor analysis as a proxy for lungworm-associated milk yield losses. Purchase of new animals (Odds Ratio (OR) = 2.68) and the proportion of the first grazing season covered by preventive anthelmintic treatment (OR up to 3.88, depending on proportion) were positively associated with lungworm-associated milk yield losses, while mowing at least 50 % of the pastures (OR = 0.57) was negatively associated with lungworm-associated milk yield losses. Our results suggest that the ELISA holds promise to identify herds with significant production losses due to lungworm infections, under the condition that BTM sampling is done repeatedly during the grazing season. Based on the confirmed risk factors, adjustments of the farm management could perhaps mitigate these losses.

Coprological survey of endoparasite infections in owned dogs and owners' perceptions of endoparasite control in Belgium and the Netherlands.

Infections with endoparasites are common in dogs. Some of these parasites are potentially zoonotic and therefore a public health concern. A survey was conducted in twenty-six small animal practices to evaluate the prevalence of endoparasites in Belgian and Dutch owned dogs older than 6 months as well as risk factors associated with infection. Out of 239 faecal samples screened (168 in Belgium and 71 in the Netherlands), 18 dogs were tested positive for at least one type of endoparasite with three dogs co-infected with two parasitic species. Toxocara sp. was the most frequently found endoparasite (4.6%). Three other dogs were positive for Angiostrongylus vasorum (1.4%) using the Baermann method and confirmed in one dog by the Angiodetect® test. Age and predation behaviour were identified as two risk factors associated with endoparasite infection. Although the majority (77%) of the owners in this study reported to administer at least one anthelmintic treatment per year, only a minority of them (24.3%) were aware of the risk to human health, indicating that owner awareness is sub-optimal. For dog owners, human toxocarosis and other potential zoonoses remain an underestimated health concern. The implementation of sustainable parasite control strategies should be promoted taking also into account the public health risk.

Association between Dictyocaulus viviparus bulk tank milk antibody levels and farmer-reported lungworm outbreaks.

The lungworm Dictyocaulus viviparus can have a major impact on bovine health and productivity. Enzyme-Linked Immunosorbent Assays (ELISA), based on a recombinant Major Sperm Protein (MSP), have been developed to detect D. viviparus-specific antibodies in bulk tank milk (BTM). The objectives of this study are to assess the association between BTM optical density ratio's (ODR) and farmer-reported lungworm outbreaks based on the clinical sign "coughing" throughout the grazing season and to compare the sensitivity and specificity of two ELISAs under field conditions. The Hannover MSP-ELISA and the prototype Svanova MSP-ELISA were used for the detection of D. viviparus antibodies in BTM samples on 717 dairy farms during the 2018 grazing season. Assuming all herds to be truly lungworm infected, the results show that the Svanova ELISA had a lower sensitivity (40-65%) and specificity (75-90%) for the detection of D. viviparus infections in BTM compared to the Hannover ELISA, which had a sensitivity of 42% and 74% and specificity of 100% and 98% at a cut-off of 0.41 ODR and 0.25 ODR, respectively. Therefore, analyses of the associations between milk antibody levels and farmer-reported outbreaks during the 2018 and 2019 grazing season were assessed using the Hannover ELISA, on 717 and 634 farms, respectively. A positive association was found between a farmer-reported outbreak and having at least two consecutive positive BTM ODR's at a cut-off of 0.41 in 2018 (Odds Ratio (OR) = 5.5) and 2019 (OR = 2.8). Furthermore, there was a significant association between a farmer-reported outbreak and having a positive BTM ODR in August (OR 2018 = 4.4; OR 2019 = 2.8) and October (OR 2018 = 3.7; OR 2019 = 1.8). On the farms with a farmer-reported outbreak and positive BTM samples, over half (2018 = 77%; 2019 = 57%) of the positive ODR's were situated before the outbreak and 47% (2018) and 42% (2019) within 12 weeks before the outbreak. In conclusion, there is a positive association between farmer-reported outbreaks and the occurrence of a positive BTM sample at the cut-off of 0.41 ODR using the Hannover ELISA.

Initial assessment of the economic burden of major parasitic helminth infections to the ruminant livestock industry in Europe.

