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Foodborne outbreaks of cryptosporidiosis are uncommon. In Denmark human cases are generally infrequently diagnosed. In 2005 an outbreak of diarrhoea affected company employees near Copenhagen. In all 99 employees were reported ill; 13 were positive for Cryptosporidium hominis infection. Two analytical epidemiological studies were performed; an initial case-control study followed by a cohort study using an electronic questionnaire. Disease was associated with eating from the canteen salad bar on one, possibly two, specific weekdays [relative risk 4.1, 95% confidence interval (CI) 2.1-8.3]. Three separate salad bar ingredients were found to be likely sources: peeled whole carrots served in a bowl of water, grated carrots, and red peppers (in multivariate analysis, whole carrots: OR 2.1, 95% CI 1.1-4.0; grated carrots: OR 2.1, 95% CI 1.2-3.9; peppers: OR 3.3, 95% CI 1.7-6.6). We speculate that a person excreting the parasite may have contaminated the salad buffet. [ABSTRACT FROM AUTHOR]

Cryptosporidium spp. are common causes of gastrointestinal disease in both humans and animals. This was a cross-sectional study conducted to determine the infection rate and genetic characteristics of Cryptosporidium infecting dogs in Jordan. A total of 249 faecal samples were collected from stray, pet, and breeding dogs from kennels (independent of their clinical condition) across three governorates in Jordan (Amman and Zarqa in Central Jordan and Irbid in Northern Jordan). Faecal samples were screened for Cryptosporidium using polymerase chain reaction (PCR) targeting the 18S rRNA gene, revealing an overall infection rate of 18.9% (47 out of 249). Cryptosporidiosis was significantly associated with indoor dogs, dogs cohabiting with other animals, and consuming raw food. Among the successfully sequenced samples, 25 (58.1%) were Cryptosporidium canis, 15 (34.9%) were Cryptosporidium parvum, and three (7.0%) were Cryptosporidium baileyi. Multiple diversity tests were employed, indicating low genetic differentiation between the studied populations of C. parvum and C. canis. Stability was observed for C. parvum, with minimal expansion observed for C. canis. Notably, each species exhibited a single dominant haplotype, consistent with the AMOVA results, where most of the variability occurred within populations. Further genotyping of C. parvum and C. canis was conducted by sequencing the gp60 gene. C. parvum isolates worldwide displayed solely the zoonotic IId genotypes, namely, IIdA20G1, IIdA22G1, IIdA18G1, and IIdA19G1. In contrast, the C. canis isolates exhibited the animal subtypes XXe and XXd. Consequently, dogs may serve as a source of infection with C. parvum and pose a public health risk in Jordan., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Mukbel et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Plant secondary metabolites (PSMs) may improve gastrointestinal health by exerting immunomodulatory, anti-inflammatory and/or antiparasitic effects. Bark extracts from coniferous tree species have previously been shown to reduce the burden of a range of parasite species in the gastrointestinal tract, with condensed tannins as the potential active compounds. In the present study, the impact of an acetone extract of pine bark ( Pinus sylvestris ) on the resistance, performance and tolerance of genetically diverse mice ( Mus musculus ) was assessed. Mice able to clear an infection quickly (fast responders, BALB/c) or slowly (slow responders, C57BL/6) were infected orally with 200 infective third-stage larvae (L 3 ) of the parasitic nematode Heligmosomoides bakeri or remained uninfected (dosed with water only). Each infection group of mice was gavaged for 3 consecutive days from day 19 post-infection with either bark extract or dimethyl sulphoxide (5%) as vehicle control. Oral administration of pine bark extract did not have an impact on any of the measured parasitological parameter. It did, however, have a positive impact on the performance of infected, slow-responder mice, through an increase in body weight (BW) and carcase weight and reduced feed intake by BW ratio. Importantly, bark extract administration had a negative impact on the fast responders, by reducing their ability to mediate the impact of parasitism through reducing their performance and tolerance. The results indicate that the impact of PSMs on parasitized hosts is affected by host's genetic susceptibility, with susceptible hosts benefiting more from bark extract administration compared to resistant ones.

The antiparasitic potential of plants could offer a vital solution to alleviating the costs of gastrointestinal nematode (GIN) infections in ruminant production globally. Leveraging known bioactive molecules, however, is complex, where plant species, extraction processes and seasonality impact bioavailability and efficacy. This study assessed the impact of a comprehensive set of factors on the antiparasitic activity of Norwegian conifers to identify bark compounds specific against GIN. Antiparasitic activity was determined using in vitro assays targeting morphologically distinct life stages of ovine GIN: the egg hatch assay and larval motility assay. In depth characterisation of the chemical composition of the bark extracts was carried out using chromatographic separation, UV-absorbance, and molecular mass profiles to identify compounds implicated in the activity. Three key findings emerged: (1) the activity of bark extracts varied markedly from 0 to 100% antiparasitic efficacy, owing to tree species, extraction solvent and seasonality; (2) the GIN exhibited species-and stage-specific susceptibility to the bark extracts; (3) the presence of condensed tannins, amongst other compounds, was associated with anthelmintic activity. These findings add new insights into urgently needed alternative parasite control strategies in livestock., (© 2023. Springer Nature Limited.)

Infections with Cryptosporidium spp. constitute a substantial public health burden and are responsible for widespread production losses in cattle herds. Reducing disease and shedding of Cryptosporidium spp. oocysts is an important One Health goal. There are very few therapeutic options available to treat cryptosporidiosis. Interest in plant bioactive compounds to mitigate the spread of anthelmintic resistance in ruminants has led to investigation of these phytocompounds against other parasitic taxa. Condensed tannins (CTs) are plant secondary metabolites that have shown potential against nematodes in vitro and in vivo but their applicability to Cryptosporidium spp. is comparatively under-explored. Cryptosporidium parvum infected human ileocecal colorectal adenocarcinoma (HCT)-8 cell cultures were treated with escalating doses of highly purified and well-characterized CTs from five plant species, big trefoil (Lotus pedunculatus), black currant (Ribes nigrum), sainfoin (Onobrychis viciifolia), white clover (Trifolium repens) and grapeseed (Vitis vinifera) for 44 h. Quantitative-PCR (qPCR) analysis revealed that none of the CTs examined demonstrated inhibitory potential against the parasite. Substantial inhibition of C. parvum by paromomycin was observed in positive controls in all assays (76.94-90.72% inhibition), proving the validity of the assay. Despite the lack of inhibition, these results represent an important step towards identifying alternative treatment options against this parasite., Competing Interests: Declaration of competing interest The authors declare they have no competing interests., (Copyright © 2022. Published by Elsevier Inc.)

Background: Eimeria spp. are widespread apicomplexan parasites known to cause coccidiosis in livestock, resulting in reduced animal welfare and productivity, particularly in sheep. The treatment options are limited, and there is an emerging development of resistance against registered pharmaceuticals. Spruce bark is rich in plant secondary metabolites (PSM), such as condensed tannins, which are bioactive compounds previously shown to have antiparasitic activity. Here, we examined the anticoccidial properties of bark extract of Norway spruce (Picea abies) against a field isolate of ovine Eimeria spp. by treating Eimeria-infected pre-ruminant lambs with water-extracted bark daily for 12 days. We hypothesised that the bark extract would reduce the faecal oocyst excretion and, consequently, the severity of diarrhoea., Results: Oral administration of spruce bark extract significantly reduced the excretion of Eimeria oocysts in milk-fed lambs post treatment till the end of the trial 22 days post infection. This difference in oocyst excretion between the treated and the untreated infected animals increased with time. Compared to the untreated and the sham-infected control group, the group treated with bark extract had softer faeces and reduced milk intake during the treatment period. After discontinuing the treatment, the treated animals got a more solid and formed faeces compared to that of the untreated control group, and the milk intake increased to the level of the sham-infected, untreated control group. The bark extract treated animals had a lower body weight and a lower mean daily body weight gain throughout the whole duration of the experiment., Conclusions: Bark extract from Norway spruce showed marked anticoccidial properties by reducing the faecal oocyst count and associated diarrhoea in young lambs. Simultaneously we experienced detrimental effects of the treatment, displayed as reduced feed intake and daily body weight gain. Therefore, we suggest conducting similar studies with lower bark extract dosage to explore the possibilities of a better trade-off to reduce the negative impact while maintaining the antiparasitic effect., (© 2022. The Author(s).)

Previous studies have illustrated that different bioactive legume fodders containing condensed tannins might represent one of the options for integrated sustainable control of gastrointestinal nematodes (GIN) in ruminants, which may help address the worldwide development of resistance to synthetic anthelmintics. More recently, impetus has been given to assess the potential antiparasitic activity of less conventional resources, represented by different agro-industrial by-products (AIBPs). This review presents in vitro and in vivo results obtained with a range of tannin-containing AIBPs of various geographical and botanical origins, namely AIBP of nuts, temperate and tropical barks, carob, coffee and cocoa. They tend to confirm the "proof of concept" for their antiparasitic effects and also for other aspects of ruminant production in an agro-ecological context. Socio-economic aspects of the exploitation of such non-conventional resources are also discussed as potential models of the circular economy, by using waste. The different modes of use of these resources are presented in this review, as well as strengths, weaknesses, opportunities, and threats (SWOT) analyses to illustrate the advantages and limitations of on-farm use., (© H. Hoste et al., published by EDP Sciences, 2022.)

