Condensed tannins, novel compounds and sources of variation determine the antiparasitic activity of Nordic conifer bark against gastrointestinal nematodes.

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.

Effects of selected condensed tannins on Cryptosporidium parvum growth and proliferation in HCT-8 cell cultures.

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.

Administration of spruce bark (Picea abies) extracts in young lambs exhibits anticoccidial effects but reduces milk intake and body weight gain.

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.

Use of agro-industrial by-products containing tannins for the integrated control of gastrointestinal nematodes in ruminants.

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.

TITLE: Utilisation de sous-produits agro-industriels contenant des tanins pour le contrôle intégré des nématodes gastro-intestinaux chez les ruminants. ABSTRACT: Plusieurs études antérieures ont illustré le fait que des légumineuses bioactives contenant des tannins condensés peuvent représenter une des alternatives à intégrer avec d'autres options pour une maitrise durable des nématodes gastro-intestinaux en réponse au développement constant et à l'expansion continue à l'échelle mondiale des résistances aux anthelminthiques de synthèse. Des recherches plus récentes se sont intéressées au potentiel d'application de ressources moins conventionnelles que représentent des coproduits agroindustriels (CPAI). Cette revue vise à présenter des résultats in vitro et in vivo obtenus avec une gamme de CPAI d'origines géographiques et botaniques diversifiées (coproduits de l'industrie des noix, du bois (en régions tempérées et tropicales), du caroubier, du café et du cacao). Ces résultats ont confirmé la preuve de concept pour les effets antiparasitaires, et aussi pour d'autres volets de la production des ruminants dans un contexte agro écologique de l'élevage. Par ailleurs, les aspects socio-économiques d'exploitation de ces ressources, considérées jusqu'à présent comme des déchets, dans un modèle de circuits courts sont aussi évoqués. Les avantages et inconvénients des différentes modalités d'exploitation des CPAI sont aussi discutés dans le cadre d'une analyse SWOT.

Extracts of pine bark (Pinus sylvestris) inhibit Cryptosporidium parvum growth in cell culture.

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 IC50 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 IC50 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.

First case of community acquired giardiasis in Nuuk, Greenland.

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.

Association between milk yield and milk anti-Fasciola hepatica antibody levels, and the utility of bulk tank milk samples for assessing within-herd prevalence on organic dairy farms.

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.

Mathematical modelling of Toxoplasma gondii transmission: A systematic review.

BACKGROUND: Toxoplasma gondii is a ubiquitous protozoan parasite that can infect virtually all warm-blooded animals. It is the causative agent of toxoplasmosis, a significant public health issue worldwide. Mathematical models are useful to study the transmission dynamics of T. gondii infection in different settings, and may be used to compare the effectiveness of prevention measures. METHODS: To obtain an overview of existing mathematical models for transmission of T. gondii, a systematic review was undertaken. The review was conducted according to an a priori protocol and the results were reported according to the PRISMA guidelines. Specific search terms were developed and used in the search of three databases (Scopus, PubMed, and Embase). RESULTS: In total, 484 unique records were retrieved from the systematic search. Among them, 15 studies that used mathematical models to study the transmission of T. gondii. These studies were categorized into four groups based on the primary aims: dynamics of transmission (n = 8), intervention (n = 5), spatial distribution (n = 1), and outbreak investigation (n = 1). CONCLUSIONS: Considering the high disease burden caused by T. gondii, the number of studies using mathematical models to understand the transmission dynamics of this parasite and to evaluate the effectiveness of intervention measures was only 15. This systematic review provides an overview of existing mathematical models and identifies the data gaps for model building. The results from this study can be helpful for further development of mathematical models and improved understanding of the transmission dynamics of T. gondii infection.

Surveillance of important bacterial and parasitic infections in Danish wild boars (Sus scrofa).

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.

Source attribution of human echinococcosis: A systematic review and meta-analysis.

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.

Giardia and Cryptosporidium infections in Danish cats: risk factors and zoonotic potential.

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.

Development of a gp60-subtyping method for Cryptosporidium felis.

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.

Assessment of differences between DNA content of cell-cultured and freely suspended oocysts of Cryptosporidium parvum and their suitability as DNA standards in qPCR.

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 (106-1); (ii) diluted template DNA from pure oocysts (×10-×106 dilution of 106 oocyst DNA template); (iii) oocysts incubated in human ileocecal adenocarcinoma (HCT-8) cells (105-1 and 5 × 104-50); and (iv) diluted DNA template (5 × 104) 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 106 oocysts exhibiting similar quantification cycle (Cq) values to those obtained from DNA template dilutions of 106 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.

Preliminary studies on in vitro methods for the evaluation of anticoccidial efficacy/resistance in ruminants.

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.

Seroprevalence of Toxoplasma gondii in domestic pigs, sheep, cattle, wild boars, and moose in the Nordic-Baltic region: A systematic review and meta-analysis.

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 (CI95%: 3-10%), 23% in sheep (CI95%: 12-36%), 7% in cattle (CI95%: 1-21%), 33% in wild boars (CI95%: 26-41%), and 16% in moose (CI95%: 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.

Prevalence of Strongylus vulgaris in horses after ten years of prescription usage of anthelmintics in Sweden.

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.

Prevalence of Strongylus vulgaris in horses after ten years of prescription usage of anthelmintics in Sweden.

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.

Anti-protozoal activity of extracts from chicory (Cichorium intybus) against Cryptosporidium parvum in cell culture.

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.

Genetic diversity of Echinococcus multilocularis in red foxes from two Scandinavian countries: Denmark and Sweden.

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.

Patterns of Fasciola hepatica infection in Danish dairy cattle: implications for on-farm control of the parasite based on different diagnostic methods.

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.