We report a European wide assessment of the economic burden of gastrointestinal nematodes, Fasciola hepatica (common liver fluke) and Dictyocaulus viviparus (bovine lungworm) infections to the ruminant livestock industry. The economic impact of these parasitic helminth infections was estimated by a deterministic spreadsheet model as a function of the proportion of the ruminant population exposed to grazing, the infection frequency and intensity, the effect of the infection on animal productivity and mortality and anthelmintic treatment costs. In addition, we estimated the costs of anthelmintic resistant nematode infections and collected information on public research budgets addressing helminth infections in ruminant livestock. The epidemiologic and economic input data were collected from international databases and via expert opinion of the Working Group members of the European Co-operation in Science and Technology (COST) action COMbatting Anthelmintic Resistance in ruminants (COMBAR). In order to reflect the effects of uncertainty in the input data, low and high cost estimates were obtained by varying uncertain input data arbitrarily in both directions by 20 %. The combined annual cost [low estimate-high estimate] of the three helminth infections in 18 participating countries was estimated at € 1.8 billion [€ 1.0-2.7 billion]. Eighty-one percent of this cost was due to lost production and 19 % was attributed to treatment costs. The cost of gastrointestinal nematode infections with resistance against macrocyclic lactones was estimated to be € 38 million [€ 11-87 million] annually. The annual estimated costs of helminth infections per sector were € 941 million [€ 488 - 1442 million] in dairy cattle, € 423 million [€ 205-663 million] in beef cattle, € 151million [€ 90-213 million] in dairy sheep, € 206 million [€ 132-248 million] in meat sheep and € 86 million [€ 67-107 million] in dairy goats. Important data gaps were present in all phases of the calculations which lead to large uncertainties around the estimates. Accessibility of more granular animal population datasets at EU level, deeper knowledge of the effects of infection on production, levels of infection and livestock grazing exposure across Europe would make the largest contribution to improved burden assessments. The known current public investment in research on helminth control was 0.15 % of the estimated annual costs for the considered parasitic diseases. Our data suggest that the costs of enzootic helminth infections which usually occur at high prevalence annually in ruminants, are similar or higher than reported costs of epizootic diseases. Our data can support decision making in research and policy to mitigate the negative impacts of helminth infections and anthelmintic resistance in Europe, and provide a baseline against which to measure future changes.

Identifying human enteric parasitic infections in Greece, with focus on Giardia and Cryptosporidium.

A study was conducted in two different areas in Greece to investigate the presence of intestinal human parasitic infections (targeting healthy and individuals with diarrhoea). In total, 876 stool samples were collected from 822 adults and 54 children. Both sedimentation (acid/ether) and concentration/flotation techniques were performed in all samples to detect intestinal parasites. Additionally, a quantitative direct immunofluorescence assay was used specifically for the detection of Giardia and Cryptosporidium. PCR followed by sequencing was applied to genotype Giardia and Cryptosporidium positive samples. Thirty-five (4%) of the individuals examined harboured at least one species of intestinal parasite, the majority of which were protozoa (3.8%). The species found were Blastocystis hominis (1.8%), Giardia duodenalis (1.3%), Cryptosporidium spp. (0.6%), Entamoeba coli (0.2%) and E. histolytica/E. dispar (0.1%). Two persons were positive for Enterobius vermicularis. Genotyping results revealed the presence of G. duodenalis sub-assemblage AII, whereas sequencing was not successful for Cryptosporidium positive samples. A novel multi-locus genotype of G. duodenalis was identified, which has not been described in humans or animals previously. Overall, in the studied population, infection rates with intestinal parasites were low and similar to previous published data. As infection levels were low, no associations could be made between infection status and clinical relevance, risk factors or indication of potential sources of infection, apart from the fact that infections with Giardia were positively correlated to diarrhoea. Based on the parasite species and genotypes detected, there was no indication that animals were an important source of infection. Thus, it is suggested that Giardia infections were more likely to be acquired via human-to-human transmission, either involving indirect pathways such as contaminated food or water, or via direct contact.

GLOWORM-PARA: a flexible framework to simulate the population dynamics of the parasitic phase of gastrointestinal nematodes infecting grazing livestock.