The Eurasian otter (Lutra lutra) is a protected species in Denmark and at present, the population is recovering due to conservation efforts. The Danish otters are mainly found in the continental part of Denmark (Jutland), but establishment in the main islands (Fyn and Zealand) has been observed. While there is a lack of systematic studies on the parasite fauna of otters in Denmark, this study aims to screen otters for their parasite fauna, especially those of zoonotic and/or veterinary importance. Thirty-three otter carcasses, road-killed (n = 30), found dead (n = 2) and shot (n = 1), were collected between June 2013 and May 2014 and examined for cardiopulmonary, urogenital, gastrointestinal, and muscle helminths by post mortem examination. Faecal samples were analysed by modified concentration McMaster technique and direct immunofluorescence test for Giardia and Cryptosporidium. At least one parasite was found in 75.8% of animals. The parasite fauna included 13 species, consisting of five nematodes: Molineus patens (30.3%), Aonchotheca putorii (27.3%), Strongyloides sp. (24.2%), Physaloptera sp. (12.1%), Eucoleus aerophilus (10.0%); one cestode: Schistocephalus solidius (6.1%); four trematodes: Metorchis bilis (33.3%), Isthimiophora melis (15.2%), Cryptocotyle sp. (3.0%), Plagiorchis sp. (3.0%); one acanthocephalan: Acanthocephalus ranae (18.2%); and two protozoans: Giardia spp. (3.1%), and Eimeria spp. (3.1%). The study showed that otters carry parasites of zoonotic and veterinary importance. Many of these parasites can also infect native carnivores and birds, and the distribution of these parasites may be affected if the otter population continue to increase in Denmark., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The widespread apicomplexan parasite Cryptosporidium parvum is responsible for severe gastrointestinal disease in humans and animals. The treatment options are limited, and the efficacy of available drugs is low. Bark contains condensed tannins (CT), which are bioactive compounds previously shown to inhibit parasite development. Here, we examined the anti-cryptosporidial properties of bark extract of Scots pine (Pinus sylvestris) against C. parvum by means of an in vitro growth inhibition test. We hypothesised that bark extracts would have dose-dependent inhibitory effects on the development of C. parvum in cell culture.Bark extracts from Scots pine extracted with acetone, methanol, and water as solvents were investigated using human colorectal adenocarcinoma cells infected with C. parvum. Oocysts were inoculated onto the cell monolayer and bark extract was added at seven different concentrations. Parasite growth inhibition was quantified by qPCR.The acetone and methanol extracts demonstrated a sigmoid dose-dependent inhibition of C. parvum. The IC 50 values were 244.6 and 279.1 µg dry matter extract/mL, and 25.4 and 24.1 µg CT/mL, for acetone and methanol extracts, respectively. The IC 50 for both extracts were similar, both with regard to the dry matter concentration of each extract and to CT concentrations.Given the limited treatment options available for Cryptosporidium spp., the evidence generated in our study encourages further investigation into the in vitro and in vivo effects of pine bark extracts against C. parvum., (© 2021. The Author(s).)

We report a case of community acquired giardiasis, in Nuuk, Greenland. Likely source of infection being consumption of untreated water from a local reservoir, alternatively through contact with sewage. Giardia is widespread worldwide but has not commonly been considered a cause of gastrointestinal distress in patients in Greenland, without relevant travel history. This may be due to under diagnosis, or historically low prevalence of Giardia in the region. Climate change with increasing temperatures, growing tourism and pet travel may influence the presence of Giardia in the region. This case highlights the need to include giardiasis as a differential diagnosis in patients presenting with suspected infectious gastroenteritis in Greenland.

Aims: To determine inactivation of Cryptosporidium parvum oocysts and reduction of Escherichia coli and enterococci in cattle slurry added aqueous ammonia., Methods and Results: Escherichia coli, enterococci and nonviable C. parvum oocysts (DAPI+PI+) were enumerated every second day for 2 weeks in cattle slurry amended with 60 mmol l -1 aq. ammonia and compared with untreated slurry at three temperatures. Regardless of temperature, the proportion of nonviable C. parvum oocysts increased significantly faster over time in slurry with added ammonia than raw slurry (P = 0·021) corresponding to 62·0% higher inactivation (P = 0·001) at day 14. Additionally, 91·8% fewer E. coli and 27·3% fewer enterococci were observed in slurry added ammonia at day 14 compared to raw slurry., Conclusion: The addition of aqueous ammonia to raw slurry significantly reduced the viability of C. parvum oocysts and numbers of bacterial indicators. Hence, ammonia is usable at lower pathogen concentrations in slurry before application to agricultural land., Significance and Impact of the Study: Livestock waste is a valuable source of plant nutrients and organic matter, but may contain high concentrations of pathogens like E. coli and Cryptosporidium sp. that can be spread in the environment, and cause disease outbreaks. However, die-off rates of pathogens in organic waste can increase following increasing ammonia concentrations., (© 2020 The Society for Applied Microbiology.)

Fasciola hepatica is an important disease of livestock that is responsible for substantial economic losses worldwide. Estimates of the impact of infection on milk yield vary, likely reflecting different geographical locations, farm-level management, and diagnostic methods. Measuring anti-Fasciola antibodies on bulk tank milk (BTM) by ELISA provides a convenient herd-level diagnosis, but the utility of this test remains unclear. Therefore, we evaluated the utility of BTM ELISA test results in Danish organic dairy farms, including estimating the association between 305 day energy corrected milk yield (305d ECM) and F. hepatica infection both at individual and herd level. BTM samples from 218 organic farms were analysed using IDEXX ELISA and subsequently the farmers were interviewed during spring 2016 with the aim of characterising their management practices. The corresponding farm-level production data covering the period 2014-2017 were collected from the Danish national cattle registry. In the following year, 284 individual milk samples (4-7 per herd) along with BTM samples were collected from a subset of the same herds (n = 55). Linear mixed models were used to estimate the association between milk production and ELISA value at both individual and farm levels, and a generalised additive model was used to assess the relationship between within-herd prevalence and BTM ELISA. A dichotomised BTM result with positive outcome was associated with a reduction of 580.5 kg in average 305d ECM, and a positive outcome on individual-level ELISA was associated with a 919.5 kg reduction in milk yield for cows in their third or later lactations. A strong relationship between quantitative BTM ELISA sample to positive percentage (S/P%) and apparent within-herd prevalence based on dichotomised individual-level milk ELISA was also observed, although this relationship was non-linear in nature. We conclude that a useful indication of the within-herd prevalence of infection can be obtained from BTM ELISA following categorisation as negative, low, medium or high according to S/P% cut-offs of approximately 30, 80, and 150. This approach represents a cheap and useful diagnostic tool for monitoring the long-term success of control strategies for F. hepatica infections on a dairy farm., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Similar to the situation in other European countries, Danish wild boars may harbour a wide range of pathogens infectious to humans and domestic pigs. Although wild boars must be kept behind fences in Denmark, hunting and consumption of the meat may cause zoonotic transmission. Moreover, most infections of wild boars are transmissible to domestic pigs, which may have important economic consequences. The aim of this study was to investigate whether Danish wild boars were infected with bacteria and parasites transmissible to humans or domestic pigs: Brucella suis, methicillin-resistant Staphylococcus aureus (MRSA), Salmonella spp., Trichinella spp., lungworms and gastrointestinal parasites, especially Ascaris suum. This is the first study to investigate the prevalence of these important pathogens in Danish wild boars., Results: Wild boars from eight enclosures were analysed over a 5-year period. All tested wild boars were negative for B. suis (n = 240), MRSA (n = 244), Salmonella spp. (n = 115) and Trichinella spp. (n = 232), while eight parasite genera were identified in the faeces (n = 254): Ascaris suum, Capillaria sp., Cystoisospora suis, Eimeria spp., Metastrongylus sp. (lungworm), Strongyloides ransomi, Trichuris suis and strongylid eggs, i.e. strongyles not identified to the genera. Eimeria spp. and Metastrongylus sp. had the highest prevalence (92.3 and 79.5%, respectively) and were identified in wild boars from all eight enclosures, while the remaining parasite genera were present more sporadically., Conclusions: Wild boars from Denmark constitute a low risk of transmitting B. suis, MRSA, Salmonella spp. and Trichinella spp. to humans or domestic pigs, while economically important parasites transmissible to domestic pigs are highly prevalent in the wild boar population.

