Occurrence of gastrointestinal nematodes in lambs in Norway, as assessed by copromicroscopy and droplet digital polymerase chain reaction.

BACKGROUND: Gastrointestinal nematodes (GINs) have a major impact on sheep production, health, and welfare worldwide. Norway is no exception, but there are only a few studies on the prevalence of GINs in Norwegian sheep. The aim of this study was to investigate the current occurrence of the most important nematodes in sheep flocks in Norway. Faecal samples were collected from flocks in 2021/2022, mainly from three geographical regions in Norway, i.e., northern, eastern, and western. In each of 134 flocks included, individual samples from 10 lambs (autumn) were pooled. Third stage larvae (L3) were cultivated and harvested (Baermann method) from the pooled samples. The DNA was then extracted and further analysed using droplet digital PCR (ddPCR). This enables assessment of the proportions of the three most important nematode species/genera, i.e., H. contortus, T. circumcincta, and Trichostrongylus. The fractional abundance/relative proportion of each species/genus was assessed by performing duplex assays with universal strongyle and species/genus-specific primers and probe sets. In addition, the occurrence of Nematodirus eggs was assessed by standard faecal egg counts (i.e., McMaster method). RESULTS: Of the 134 flocks sampled, 24 were from the northern region, 31 from eastern, and 71 from western Norway. In addition, some flocks from central (n = 7), and southern (n = 1) Norway were included. Among the sampled flocks, T. circumcincta occurred most commonly (94%), followed by H. contortus (60%) and Trichostrongylus (55%), and Nematodirus (51%). In general, mixed infections were observed, with 38% and 18% of flocks infected with three or all four genera, respectively. CONCLUSIONS: The results of this study indicate that GINs are widespread in Norway. Teladorsagia circumcincta seems to be present in most flocks based on this screening. Moreover, the results show that Nematodirus spp. infect lambs throughout the country, predominantly N. battus, and indicate that this nematode has become more abundant, which could lead to an increase in nematodirosis.

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.

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.

Echinococcus granulosus sensu lato and Echinococcus multilocularis: A review.

Echinococcus spp. have a global distribution and are found on every continent except Antarctica. Infections with these parasites are considered extremely serious, contributing to significant morbidity and mortality in addition to substantial economic losses to the livestock industry. Echinococcus granulosus sensu lato (s.l.) and Echinococcus multilocularis, causing cystic echinococcosis (CE) and alveolar echinococcosis (AE) respectively, are the two main species of interest from a human and veterinary perspective. This review collates the current state-of-the-art understanding of these two parasites within four key areas of relevance to human and veterinary professionals: transmission and epidemiology, clinical signs and pathogenesis, diagnosis, and treatment and prevention. This review should serve as a broad introduction to the most important Echinococcus spp. The reader is advised to seek out specific literature on individual diseases and their causative parasites for a deeper understanding.

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.

Parasites in Myodes glareolus and their association with diet assessed by stable isotope analysis.

Vertebrates are hosts to numerous parasites, belonging to many different taxa. These parasites differ in transmission, being through either direct contact, a faecal-oral route, ingestion of particular food items, vertical or sexual transmission, or by a vector. Assessing the impact of diet on parasitism can be difficult because analysis of faecal and stomach content are uncertain and labourious; and as with molecular methods, do not provide diet information over a longer period of time. We here explored whether the analysis of stable isotopes in hair provides insight into the impact of diet and the presence of parasites in the rodent Myodes glareolus. Twenty-one animals were examined for parasites and their hair analysed for stable isotopes (C and N). A positive correlation between δ15N and one species of intestinal parasite was observed in females. Furthermore, several ectoparasites were negatively correlated with δ15N, indicating that infections are further associated with foraging habits (size and layout of the home range, length and timing of foraging, interaction with other rodents, etc.) that set the rodents in direct contact with infected hosts. Although a limited number of animals were included, it seemed that the isotope values allowed for identification of the association between diet and parasite occurrence in this rodent. We therefore propose that this method is useful in providing further insight into host biology, feeding preferences and potential exposure to parasites species, contributing to the understanding of the complex relationship between hosts and parasites.

Repeated inoculations with the lung and heartworm nematode Angiostrongylus vasorum result in increasing larval excretion and worm burden in the red fox (Vulpes vulpes).