Gastrointestinal nematodes are a significant threat to the economic and environmental sustainability of keeping livestock, as adequate control becomes increasingly difficult due to the development of anthelmintic resistance in some systems and climate-driven changes to infection dynamics. To mitigate any negative impacts of climate on gastrointestinal nematode epidemiology and slow anthelmintic resistance development, there is a need to develop effective, targeted control strategies that minimise the unnecessary use of anthelmintic drugs and incorporate alternative strategies such as vaccination and evasive grazing. However, the impacts climate and gastrointestinal nematode epidemiology may have on the optimal control strategy are generally not considered, due to lack of available evidence to drive recommendations. Parasite transmission models can support control strategy evaluation to target field trials, thus reducing the resources and lead-time required to develop evidence-based control recommendations incorporating climate stochasticity. Gastrointestinal nematode population dynamics arising from natural infections have been difficult to replicate and model applications have often focussed on the free-living stages. A flexible framework is presented for the parasitic phase of gastrointestinal nematodes, GLOWORM-PARA, which complements an existing model of the free-living stages, GLOWORM-FL. Longitudinal parasitological data for two species that are of major economic importance in cattle, Ostertagia ostertagi and Cooperia oncophora, were obtained from seven cattle farms in Belgium for model validation. The framework replicated the observed seasonal dynamics of infection in cattle on these farms and overall, there was no evidence of systematic under- or over-prediction of faecal egg counts. However, the model under-predicted the faecal egg counts observed on one farm with very young calves, highlighting potential areas of uncertainty that may need further investigation if the model is to be applied to young livestock. The model could be used to drive further research into alternative parasite control strategies such as vaccine development and novel treatment approaches, and to understand gastrointestinal nematode epidemiology under changing climate and host management.

Beliefs, intentions, and beyond: A qualitative study on the adoption of sustainable gastrointestinal nematode control practices in Flanders' dairy industry.

Emerging anthelmintic resistance emphasizes the need for sustainable control approaches against gastrointestinal nematode (GIN) infections in cattle. The uptake of diagnostic methods for sustainable control could enable more informed treatments and reduce excessive anthelmintic use. Unfortunately, the adoption of such methods remains relatively poor. A better understanding of farmers' motivations and behaviour would help to develop applicable advises and communication strategies for sustainable worm control strategies. A previous study created a general model for adoption intention of GIN diagnostics on dairy farms and measured the most important factors driving this intention (Vande Velde et al., 2015). The current research aimed to dig deeper into this model for the beliefs underlying these factors, and to identify additional factors impelling this specific behaviour. Data were collected through 22 semi-structured interviews with dairy farmers. Using analytic induction analysis, data were moved between deduction and induction. Results show that the adoption process of diagnostic methods for GIN occurs through three different phases: adoption intention, actual adoption and maintenance. Low infection awareness and low priority ('top of mind') of the disease are important barriers for adopting sustainable GIN control. Secondly, farmer behaviour is guided by two important social norms: the opinion of their veterinarian and their fellow farmers. However, farmers hold an incongruent relationship with both norms throughout different stages of behaviour: they do not value other farmers' opinions as a positive reference (intention phase), but follow and mimic their behaviour as a group (action phase). The veterinarian is seen as the most important positive reference, but also the responsible actor for GIN control. As such, the farmers do not hold themselves responsible for implementing sustainable control strategies. Thirdly, different types of motivations influence different sorts of behaviours. Sustainable behaviour such as use of diagnostics will be influenced by moral motives, while management behaviour such as treatment is raised by more economic motives. Finally, not only performing, but also maintaining behaviour is important to fully address the adoption of sustainable control. As such, to maintain the adoption on farm, planning could be an important contribution. These insights can be used by animal health organizations and industry by exploiting motivations, social norms and planning to encourage the uptake of diagnostic approaches in GIN control.

Mind the gaps in research on the control of gastrointestinal nematodes of farmed ruminants and pigs.