Giardia and Cryptosporidium infections are common in cats, but knowledge is limited about their clinical importance, risk factors, and the role of cats as a reservoir for human infections. Here, we collected faeces and questionnaire data from 284 cats from shelters and veterinary clinics in the Copenhagen Metropolitan Region (= study population). Additionally, 33 samples were analysed separately from catteries with gastrointestinal clinical signs (= cases). (Oo-)cysts were quantified by immunofluorescence microscopy. All Giardia (n = 34) and Cryptosporidium (n = 29) positive samples were analysed by sequencing of the 18S rRNA, gdh and hsp70 loci, and co-infections were detected by McMaster/inverted microscopy. In the study population, 7.0% and 6.7% were positive for Giardia and Cryptosporidium respectively; 48.5% and 36.4% of the breeder cats (cases) were infected. Increased odds of diarrhoea were demonstrated in Giardia (p = 0.0008) and Cryptosporidium (p = 0.034) positive cats. For Giardia, the odds were positively correlated with infection intensity. Co-infection with Cryptosporidium (OR 12.79; p < 0.001), parasitic co-infections other than Cryptosporidium (OR 5.22; p = 0.009), no deworming (OR 4.67; p = 0.035), and male sex (OR 3.63; p = 0.025) were risk factors for Giardia. For Cryptosporidium, co-infection with Giardia was the only risk factor (OR 11.93; p < 0.0001). Genotyping revealed G. duodenalis assemblages A and F, and C. felis, all of them previously detected in humans. In conclusion, excretion of Giardia and Cryptosporidium was associated with clinical disease. Although a public health risk is likely, studies including larger sample sizes, more discriminatory markers and samples from other animals and humans are needed to reveal the full zoonotic potential.

Background: A substantial proportion of echinococcosis transmission to humans via contamination of food has been assumed. However, the relative importance of food as a transmission vehicle has previously been estimated through expert opinion rather than empirical data., Objective: To find and evaluate empirical data that could be used to estimate the source attribution of echinococcosis, in particular the proportion that is transmitted through contaminated food., Methods: A systematic review was undertaken to identify reports on the risk factors for human cystic (CE) and alveolar (AE) echinococcosis. Data bases searched included PubMed, Scopus, Web of Knowledge, Cab Direct, Science Direct, Google Scholar, eLIBRARY.RU, CyberLeninka, CNKI and VIP. Search terms included Echinococc*, hydatid, epidemiology, logistic regression, risk factors, odds ratio, relative risk, risk factors. Reports, including grey literature where available, that had suitable data were selected and data were extracted. The main pathways of transmission were hypothesised to be contact with the definitive host, contaminated water, contaminated food and contaminated environment (other than food). For each study the attributable fraction for these potential sources of infection was calculated from the data presented. A meta-analysis was then undertaken to obtain pooled estimates for the relative contribution of these transmission pathways., Results: Data from 28 cross-sectional studies and 14 case-control studies were extracted. There was strong evidence for transmission by direct contact with dogs for both CE and AE. The estimated attributable fractions were 26.1% (CI 13.8%-39.6%) and 34.4% (CI 20.7% -48.2%) respectively. Transmission through contaminated water was estimated to be responsible for approximately 29.4% (CI 12.1%-51.7%) for CE and 24.8% (CI 10.6% to 42.6%) for AE. Contaminated food may be responsible for approximately 23.4% of CE cases (CI 2.1%-47.3%). Globally, there was insufficient evidence to conclude AE can be transmitted by food, although case control studies from low human incidence areas suggested that possibly 32.5% (CI 10.0%-53.2%) could be transmitted by food. There was also insufficient evidence that direct contact with foxes was a significant source of human disease. There were no suitable studies with a risk of environmental contact reported, but the residual attributable fraction thatwould likely include this pathway was approximately 21.1% for CE and 11.1% for AE., Conclusions: The results support the hypothesis that dog contact and drinking contaminated water are major pathways of transmission of both CE and AE. For contaminated food, the results are less consistent, but suggest that it is an important transmission pathway and provide better evidence than expert elicitations as previously used., Competing Interests: The authors have declared that no competing interests exist.

Background: Feline cryptosporidiosis is an increasing problem, especially in catteries. In humans, close contact with cats could be a potential source of infection although the risk of contracting cryptosporidiosis caused by Cryptosporidium felis is considered to be relatively low. Sequencing of the 60-kDa glycoprotein gene is a commonly used tool for investigation of the genetic diversity and transmission dynamics of Cryptosporidium species. However, until now the sequence of gp60 from C. felis has not been available and genotyping has been limited to less discriminatory markers, such as 18S rRNA, COWP and HSP70., Methods: We have identified the gp60 orthologue within the genome sequence of C. felis, and used the sequence to design a nested PCR for subtyping purposes. A total of 128 clinical isolates of both feline and human origin, were used to evaluate the marker., Results: Sequence analysis revealed large variations between the different samples. The C. felis gp60 lack the characteristic serine-tract found in many other cryptosporidian orthologues, instead it has an insertion of variable length (361-742 nt). Also, two cases of suspected zoonotic transmission of C. felis between cats and humans were successfully confirmed., Conclusions: We have identified the gp60 gene in C. felis and show how this highly variable marker can be used in epidemiological investigations.

Cryptosporidium spp. are responsible for severe public health problems and livestock production losses. Treatment options are limited to only one drug available for human and bovine cryptosporidiosis, respectively, and both drugs exhibit only partial efficacy. Sesquiterpene lactones (SL) are plant bioactive compounds that function as a defence mechanism against herbivores. SL have demonstrated anti-parasitic properties against a range of parasitic taxa but knowledge about their anti-Cryptosporidium efficacy is limited. The effect of SL-rich leaf and root extracts from chicory (Cichorium intybus cv. Spadona) was investigated using human colon adenocarcinoma (HCT-8) cells infected with Cryptosporidium parvum. C. parvum oocysts were inoculated onto the cell monolayer and i) incubated for 4 hours with extracts (leaf and root extracts 300, 150, 75, 37.5, 18.75 and 9.375 μg/mL) in triplicates followed by incubation in bioactive free media (sporozoite invasion assays) or ii) incubated for 4 hours in bioactive free media followed by 48-hours incubation with extracts (growth inhibition assays). Extract toxicity on HCT-8 cells was assessed via water-soluble tetrazolium (WST)-1 assay prior to quantifying parasitic growth via immunofluorescence. Both extracts demonstrated dose-dependent inhibition in the growth inhibition assays (p = < 0.0001 for both extracts) but not in the invasion assays. Anti-parasitic activity did not appear to be solely related to SL content, with the extract with lower SL content (leaf) exhibiting higher inhibition at 300 μg/ml. However, given the limited treatment options available for Cryptosporidium spp., our study encourages further investigation into the use of chicory extracts to identify novel active compound(s) inhibiting these protozoa.

Background: Although more modern methods are available, quantitative PCR (qPCR) is reproducible, sensitive and specific with instruments and expertise readily available in many laboratories. As such, the use of qPCR in Cryptosporidium research is well established and still widely used by researchers globally. This method depends upon the generation of standards at different concentrations to generate standard curves subsequently used for the quantification of DNA., Methods: We assessed four types of DNA template used to generate standard curves in drug screening studies involving Cryptosporidium spp.: (i) serially diluted Cryptosporidium parvum oocysts (10 6 -1); (ii) diluted template DNA from pure oocysts (×10-×10 6 dilution of 10 6 oocyst DNA template); (iii) oocysts incubated in human ileocecal adenocarcinoma (HCT-8) cells (10 5 -1 and 5 × 10 4 -50); and (iv) diluted DNA template (5 × 10 4 ) from cell culture incubated parasites (×10-×1000)., Results: Serial dilutions of both cell culture and pure oocyst suspension DNA template yielded better linearity than cell culture derived standards, with dilutions of 10 6 oocysts exhibiting similar quantification cycle (Cq) values to those obtained from DNA template dilutions of 10 6 oocysts. In contrast, cell culture incubated oocysts demonstrated significantly higher DNA content than equivalent freely suspended oocysts and diluted DNA template from both cell culture derived and freely suspended oocysts across numerous concentrations., Conclusions: For many studies involving Cryptosporidium, only relative DNA content is required and as such, the superior linearity afforded by freely suspended oocysts and diluted DNA template (from either cell culture derived standards or freely suspended oocysts) will allow for more accurate relative quantification in each assay. Parasite division in the cell culture standards likely explains the higher DNA content found. These standards, therefore, have the potential to more accurately reflect DNA content in cell culture assays, and despite more modern methods available for absolute quantification, i.e. droplet digital PCR (ddPCR), the ubiquity of qPCR for the foreseeable future encourages further investigation into the reduced linearity observed in these standards such as varying oocyst seeding density, non-linear growth rates and assay efficiency.

Ovine Eimeria spp. infections cause increased mortality, reduced welfare and substantial economic losses, and anticocccidials are important for their control. Recent reports of anticoccidial resistance against ovine Eimeria spp. necessitate the development of in vitro methods for the detection of reduced anticoccidial efficacy, especially since the in vivo methods are both expensive, time consuming and requires the use of otherwise healthy animals. The aim of the present study was therefore to approach a preliminary standardization of in vitro assays for evaluation of the efficacy of the most commonly used anticoccidials in ruminants. For this purpose, apart from the evaluation of inhibition of oocyst sporulation, most effort was concentrated on assessment of the capacity of the different anticoccidials to inhibit both the invasion and further development (up to the first schizogony) of E. ninakohlyakimovae sporozoites in bovine colonic epithelial cells (BCEC). For this purpose, infected cultures were monitored 1, 8 and 15 days post infection to determine the infection rate, number of immature schizonts and number, size and appearance of mature schizonts, respectively. No clear inhibitory effect was found with any of the anticoccidial formulations tested, and we could not identify why there were no measurable effects from the different anticoccidials. Despite the lack of positive results, further investigations should be encouraged, as this could decrease the need for animal experiments and could be used in the initial assessment of anticoccidial efficacy of new drugs., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Strongylus vulgaris is considered the most pathogenic nematode parasite of the horse. Frequent deworming programs since the 1970s have reduced the prevalence of S. vulgaris to low levels, but to the price of widespread benzimidazole resistance in the small strongyles (cyathostominae) and ivermectin resistance especially in the equine roundworm Parascaris spp. To slow down the progression of anthelmintic resistance in Sweden, horse anthelmintics were made prescription only medicine in 2007 and selective therapy principles were introduced. This means that only individuals with high egg excretion or clinical signs of helminth infection were treated instead of blanket treatment of all horses on a farm. The aims of this study were to investigate the prevalence and risk factors associated with S. vulgaris infection in Sweden ten years after the introduction of a selective therapy regime. A total of 529 faecal samples from 106 farms were collected during March to June in 2016 and 2017. A web-based questionnaire was used to collect information about deworming routines. Strongyle faecal egg counts (FEC) were performed by McMaster and presence of S. vulgaris was demonstrated with a specific PCR on individual larval cultures. Results were analysed by mixed logistic ( S. vulgaris prevalence) or linear (FEC) regression models, where farm was included as random factor. The overall prevalence of S. vulgaris at individual and farm levels was 28% and 61%, respectively. We observed a 2.9 increased odds risk of S. vulgaris infection on farms that based their treatment on strongyle FEC only as compared to farms that complemented strongyle FECs with larval cultures or dewormed regularly, 1-4 times per year, without prior diagnosis. We found no association between the prevalence of S. vulgaris and strongyle FEC level, horse age, geographical region or signs of colic. The prevalence of S. vulgaris was 25% in horses shedding ≤150 eggs per gram. Thus horses with low strongyle FECs that are left untreated could be an important source of S. vulgaris infection. This may be an important reason for the approximately three-fold increase in S. vulgaris prevalence since 1999 in Sweden. However, our combined results indicate that selective therapy based on a combination of strongyle FECs and larval cultivation was not associated with an increased risk of S. vulgaris infection. Still, S. vulgaris needs to be monitored continuously and should be taken into careful consideration when the treatment frequency is reduced., (© 2019 The Authors.)

Background: Toxoplasma gondii is an important foodborne zoonotic parasite. Meat of infected animals is presumed to constitute a major source of human infection and may be a driver of geographical variation in the prevalence of anti- T. gondii antibodies in humans, which is substantial in the Nordic-Baltic region in northern Europe. However, data on seroprevalence of T. gondii in different animal species used for human consumption are scattered., Methods: We conducted a systematic review of seroprevalence studies and meta-analysis to estimate the seroprevalence of T. gondii in five animal species that are raised or hunted for human consumption in the Nordic-Baltic region: domestic pigs ( Sus scrofa domesticus ), sheep ( Ovis aries ), cattle ( Bos taurus ), wild boars ( Sus scrofa ), and moose ( Alces alces ). We searched for studies that were conducted between January 1990 and June 2018, and reported in articles, theses, conference abstracts and proceedings, and manuscripts. Subgroup analyses were performed to identify variables influencing the seroprevalence., Findings: From a total of 271 studies identified in the systematic review, 32 were included in the meta-analysis. These comprised of 13 studies on domestic pigs, six on sheep, three on cattle, six on wild boars, and four on moose. The estimated pooled seroprevalence of T. gondii was 6% in domestic pigs (CI 95% : 3-10%), 23% in sheep (CI 95% : 12-36%), 7% in cattle (CI 95% : 1-21%), 33% in wild boars (CI 95% : 26-41%), and 16% in moose (CI 95% : 10-23%). High heterogeneity was observed in the seroprevalence data within each species. In all host species except wild boars, the pooled seroprevalence estimates were significantly higher in animals >1 year of age than in younger animals. Not all studies provided information on animal age, sensitivity and specificity of the serological method employed, and the cut-off values used for defining an animal seropositive., Conclusions: A substantial proportion of animals raised or hunted for human consumption in the region had tested positive for T. gondii . This indicates widespread exposure to T. gondii among animals raised or hunted for human consumption in the region. Large variations were observed in the seroprevalence estimates between the studies in the region; however, studies were too few to identify spatial patterns at country-level.

Echinococcus multilocularis is an endemic parasite of red foxes in several European countries. This parasite has been present for decades in central Europe i.e. Switzerland, Eastern France, Southern Germany and Austria, which constitute the core endemic area of Europe. In the Scandinavian countries Sweden and Denmark, several recent findings were made in foxes. To better understand the dynamics and geographic spread of E. multilocularis in Europe, genetic studies have been undertaken using the DNA microsatellite marker EmsB. In Europe, the parasite spread in hitherto non-endemic areas was suspected to take place after founder events, in which the core endemic area presents a wider genetic diversity in comparison to newly endemic areas. However, identical parasite profiles can be shared between them, highlighting the parasite spreading in a mainland-island system. In this study, Swedish (27 adult worms from seven red foxes) and Danish (38 adult worms from nine red foxes) isolates were examined using fragment size analyses of the tandemly repeated microsatellite EmsB in order to compare the genetic profiles of the Scandinavian worms with a reference collection of European worm isolates from seven countries. Six EmsB profiles were detected in the Scandinavian panel. Three profiles were described in Denmark and four in Sweden. Only one of these profiles was detected in both countries. All profiles identified in the present study have previously been found in other European countries, suggesting an epidemiological link. Due to the relatively low number of Scandinavian E. multilocularis isolates analysed so far, firm conclusions cannot be made regarding the true genetic diversity. Nevertheless, the low genetic variation detected in Sweden and Denmark in this study is similar to the values obtained from peripheral areas of the main European endemic focus, which were more recently colonized by E. multilocularis ; and continuous surveillance of this parasite is warranted to provide further insight into its epidemiology in Scandinavia., (© 2019 Norwegian Veterinary Institute. Published by Elsevier Inc. on behalf of International Association of Food and Waterborne Parasitology.)

Background: Bovine fasciolosis is an economically important livestock disease in Europe, and represents a particular challenge for organic farms, where cattle are grazed extensively and the use of anthelmintic is limited. A two-year longitudinal study was conducted on two conventional and two organic Danish dairy farms to examine the current temporal trend of F. hepatica infection on-farm, and to gather data of practical relevance for parasite control. Data were collected both at the herd and individual level using currently available diagnostic methods: a commercial serum antibody ELISA, a commercial copro-antigen ELISA, faecal egg counts, and monthly bulk tank milk (BTM) ELISA. The temporal patterns (animal age, farm-level temporal trends and seasonality) in the animal-level test results were analysed by generalised additive mixed models (GAMM)., Results: Patterns of infection differed substantially between the farms, due to different grazing management and anthelmintic use. However, animals were first infected at the age of 1.5-2 years (heifers), and most at-risk animals sero-converted in autumn, suggesting that summer infections in snails prevail in Denmark. Our results also suggest that the lifespan of the parasite could be over 2 years, as several cows showed signs of low grade infection even after several years of continuous indoor housing without access to freshly-cut grass. The serum antibody ELISA was able to detect infection first, whereas both copro-antigen ELISA and faecal egg counts tended to increase in the same animals at a later point. Decreasing BTM antibody levels were seen on the two farms that started anthelmintic treatment during the study., Conclusions: While important differences between farms and over time were seen due to varying grazing management, anthelmintic treatment and climatic conditions, the young stock was consistently seen as the main high-risk group and at least one farm also had suspected transmission (re-infection) within the lactating herd. Careful interpretation of test results is necessary for older cows as they can show persistent infections several years after exposure has stopped. Rigorous treatment regimens can reduce BTM ELISA values, but further research is needed to develop a non-medicinal approach for sustainable management of bovine fasciolosis.

Background: Iron is an essential nutrient, and iron supplementation has been shown to reduce the incidence of abomasal bloat in lambs. Additionally, iron deficiency is linked to pica, which may increase uptake of Eimeria oocysts. Coccidiosis in sheep, caused by Eimeria spp., is an important infection, leading to reduced welfare and economic losses. The aims of our study were to investigate: (1) the use of iron supplementation in Norwegian sheep flocks using a questionnaire survey, and (2) whether iron supplementation reduced excretion of Eimeria oocysts and increased the growth rates of young lambs., Results: A questionnaire regarding the use of iron supplementation, sent to all members of the Norwegian Sheep Recording System (n = 4993), showed that 152/1823 farmers iron-supplemented lambs, either orally (56.7%) or by injection (43.3%). The main purpose of supplementation was to prevent abomasal bloat (38.4%), coccidiosis (9.3%), or both (27.8%). In the field study, 102 twin lambs from five flocks were included: one twin (treated) received 600 mg of gleptoferron subcutaneously within 3 days of birth, whereas the control was given saline. McMaster analysis of individual faecal samples obtained at weekly intervals (n = 4 per lamb, starting at turnout) showed no significant difference in oocyst excretion between treatment groups at any sampling, except for one flock 14 days after turnout. Mean growth rates, measured at iron injection, 21 days after turnout, and in the autumn, differed significantly between treated and untreated lambs from iron injection to 21 days after turnout, however, no difference in growth rates was observed in the overall period from iron injection to autumn. Blood analysis suggested that the controls were at risk of developing iron deficiency anaemia during the housed period, but signs of anaemia were not observed., Conclusion: Iron supplementation of lambs was used by 8.3% of the farmers responding to the questionnaire, mainly with the intention to prevent abomasal bloat, coccidiosis, or both. The field trial results indicate that iron supplementation of young lambs do not reduce oocyst excretion and only induced a transitory increase in weight gain. However further studies, including more flocks and possibly repeated iron injections, would provide more definitive information.