Gastrointestinal (GI) nematode control has an important role to play in increasing livestock production from a limited natural resource base and to improve animal health and welfare. In this synthetic review, we identify key research priorities for GI nematode control in farmed ruminants and pigs, to support the development of roadmaps and strategic research agendas by governments, industry and policymakers. These priorities were derived from the DISCONTOOLS gap analysis for nematodes and follow-up discussions within the recently formed Livestock Helminth Research Alliance (LiHRA). In the face of ongoing spread of anthelmintic resistance (AR), we are increasingly faced with a failure of existing control methods against GI nematodes. Effective vaccines against GI nematodes are generally not available, and anthelmintic treatment will therefore remain a cornerstone for their effective control. At the same time, consumers and producers are increasingly concerned with environmental issues associated with chemical parasite control. To address current challenges in GI nematode control, it is crucial to deepen our insights into diverse aspects of epidemiology, AR, host immune mechanisms and the socio-psychological aspects of nematode control. This will enhance the development, and subsequent uptake, of the new diagnostics, vaccines, pharma-/nutraceuticals, control methods and decision support tools required to respond to the spread of AR and the shifting epidemiology of GI nematodes in response to climatic, land-use and farm husbandry changes. More emphasis needs to be placed on the upfront evaluation of the economic value of these innovations as well as the socio-psychological aspects to prioritize research and facilitate uptake of innovations in practice. Finally, targeted regulatory guidance is needed to create an innovation-supportive environment for industries and to accelerate the access to market of new control tools.

Attitudes towards worm egg counts and targeted selective treatment against equine cyathostomins.

Gastrointestinal nematodes present a major threat to the health and welfare of equids worldwide. Anthelmintic resistance (AR) is increasingly reported and challenges effective control in horses and ponies in many regions. The use of faecal worm egg counts (FECs) to support targeted treatment (FEC-TT) and targeted selective treatment (FEC-TST) has been promoted as an effective deworming strategy that may prolong the useful life of anthelmintics and reduce the costs associated with parasite control. However, treatment applied at set intervals or on pre-determined dates remains common. A structural equation model was developed to identify factors influencing the uptake of FEC-directed treatment strategies, based on well-established socio-psychological theories of intentional health-related behaviours: the Theory of Planned Behaviour and the Health Belief Model. More than 850 valid responses were received from horse owners in the UK via an online survey. The intention to use FECs prior to deworming was not influenced by the perceived risk of anthelmintic resistance or that of gastrointestinal nematode infection but was positively influenced by a negative attitude towards anthelmintics, a positive attitude towards FECs, an increase in social pressure (e.g. significant others think the respondent should use FECs) and an increase in perceived control over their deworming programme. The results were consistent with a similar study conducted on FEC-TT in cattle in Belgium. An increase in respondents' self-perceived level of knowledge significantly increased the intention to use FECs via mediating factors. These results suggest that knowledge transfer activities aimed at increasing awareness and understanding of sustainable nematode control practices may be more effective at encouraging behaviour change than emphasising the dangers of nematodes and AR, which had limited influence on behaviour intention in this study population.

Progress in the development of subunit vaccines for gastrointestinal nematodes of ruminants.

The global increase in anthelmintic resistant nematodes of ruminants, together with consumer concerns about chemicals in food, necessitates the development of alternative methods of control for these pathogens. Subunit recombinant vaccines are ideally placed to fill this gap. Indeed, they are probably the only valid option for the long-term control of ruminant parasitic nematodes given the increasing ubiquity of multidrug resistance in a range of worm species across the world. The development of a subunit multicellular parasite vaccine to the point of practical application would be a groundbreaking step in the control of these important endemic infections of livestock. This review summarizes the current status of subunit vaccine development for a number of important gastrointestinal nematodes of cattle and sheep, with a focus on the limitations and problems encountered thus far, and suggestions as to how these hurdles might be overcome.

Diagnosis before treatment: Identifying dairy farmers' determinants for the adoption of sustainable practices in gastrointestinal nematode control.