Increasing drug resistance in gastrointestinal (GI) parasites of livestock and concerns about chemical residues in animal products and the environment are driving the development of alternative control strategies that are less reliant on the use of synthetic drugs. An increasingly investigated approach is the use of bioactive forages with antiparasitic properties as part of the animal's diet (nutraceuticals) or as potential sources of novel, natural parasiticides. Chicory (Cichorium intybus) is a multi-purpose crop and one of the most promising bioactive forages in temperate regions, and numerous in vivo trials have explored its potential against parasitic nematodes in livestock. However, it is unclear whether chicory can induce a direct and broad activity against various GI parasites in different livestock species, and the levels of chicory in the diet that are required to exert an efficient antiparasitic effect. Moreover, the mechanisms leading to the reported parasiticidal activity of chicory are still largely unknown, and its bioactive phytochemicals have only recently been investigated. In this review, we summarise the progress in the study of the antiparasitic activity of chicory and its natural bioactive compounds against GI parasites in livestock, through examination of the published literature. The available evidence indicates that feeding chicory can reduce faecal egg counts and/or worm burdens of abomasal nematodes, but not infections with intestinal worms, in ruminants. Highly chicory-rich diets (≥ 70% of chicory dry matter in the diet) may be necessary to directly affect abomasal parasitism. Chicory is known to synthesise several bioactive compounds with potential antiparasitic activity, but most research has been devoted to the role of sesquiterpene lactones (SL). Recent in vitro studies have confirmed direct and potent activity of SL-rich extracts from chicory against different GI helminths of livestock. Chicory SL have also been reported to exhibit antimalarial properties and its potential antiprotozoal activity in livestock remains to be evaluated. Furthermore, the detailed identification of the main antiparasitic metabolites of chicory and their pharmacokinetics need further confirmation. Research gaps and perspectives on the potential use of chicory as a nutraceutical forage and a source of bioactive compounds for parasite control in livestock are discussed.

In Western Europe, the Echinococcus multilocularis lifecycle is predominantly sylvatic, typically involving red foxes (Vulpes vulpes) as the main definitive hosts with Microtus spp. and Arvicola spp. as intermediate hosts. During a 4-year surveillance study (2012-2015), Danish red foxes and raccoon dogs (n = 1345) were examined for E. multilocularis. Moreover, 134 insectivores and rodents collected in South Jutland during spring and summer 2016 were examined for the presence of metacestodes. The sedimentation and counting technique and molecular typing were used to identify E. multilocularis infections in the carnivores, while the rodent livers were examined macro- and microscopically for parasite lesions. Following morphological identification of E. multilocularis adult worms, the identity was verified by sequence analysis of the 12S rRNA gene in most cases (n = 13). Echinococcus multilocularis infection was demonstrated in 19 red foxes (Vulpes vulpes) originating from only two specific areas of South Jutland, namely Højer and Grindsted, and in two raccoon dogs (Nyctereutes procyonoides), originating from Højer. In Højer, 28.5% (CI 95% 11.7-45.3) of the examined red foxes were E. multilocularis positive per year. Moreover, positive red foxes were identified each year from 2012 to 2015, while E. multilocularis positive red foxes were only identified in Grindsted in 2013 (4.0%) and 2014 (6.4%). In contrast, all collected rodents were negative for E. multilocularis. We conclude that E. multilocularis is locally endemic in South Jutland with a high local prevalence in Højer.

Ovine Eimeria spp. infections cause reduced welfare, increased mortality, and substantial economic losses, and anticoccidials are crucial for their control. Recent reports of toltrazuril resistance in pigs, and anecdotal reports of reduced anticoccidial efficacy in lambs, necessitate evaluation of anticoccidial efficacy. Due to the substantial lifecycle differences between nematodes and coccidia, current WAAVP methods for assessing anthelmintic efficacy are not suitable for such evaluations. Faecal samples were collected from 8 pairs of twin lambs from 36 Norwegian sheep farms 6-8 days after turnout. One twin of each pair was then treated with 20 mg/kg toltrazuril and a second faecal sample from all lambs was collected 7-11 days later. Oocyst excretion rate in all samples was determined using McMasters. Suitability of treatment timing was investigated by evaluating the increase in mean log oocyst excretion in untreated lambs. Based on comparisons between groups, a threshold of ≥0.75 (13 farms) was used to identify farms where drug efficacy could be assessed with confidence, drug efficacy on farms with increases of ≥0.5 but <0.75 (7 farms) were evaluated with caution, and drug efficacy on farms with increases of <0.5 (16 farms) was not estimated. Reduction in oocyst excretion between samples from treated lambs compared with controls from the 20 farms with a threshold of ≥0.5 were then analysed using a generalised linear mixed model. The results were classified based on 95% CI obtained using parametric bootstrapping. Among these 20 farms, two exhibited reduced drug efficacy (upper 95% CI < 95%), 13 had good efficacy (lower 95% CI > 90%), and for 5 the results were inconclusive. This is the first evidence-based report of reduced anticoccidial efficacy in ovine Eimeria spp. Additionally, we highlight the problem of sub-optimal timing of treatment (16/36 farms), which could potentially result in incorrect conclusions being reached regarding lack of drug efficacy., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Coccidiosis due to Eimeria spp. infections in lambs causes increased mortality and substantial production losses, and anticoccidials are important for control of the infection. Anticoccidial resistance has been reported in poultry and swine, and we recently described reduced toltrazuril efficacy in ovine Eimeria spp. in some Norwegian sheep farms using a newly developed faecal oocyst count reduction test (FOCRT). The aim of the present study was to use a controlled efficacy trial to assess the efficacy of toltrazuril against a field isolate suspected of being resistant., Methods: Twenty lambs, 17-22 days old and raised protected against exposure to coccidia, were infected with a field isolate of 100,000 Eimeria spp. oocysts. This isolate was obtained from a farm with a previously calculated drug efficacy of 56% (95% confidence interval: -433.9 to 96.6%). At day 7 post-infection, 10 of the lambs were orally treated with 20 mg/kg toltrazuril (Baycox Sheep vet., Bayer Animal Health), while the other 10 lambs (controls) were given physiological saline. Clinical examinations were conducted, and weight gains recorded. Daily faecal samples were scored for diarrhoea on a scale from 1 to 5, and oocyst excretion was determined using a modified McMaster technique. Oocysts were morphologically identified to species level. At 17-24 days post-infection, the lambs were euthanized and necropsied., Results: The tested Eimeria isolate was resistant against toltrazuril, and resistance was seen in both pathogenic and non-pathogenic species. In addition, no significant differences in faecal score, growth, gross pathology or histological changes were identified between the two groups. The pathogenic E. ovinoidalis was the dominant species, and no significant difference in the individual prevalence of E. ovinoidalis post-treatment was found between treated (66.9%) and control lambs (61.9%). Other species identified included E. crandallis/weybridgensis, E. parva, E. marsica, E. faurei, E. pallida, E. ahsata and E. bakuensis., Conclusions: This study confirms toltrazuril resistance in ovine Eimeria spp.; in addition, the data support the use of FOCRT as an appropriate tool for field evaluation of anticoccidial efficacy. Due to limited anticoccidial treatment alternatives, these findings may have important implications for the sheep industry, particularly in northern Europe.

Viability assessment of Cryptosporidium parvum oocysts is crucial for evaluation of the public health significance of this important zoonotic protozoon. Viability is commonly assessed in wet mounts after acid pretreatment and staining with fluorogenic vital dyes. However, in some studies, oocyst viability is evaluated in dry mounts after staining in suspension. Here, we evaluate the effect of acid pretreatment in nine replicate samples and compare the assessment of oocyst viability after evaluation in wet and dry mounts, respectively. Although acid pretreatment had no significant effect on the viability scores, data obtained by scoring oocysts in dry mounts resulted in ∼25% underestimation of the proportion of viable oocyst (82.5% ± 0.9% [wet mount +acid], 57.7% ± 2.3% [dry mount, ÷ acid], 76.0% ± 1.7% [wet mount, ÷ acid]), while the proportions of nonviable oocysts (DAPI+/PI+) were comparable for wet and dry mounts (9.7% ± 0.4% [wet mount +acid], 12.1 ± 1.5% [dry mount, ÷ acid], 15.5% ± 1.1% [wet mount, ÷ acid]).

Background: The prevalence of bovine fasciolosis in Denmark is increasing but appropriate guidelines for control are currently lacking. In order to help develop a control strategy for liver fluke, a risk factor study of farm management factors was conducted and the utility of bulk tank milk (BTM ELISA) as a tool for diagnosis in Danish dairy cattle farms was assessed., Methods: This case-control study aimed to identify farm-level risk factors for fasciolosis in Danish dairy farms (> 50 animals slaughtered in 2013) using two diagnostic methods: recordings of liver condemnation at slaughter, and farm-level Fasciola hepatica antibody levels in BTM. A case farm was defined as having a minimum of 3 incidents of liver condemnation due to liver fluke at slaughter (in any age group) during 2013, and control farms were located within 10 km of at least one case farm and had no history of liver condemnation due to liver fluke during 2011-2013. The selected farmers were interviewed over telephone about grazing and control practices, and BTM from these farms was collected and analysed by ELISA in 2014. The final complete dataset consisting of 131 case and 63 control farms was analysed using logistic regression., Results: Heifers grazing on wet pastures, dry cows grazing on wet pastures, herd size, breed and concurrent beef cattle production were identified as risk factors associated with being classified as a case farm. With the categorised BTM ELISA result as the response variable, heifers grazing on wet pastures, dry cows grazing on wet pastures, and purchase of cows were identified as risk factors. Within the case and control groups, 74.8 and 12.7% of farms were positive for fasciolosis on BTM ELISA, respectively. The differences are likely to be related to the detection limit of the farm-level prevalence by the BTM ELISA test, time span between slaughter data and BTM, and the relatively low sensitivity of liver inspection at slaughter., Conclusions: Control of bovine fasciolosis in Denmark should target heifers and dry cows through grazing management and appropriate anthelmintic treatment, and BTM ELISA can be a useful diagnostic tool for fasciolosis in Danish dairy farms.

Chicory shows great promise as an anthelmintic forage for grazing ruminants that can reduce reliance on anti-parasitic drugs. Recently, we reported potent anthelmintic effects of chicory-based diets in infected cattle with significant reductions in worm burdens of the abomasal nematode Ostertagia ostertagi, whilst no apparent activity was observed against the small intestinal parasite Cooperia oncophora. To explore this discrepancy, we investigated direct anthelmintic effects of forage chicory against C. oncophora in vitro. Chicory leaves (cultivar 'Spadona') were extracted with methanol in a Soxhlet apparatus and the resulting extract was purified by solid-phase extraction to concentrate bioactive phytochemicals such as sesquiterpene lactones. C. oncophora eggs and adult worms from mono-infected donor calves were exposed to decreasing concentrations of the chicory extract. In an egg hatch assay, the chicory extract induced a marked and dose-dependent inhibition of egg hatching, with 95% inhibition at 2500μg extract/mL (EC 50 =619 [95% CI: 530-722] μg extract/mL). In the adult motility inhibition assays, the chicory extract induced a potent and dose-dependent worm paralysis. At 12h of incubation, worms exposed to chicory showed a total paralysis at ≥500μg extract/mL, while after 48h of incubation a complete inhibition of worm motility was observed at ≥250μg extract/mL (EC 50 =80 [95% CI: 67-95] μg extract/mL). We have demonstrated that forage chicory can induce potent inhibitory effects on the egg hatching and exert direct anthelmintic activity against parasitic stages of C. oncophora. These results suggest that the previously reported absence of in vivo effects of chicory towards C. oncophora in infected animals may be related with host-mediated factors and/or inhibitory digestive conditions, rather than an inherent inactivity of chicory and its bioactive phytochemicals., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The objectives of this study were to investigate the use of anticoccidials in Norwegian sheep flocks and identify farms with management procedures likely to select for drug resistance. Data were obtained by a questionnaire sent to all members of the Norwegian Sheep Recording System in October 2015. The data set consisted of 1215 answers, corresponding to 8.5% of Norwegian sheep flocks. Anticoccidials were used in 82.7% of flocks. Main treatment was at turnout (38.6% of treated flocks) or 1 week after turnout (32.4%). Interestingly, clinical signs possibly related to coccidiosis were observed by almost 40% of the farmers after treatment, which might be an indication of drug resistance. Correlations between the apparently reduced anticoccidial efficacy and management conditions, such as the size of the farms, were found. From the farmers' perspective, metaphylactic treatment was used in 88.5% of treated flocks, of which approximately one third had no history of clinical coccidiosis. Even though farmers seem aware of the importance of good drenching routines based on reliable estimates of weights and calibration of drench guns, drench gun used for anticoccidial administration was never calibrated in 12.1% of the flocks. Finally, dose estimation was made by visual appraisal in 27.5% of the flocks, which can lead to incorrect dosing. Based on the present study, it cannot be determined whether the apparent treatment failure was related to management practises, incorrect administration of the drug, other infections or actual anticoccidial drug resistance.

Condensed tannins' (CTs) fate along the digestive tract of ruminants may account for the variable efficacy of CTs against gastrointestinal nematodes. We analyzed CTs in the digesta of cattle fed sainfoin. With the acetone-butanol-HCl assay, the total CTs concentrations in the digesta were close to those in the diets (6.3 and 1.5% of DM in experiments 1 and 2, respectively); thus, CTs remained potentially largely undegraded/unabsorbed. With the thiolysis assay, CTs concentration was much higher in the abomasum (2.3% of DM; expt 1) compared with the rumen and intestines, along with higher mean size and prodelphinidins percentage, corroborating CTs efficacy reported only against Ostertagia ostertagi in the abomasum. In expt 2, the dietary levels of CTs were probably too low to demonstrate anthelmintic effects in the rumen. Overall, the level of CTs accessible to thiolysis is favored under the acidic conditions of the abomasum, which seems critical for anthelmintic activity.

Setaria tundra is a mosquito-borne filarial nematode of cervids in Europe. It has recently been associated with an emerging epidemic disease causing severe morbidity and mortality in reindeer and moose in Finland. Here, we present the first report of S. tundra in six roe deer ( Capreolus capreolus ) collected between October 2010 and March 2014 in Denmark. The deer originated from various localities across the country: the eastern part of the Jutland peninsular and four locations on the island Zealand. With the exception of one deer, with parasites residing in a transparent cyst just under the liver capsule, worms (ranging from 2 to >20/deer) were found free in the peritoneal cavity. The worms were identified as S. tundra by morphological examination and/or molecular typing of the mitochondrial 12S rRNA and cox1 genes, which showed 99.1-99.8% identity to previously published S. tundra isolates from Europe. Roe deer are generally considered as asymptomatic carriers and their numbers in Denmark have increased significantly in recent decades. In light of climatic changes which result in warmer, more humid weather in Scandinavia greater numbers of mosquitoes and, especially, improved conditions for development of parasite larvae in the mosquito vectors are expected, which may lead to increasing prevalence of S. tundra . Monitoring of this vector-borne parasite may thus be needed in order to enhance the knowledge of factors promoting its expansion and prevalence as well as predicting disease outbreaks.

The efficacy of ivermectin (IVM) against gastrointestinal nematodes in Danish cattle was assessed by faecal egg count reduction test (FECRT). Six cattle farms with history of clinical parasitism and avermectin use were included. On the day of treatment (Day 0), 20 naturally infected calves per farm (total n = 120) were stratified by initial faecal egg counts (FEC) and randomly allocated to a treatment group dosed with 0.2 mg IVM kg -1 body weight s.c. (IVM; n = 10) or an untreated control group (CTL; n = 10). Individual FEC were obtained at Day 0 and Day 14 post-treatment and pooled faeces by group were cultured to isolate L3 for detection of Ostertagia ostertagi and Cooperia oncophora by qPCR. Treatment efficacies were analysed using the recommended WAAVP method and two open-source statistical procedures based on Bayesian modelling: 'eggCounts' and 'Bayescount'. A simulation study evaluated the performance of the different procedures to correctly identify FEC reduction percentages of simulated bovine FEC data representing the observed real data. In the FECRT, reduced IVM efficacy was detected in three farms by all procedures using data from treated animals only, and in one farm according to the procedures including data from treated and untreated cattle. Post-treatment, O. ostertagi and C. oncophora L3 were detected by qPCR in faeces of treated animals from one and three herds with declared reduced IVM efficacy, respectively. Based on the simulation study, all methods showed a reduced performance when FEC aggregation increased post-treatment and suggested that a treatment group of 10 animals is insufficient for the FECRT in cattle. This is the first report of reduced anthelmintic efficacy in Danish cattle and warrants the implementation of larger surveys. Advantages and caveats regarding the use of Bayesian modelling and the relevance of including untreated cattle in the FECRT are discussed., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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

Background: The Internal Transcribed Spacer 2 (ITS2) is a candidate diagnostic marker of the pathogenic cattle nematode Ostertagia ostertagi. The aims of this study were: (i) to document and quantify how the development of O. ostertagi eggs affects ITS2 copies under different storage conditions, and (ii) to suggest optimal storage conditions for faecal samples in a diagnostic pipeline that involves detection and semi-quantification by real-time semi-quantitative polymerase chain reaction (qPCR)., Findings: Eggs of Ostertagia ostertagi were obtained from fresh faeces and stored at 4 °C or 25 °C under aerobic or anaerobic (vacuum packing) conditions. Development was monitored by microscopy for up to 336 h, and the ITS2 copies were determined by qPCR from a fixed number of parasites. Under aerobic conditions at 25 °C, embryonation and a significant increase of ITS2 copies (P < 0.0001) were observed after 12 h. At 4 °C, embryonation occurred after 168 h with a trend towards increased ITS2 copies. Anaerobic conditions inhibited egg development at both temperatures and no significant increase in ITS2 copies was noticed (P = 0.90). ITS2 copies were analysed for each parasite stage: first-stage larvae (L1) exhibited significantly higher copy numbers (20,353 ± 1,950) than unembryonated eggs (568 ± 168; P < 0.0001) with lower coefficient of variation (33 vs 266 %)., Conclusions: Aerobic storage of O. ostertagi eggs at 25 °C led to a significant increase in ITS2 copies after 12 h due to embryonation and subsequent hatching. In contrast, anaerobic storage (vacuum packing) at 25 °C completely inhibited egg development and any undesirable semi-quantification bias for up to 336 h. Hence, vacuum packing is an optimal storage strategy prior to molecular diagnostic analyses. Alternatively, aerobic storage at 4 °C for up to 72 h can be used. Due to high copy numbers and lower genetic variation, the L1 stage may be considered for diagnostics and further molecular research.