Anthelmintic resistance is emerging in dairy cattle and this can result in a lack of effective control and production losses. Therefore, sustainable control strategies, such as targeted treatments (TT) and targeted selected treatments (TST), should be adopted by the industry. TT and TST approaches require the use of diagnostic methods to take informed treatment decisions. To understand the factors affecting the farmers' intention to adopt diagnostic methods before implementing anthelmintic drugs ('adoption intention'), a cross-sectional survey was carried out in dairy farms in Belgium (Flanders). A framework was constructed to predict adoption intentions based on two fundamental theories in the field of behavioural psychology and health psychology: the Theory of Planned Behaviour and the Health Belief Model. In the tested model, adoption intentions were predicted based on attitudes towards anthelminthics, attitudes towards diagnostic methods, subjective norms, behavioural control and perceived risk. Structural equation modelling was used for analyses. The model fitted the data well and explained 46% of the variance in adoption intention of diagnostics. The factors 'attitude towards diagnostic methods' and 'subjective norm'; i.e. the influence of significant others, had the strongest, positive influence on adoption intention of diagnostic methods. 'Perceived behavioural control' had a weak, positive effect on intention. Further, 'attitude towards the use of anthelmintic drugs' had a negative effect on adoption intention of the diagnostic methods. This implicates an effect of current behaviour on future adoption, which should be considered in future research. Factors measuring risk perception of anthelmintic resistance; perceived severity and perceived susceptibility, had no effect on the adoption intention of diagnostic methods. The threat of anthelmintic resistance is perceived to be low for dairy herds. The study further did not find any differences in the effects of the predictors for young stock and adult dairy cows. The results of this study can be used to develop communication strategies to advertise sustainable nematode control on dairy farms.

The occurrence and genetic characterization of Cryptosporidium and Giardia species in foals in Belgium, The Netherlands, Germany and Greece.

Faecal samples were collected from foals between the age of 1 week and 6 months in Belgium, The Netherlands, Germany and Greece. A quantitative direct immunofluorescence assay based on the commercial MERIFLUOR Cryptosporidium/Giardia kit was performed to evaluate the presence of (oo) cysts. Parasite positive samples were genotyped, based on the 18S ribosomal DNA gene and the heat shock protein (HSP70) gene for Cryptosporidium and on the ß-giardin gene and the triose phosphate isomerase (TPI) gene for Giardia. In total, 134 foals from Belgium, 44 foals from The Netherlands, 30 foals from Germany and 190 foals from Greece were examined. No Cryptosporidium oocysts were identified in faecal samples from foals in Germany and The Netherlands. In Belgium and Greece, 4.5% and 1.1% of the foals examined were Cryptosporidium positive, respectively, all with a low oocyst excretion ranging from 100 to 2450 oocysts per gram of faeces. For Giardia, 14.2%, 11.4%, 10.0% and 11.6% of the foals in Belgium, The Netherlands, Germany and Greece, respectively, were found to excrete cysts, with a range of 50 up to 4,000,000 cysts per gram of faeces. Younger animals secreted significantly more Giardia cysts than older horses (p<0.05), but no significant correlation between Giardia infection and diarrhoea was observed. Most Giardia positive samples belonged to assemblage AI and/or BIV, but also assemblage E was detected in two samples. Together with the identification of Cryptosporidium horse genotype, this suggests only a low risk for zoonotic transmission.

Analysis of the protective immune response following intramuscular vaccination of calves against the intestinal parasite Cooperia oncophora.

Recently we reported the successful vaccination of calves against Cooperia oncophora with a double domain activation-associated secreted protein, purified from the excretory-secretory material of adult stage parasites. In an attempt to elucidate the immune mechanisms involved in protection, the humoral and cell-mediated immune responses following vaccination and infection were compared with non-vaccinated control animals. Antigen-specific IgG1, IgG2 and IgA levels were significantly increased in sera of vaccinated animals post vaccination, whereas no effect was observed for IgM. Antigen-specific intestinal IgG1 levels were significantly increased in the vaccinated animals, whereas no differences were observed for antigen-specific IgA, IgM and IgG2 levels. Upon re-stimulation in vitro with the vaccine antigen, a significant proliferation of both αß- and γδ-T cells, and B cells, collected from mesenteric lymph nodes, was only observed in vaccinated animals. RNA-seq analysis of intestinal tissue yielded a list of 67 genes that were differentially expressed in vaccinated animals following challenge infection, amongst which were several cell adhesion molecules, lectins and glycosyl transferases. A correlation analysis between all immunological and parasitological parameters indicated that intestinal anti-double domain activation-associated secreted protein IgG1 levels correlated negatively with cumulative faecal egg counts and positively with the proportion of L4s and L5s. The proportion of immature stages was also positively correlated with the proliferation of αß T cells. Worm length was negatively correlated with the transcript levels of several lectins and cell adhesion molecules. Overall, the results indicate that intramuscular administration of the vaccine resulted in an immune memory response particularly characterised by increased antigen-specific IgG1 levels in the intestinal mucosa.