Background: Increasing anthelmintic-resistance in nematodes of ruminants emphasises the need for sustainable parasite control. Condensed tannin-containing legume forages such as sainfoin (Onobrychis viciifolia) have shown promising anthelmintic properties in small ruminants but this has never been explored in cattle. Therefore, our aim was to examine the efficacy of sainfoin against cattle nematodes in vivo., Methods: Fifteen Jersey male calves (2-4 month-old) were allocated into two groups and fed isoproteic and isoenergetic diets mainly composed of sainfoin pellets (Group SF; n = 9, three pens) or concentrate and grass-clover hay (Group CO; n = 6, two pens). After 16 days of adaptation, all animals were experimentally infected with 10,000 and 66,000 third-stage larvae of Ostertagia ostertagi and Cooperia oncophora, respectively. Egg excretion, blood parameters and bodyweights were recorded throughout the study. Worms were harvested by sieving for quantification and scanning electron microscopy (SEM) 42 days post-infection (dpi) when the calves were necropsied., Results: The number of O. ostertagi adults in the abomasum was reduced by 50 % in Group SF compared with Group CO (P < 0.05). This was further reflected in higher albumin (P < 0.1) and lower pepsinogen levels (P < 0.05) in Group SF at 21 dpi, and structural damage of the worm cuticle could be visualised by SEM. Yet, the nematode egg excretion in Group SF was not significantly different from that of the controls (P > 0.05). Likewise, no statistical difference in total worm burdens of C. oncophora was found between the groups. Weight gains were lower for Group SF (P < 0.05), which may reflect lower digestibility and phosphorus levels in the SF diet, despite similar feed intake at pen-level., Conclusions: Overall, the effect of sainfoin on abomasal nematodes corroborates results from studies with small ruminants and encourages further investigations of the use of this crop for control of cattle nematodes.

Chicory is a perennial crop that has been investigated as a forage source for outdoor-reared ruminants and pigs, and has been reported to have anthelmintic properties. Here, we investigated in vitro anthelmintic effects of forage chicory-extracts against the highly prevalent swine parasites Ascaris suum and Oesophagostomum dentatum. Methanol extracts were prepared and purified from two different cultivars of chicory (Spadona and Puna II). Marked differences were observed between the anthelmintic activity of extracts from the two cultivars. Spadona extracts had potent activity against A. suum third (L3) and fourth (L4) - stage larvae, as well as O. dentatum L4 and adults, whereas Puna II extracts had less activity against A. suum and no activity towards O. dentatum L4. Transmission-electron microscopy of A. suum L4 exposed to Spadona extracts revealed only subtle changes, perhaps indicative of a specific anthelmintic effect rather than generalized toxicity. Ultra-high liquid chromatography-mass spectrometry analysis revealed that the purified extracts were rich in sesquiterpene lactones (SL), and that the SL profile differed significantly between cultivars. This is the first report of anthelmintic activity of forage chicory towards swine nematodes. Our results indicate a significant anthelmintic effect, which may possibly be related to SL composition.

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.

Halicephalobus gingivalis is an opportunistic parasite which is known to cause fatal meningoencephalomyelitis primarily in equines but sporadically also in humans. In April 2014, laboratory examination of the head of a young dairy calf, euthanized due to severe central nervous system symptoms, revealed the presence of granulomatous to necrotizing encephalitis and myriads of nematodes in the brain lesion. Morphologically the parasites were identified as H. gingivalis. The diagnosis was confirmed by molecular analysis of the large subunit (LSU) rRNA and the small subunit (SSU) rRNA genes, revealing genetic variations of 0.5-4.4% and 0.7-8.6%, respectively, between the H. gingivalis isolated from the Danish calf and published isolates, collected worldwide from free-living and parasitic stages of the nematode. Clinical symptoms and histological changes indicated infection with H. gingivalis from another three calves in the herd. This is the first scientific publication of H. gingivalis induced meningoencephalomyelitis in ruminants. As ante mortem diagnosis is a major challenge, the infection may easily remain undiagnosed in cattle., (Copyright © 2016 Elsevier B.V. All rights reserved.)

For decades, microscopy of feces after formol-ethylacetate (FEA) concentration and iodine staining has been the routine test for intestinal protozoa. Lately, polymerase chain reaction or fluorescence-labeled parasite-specific antibodies have been introduced, but their place in everyday routine diagnostics has not yet been established. We compared FEA and salt-sugar flotation (SSF) concentration followed by microscopy of iodine-stained concentrate and immunofluorescence assay (IFA) and real-time polymerase chain reaction (qPCR) for detection of Giardia duodenalis in human feces. The median number of Giardia cysts found by FEA in 19 Giardia-positive samples was 50 cysts per gram (CPG), by SSF 350 CPG, by IFA 76,700 CPG, and by qPCR 316,000 CPG. We next tested 455 consecutive samples for presence of Giardia cysts. Using IFA as reference, qPCR had a sensitivity of 91%, specificity of 95.1%, a false-positive rate of 50%, a false-negative rate of 0.48%, a positive predictive value of 50%, and a negative predictive value of 99.5%. In conclusion, qPCR and IFA were significantly more sensitive than microscopy of iodine-stained concentrates using either FEA or SSF. We suggest, when using qPCR, that positive samples are verified by IFA to prevent false-positive results., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The large population of feral cats in Denmark may potentially transmit pathogens to household cats and zoonotic parasites to humans. A total of 99 euthanized cats; feral cats (n=92) and household cats with outdoor access (n=7), were collected from March to May 2014 from the Zealand region, Denmark. The sedimentation and counting technique (SCT) was used to isolate helminths and coproscopy was done by concentration McMaster technique (c-McMaster). Overall, 90.1% of the cats were infected and a total of 10 species were recorded by SCT: 5 nematode species: Toxocara cati (84.8%), Ollulanus tricuspis (13.1%), Aonchotheca putorii (7.1%), Paersonema spp. (3.0%), Strongyloides spp. (1.0%); 3 cestodes: Hydatigera taeniaeformis (36.4%), Mesocestoides sp. (3.0%), Dipylidium caninum (1.0%); and 2 trematodes: Cryptocotyle spp. (5.1%) and Pseudamphistomum truncatum (1.0%). O. tricuspis was the second most common gastrointestinal nematode of cats but had the highest intensity of infection. For T. cati, prevalence and worm burden were significantly higher in feral than household cats. No juvenile cats were infected with H. taeniaeformis, and age thus had a significant effect on prevalence and worm burdens of this species. Rural cats had a higher prevalence and worm burden of A. putorii than urban cats. By c-McMaster, ascarid, capillarid, strongylid or taeniid type eggs were found in 77.9% of the cats while Cystoisospora felis was found in 2.1%. The sensitivity of the c-McMaster was 82.5% for T. cati but 26.5% for taeniid eggs, using the SCT as gold standard. A positive correlation between faecal egg counts and worm burdens was seen for T. cati, but not for taeniid eggs (assumed to be H. taeniaeformis). Coprological examination also detected the eggs of extraintestinal Capillariidae species including Eucoleus aerophilus and Eucoleus boehmi, but further necropsy studies are needed to confirm these findings., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The study investigated direct anthelmintic effects of sesquiterpene lactones (SL)-containing extracts from forage chicory against free-living and parasitic stages of Ostertagia ostertagi. Freeze-dried leaves from chicory cultivars 'Spadona' and 'Puna II' were extracted using methanol/water. Total SL were further fractionated by solid-phase extraction and resulting extracts were characterised by high-performance liquid chromatography (HPLC). O. ostertagi eggs from faeces of mono-infected calves were hatched and L1 were used in a larval feeding inhibition assay (LFIA), while cultured L3 were used in a larval exsheathment inhibition assay (LEIA). Adult worms were immediately recovered after slaughter and used for motility inhibition assays (AMIA). Electron microscopy (EM) was performed on adult O. ostertagi exposed to 1000 μg extract mL(-1) of both chicory cultivars. In all assays, decreasing concentrations of SL-containing extracts in PBS (1% DMSO) were tested in replicates with 1% DMSO in PBS as negative controls. HPLC demonstrated similar concentrations of most SL in both extracts. However, Spadona-extract contained significantly higher concentrations of 11, 13-dihydro-8-deoxylactucin (P = 0.01), while Puna II-extract had increased levels of 11, 13-dihydrolactucin (P < 0.0001). In the LFIA, both extracts reduced larval feeding at increasing concentrations, but Spadona-extract showed higher potency confirmed by significantly lower EC50 (P < 0.0001). In the LEIA, neither of the two extracts interfered with the exsheathment of L3 (P > 0.05). In the AMIA, both SL-containing extracts induced a dose-dependent effect but Spadona-extract showed greater activity and exerted faster worm paralysis than Puna II-extract with significantly lower EC50 (P < 0.0001). No cuticular damage was observed by EM in worms exposed to any of the extracts. We have demonstrated that SL-containing extracts from forage chicory can inhibit feeding of free-living larvae and exert direct effects against parasitic stages of O. ostertagi. Our results may contribute to the identification of natural anti-parasitic compounds and to interpret the in vivo anthelmintic effects of forage chicory., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The nematode Baylisascaris procyonis, which may cause severe clinical disease in humans and animals, is emerging in Europe after its introduction with raccoons (Procyon lotor) from North America. B. procyonis has a broad spectrum of paratenic hosts, including rodents, birds, wild carnivores and primates, which are severely affected by the migrating larval stages of the parasite. We report here the recovery of B. procyonis from two out of 18 examined wild raccoons in Denmark. The parasites were identified based on morphology and their identity was confirmed by partial sequencing of the 18S rRNA gene. Follow-up telephone interviews of staffs in nine zoos housing captive raccoons and veterinarians supervising these zoos showed that knowledge of B. procyonis and its zoonotic potential were sparse. Eggs of B. procyonis were detected in two raccoons kept in one of three zoos that submitted fecal samples following the telephone interviews. Continuous monitoring and increased awareness are needed to reduce further spread of the parasite and to limit the public health risks associated with baylisascariasis., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Although pigs are commonly infected with Cryptosporidium spp. and Giardia duodenalis, including potentially zoonotic species or genotypes, little is known about age-related infection levels, seasonal differences and genetic variation in naturally infected pigs raised in organic management systems. Therefore, the current study was conducted to assess seasonal and age-related variations in prevalence and infection intensity of Cryptosporidium and Giardia, evaluate zoonotic potential and uncover correlations between species/genotypes, infection intensity and faecal consistency. Shedding of oocysts and cysts ((oo-)cysts) was monitored at quarterly intervals (September 2011-June 2012) in piglets (n = 152), starter pigs (n = 234), fatteners (n = 230) and sows (n = 240) from three organic farms in Denmark. (oo-)Cysts were quantified by immunofluorescence microscopy; and 56/75 subsamples from Cryptosporidium infected pigs were successfully analysed by PCR amplification and partial sequencing of the small subunit (SSU) 18S rRNA and hsp70genes, while 13/67 Giardia subsamples were successfully analysed by amplification and partial sequencing of the 18S rRNA and the gdh genes. Altogether, Cryptosporidium or Giardia infections were observed in 40.9% (350/856) and 14.0% (120/856) of the pigs, respectively, including 8.2% (70/856) infected with both parasites. Prevalence, intensity of infections and presence of Cryptosporidium species varied significantly between age-groups; 53.3% piglets, 72.2% starter pigs, 40.4% fatteners and 2.9% sows were infected with Cryptosporidium, whereas 2.0% piglets, 27.4% starter pigs, 17.8% fatteners and 5.0% sows were infected with Giardia. The overall prevalence was stable throughout the year, except for dual-infections that were more prevalent in September and December (p < 0.05). The infection intensity was age-related for both parasites, and dual-infected pigs tended to excrete lower levels of oocysts compared to pigs harbouring only Cryptosporidium. Likewise, pigs infected with Cryptosporidium scrofarum excreted fewer oocysts (mean CPG: 54,848 ± 194,508CI: 9085-118,781) compared to pigs infected with Cryptosporidium suis (mean OPG: 351,035 ± 351,035CI: 67,953-634,117). No correlation between faecal consistency and (oo-)cyst excretion levels was observed. Of the successfully genotyped isolates, 38/56 (67.9%) were C. scrofarum and 18/56 (32.1%) were C. suis, while the livestock specific G. duodenalis Assemblage E was detected in 11/13 (84.6%) isolates and the potentially zoonotic Assemblage A was identified in 2/13 (15.4%) isolates. Piglets exclusively hosted C. suis, with one exception, while starter pigs and fatteners predominantly hosted C. scrofarum. As organic pigs are partly reared outdoors, environmental contamination with Cryptosporidium and Giardia is inevitable. Nevertheless, the present data indicate that the potential public health risk associated with both of these parasites in Danish organic pig production seems to be negligible., (Copyright © 2015 Elsevier B.V. All rights reserved.)

When Echinococcus (E.) multilocularis was first detected in mainland Scandinavia in Denmark in 2000, surveillance was initiated/intensified in Sweden, mainland Norway and Finland. After 10 years of surveillance these countries all fulfilled the requirements of freedom from E. multilocularis as defined by the EU, i.e. a prevalence in final hosts <1% with 95% confidence level. However, in 2011 E. multilocularis was detected in Sweden for the first time and surveillance was increased in all four countries. Finland and mainland Norway are currently considered free from E. multilocularis, whereas the prevalence in foxes in Sweden and Denmark is approximately 0.1% and 1.0%, respectively. E. multilocularis has been found in foxes from three different areas in Denmark: Copenhagen (2000), Højer (2012-14) and Grindsted (2014). Unlike Sweden, Norway and Finland, human alveolar echinococcosis (AE) is not notifiable in Denmark, and the number of human cases is therefore unknown. In Sweden, E. multilocularis has been found in foxes in four counties, Västra Götaland, Södermanland, Dalarna (2011) and Småland (2014). E. multilocularis has also been found in an intermediate host in Södermanland (2014). Two cases of AE have been reported in humans (2012), both infected abroad. No cases of E. multilocularis or AE have been reported in Finland and Norway. Recommendations and future considerations are discussed further., (Copyright © 2015. Published by Elsevier B.V.)

Aims: To develop a filtration unit for efficient recovery of waterborne Cryptosporidium oocysts and Giardia cysts ((oo-)cysts) in drinking water., Methods and Results: This unit utilizes a metallic filter and an ultrasound transducer for eluting (oo-)cysts, with a fixed retentate backwash volume; approx. 400 μl. Changes in the viability was evaluated by seeding wild type (oo-)cysts (1 × 10(4)) followed by sonication for 5, 10, 20 or 40 s (five replicates for each period). Flow cytometry analysis showed negligible increase in the mortality of (oo-)cysts exposed to 5-10 s of sonication. Recovery rate was assessed by seeding ColorSeed(™) (10 replicates) into the filter unit followed by air backwash to a glass slide and counting of (oo-)cysts by epifluorescent microscopy. High recovery rates (mean ± SD) were found: 84·9% ± 4·8 for Giardia cysts and 70% ± 6·5 for Cryptosporidium oocysts. DNA of seeded wild type (oo-)cysts (1 × 10(2); 10 replicates) was successfully amplified using real-time PCR., Conclusions: The use of a metallic filter, sonication and 'air backwash' were key factors for creating a highly efficient system for recovery of apparently undamaged protozoa., Significance and Impact of the Study: This reagent-less system can be used for monitoring of parasite contamination in drinking water., (© 2015 The Society for Applied Microbiology.)

In this multicentric, randomised, blinded and placebo-controlled field study, the effect of treatment with toltrazuril (Baycox(®) Bovis, Bayer) on oocyst excretion, diarrhoea score and weight gain was studied in Danish dairy herds with confirmed history of eimeriosis (coccidiosis) and prevalence of Eimeria bovis and Eimeria zuernii. Three commercial herds and a total of 71 calves, aged 48 - 135 days, were included. Treatment with a single oral dose of toltrazuril (15 mg/kg) was given after relocation to common pens and one week before expected outbreak of eimeriosis. The effect of treatment was followed by weekly faecal sampling and weighing initially and at the end of a study period of 8 weeks. In Herd 2 and 3 toltrazuril treated calves gained on average 7.95 kg more than placebo treated calves (p = 0.007), and both oocyst excretion and prevalence of Eimeria spp. were significantly reduced the first weeks post treatment. In Herd 1, by contrast, the farmer made some unforeseen changes in the management which entailed relocation to large deep-litter pens 3 - 6 weeks post treatment. In addition, many calves were not treated metaphylactically while few calves excreted oocysts when the trial was initiated. Thus, no significant difference in weight gain was found between toltrazuril and placebo treated calves (p = 0.523), and the oocyst excretion of toltrazuril treated calves was significantly higher during week 7 and 8. Significant differences in faecal scores were observed between the herds (p<0.002) but not between treatment groups in any of the herds. In conclusion, timing of treatment is crucial for optimal effect of metaphylactic toltrazuril treatment on weight gain and oocyst excretion.