The French heartworm Angiostongylus vasorum is found in European red fox (Vulpes vulpes) and dog populations, where it appears to be spreading geographically. Once introduced into new areas, it establishes in local fox populations, typically to over 50% prevalence in a few years. High susceptibility and constant excretion of first stage larvae (L1) by the definitive hosts are prerequisites for sustaining high parasite biomass in a particular habitat. The present study explores the hypothesis that repeated ingestion of gastropods in nature will result in accumulation of adult worms and elevated excretion of L1 in feces. Experimentally infected foxes were subsequently inoculated via stomach tube once (9 weeks post initial inoculation) or twice (9 and 13 weeks post inoculation (wpi)) with 100 third stage A. vasorum larvae (L3) previously isolated from aquatic snails infected with L1 from a naturally infected dog. Despite large variation in fecal larval excretion for the individual animals within the groups, excretion of L1 was significantly higher in foxes twice inoculated as compared to foxes inoculated only once. With an outlier in the once inoculated group removed, excretion became significantly higher in the three times inoculated group. Establishment of adult worms varied and only a trend to higher worm burdens was found in the group of foxes inoculated three times. However, this became significant with the same single outlier removed. Overall, it appears that protective immunity to A. vasorum does not appear to occur in V. vulpes with animals exhibiting high infection intensities without obvious clinical signs. The increasing larval excretion in foxes being repeatedly exposed to A. vasorum L3 support the hypothesis that foxes under natural conditions may repeatedly ingest infected gastropods and remain a source of environmental contamination for several months, potentially contributing to the establishment of endemic foci through increasing L1 excretion.

Detection and molecular characterization of the mosquito-borne filarial nematode Setaria tundra in Danish roe deer (Capreolus capreolus).

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.

Peroral Echinococcus multilocularis egg inoculation in Myodes glareolus, Mesocricetus auratus and Mus musculus (CD-1 IGS and C57BL/6j).

Echinococcus multilocularis transmission predominantly occurs in Europe between the red fox (Vulpes vulpes) and various species of rodent intermediate hosts. We infected 3 species of rodent, Myodes glareolus (n = 47), Mesocricetus auratus (n = 11) and outbred Mus musculus (CD-1 IGS) (n = 9) with an E. multilocularis egg suspension that contained 100 eggs with viable oncospheres and performed post mortem examination 6, 8 (M. glareolus) and 10 weeks post inoculation (wpi). C57BL/6j mice (n = 4) were used as positive controls as they have been shown to exhibit macroscopic liver lesions 4 wpi. To the best of our knowledge, this is the first study to experimentally assess susceptibility in the ostensibly competent host M. glareolus. Lesions were only detected in 2 of 47 M. glareolus (4.3%) at 8 and 10 wpi and although both contained protoscolices (1675 at 8 wpi and 88 at 12 wpi) the low percentage of infected animals brings into question their role as transmitters of the parasite. Significant differences were observed between inbred and outbred mice with E. multilocularis infection in the former demonstrating increased establishment (p ≤ 0.0001) and growth (p ≤ 0.0001). No lesions were found in all 11 M. auratus.

Fresh fruit, vegetables, and mushrooms as transmission vehicles for Echinococcus multilocularis in Europe: inferences and concerns from sample analysis data from Poland.

Fresh fruit, vegetables, mushrooms, and other fresh produce are recognised as important vehicles of infection for several foodborne parasites, particularly those with a faecal-oral transmission route and robust environmental transmission stages. Nevertheless, analysis of such foods for parasite transmission stages, even during outbreaks, tends to show only low contamination. Echinococcus multilocularis is considered one of the most important foodborne parasites, but there are few studies in which fresh produce or like foods collected in their natural habitat is analysed for contamination with E. multilocularis eggs. In this article, we question a recent study from Poland reporting over 23 % of fresh berries, vegetables, and mushroom being highly contaminated with E. multilocularis eggs. In particular, it appears unlikely that 20 % of raspberries, which are elevated from ground level, should be exposed to faecal contamination. Additionally, the similar egg contamination of vegetation in forest and plantation environments is surprising considering the preference of the parasite's most competent intermediate hosts for the latter environment. Furthermore, a lack of specific temporal information is concerning due to the varying infection pressure (and therefore environmental contamination) occurring in definitive hosts over the course of the year. Several important aspects of the study seem to us to have been neglected, and we are concerned that the published data might, if not questioned, lead to incorrect interpretation, and unnecessary losses in the agricultural sector.

Establishment and development of Echinococcus multilocularis metacestodes in the common vole (Microtus arvalis) after oral inoculation with parasite eggs.

Transmission of the zoonotic tapeworm, Echinococcus multilocularis mainly occurs between the red fox (Vulpes vulpes) and various species of vole. Microtus arvalis is considered one of the key intermediate hosts in Europe. We infected 21 M. arvalis aged 35 days (n=2), 56 days (n=6), 84 days (n=4) and 263 days (n=9) with 100 E. multilocularis eggs. Four voles aged 263 days were euthanized at 6 weeks post inoculation (wpi) with the remainder euthanized 10 wpi for analysis of metacestode growth and protoscolex development. Eight C57BL/6j mice (age 35-231 days) were included as controls for egg viability (they have been shown to exhibit visible infection after 4 wpi) and dissected at 6 (n=2) and 10 (n=6) wpi. M. arvalis had significantly higher metacestode establishment (p=0.008) 6 wpi with 27.5±6.63S.D. compared to C57BL/6j with 15.5±0.71S.D. Multivesiculation precluded enumeration at 10 wpi in M. arvalis. No protoscolices were found in metacestodes in M. arvalis 6 wpi or C57BL/6j at any time point but were found in all infected voles 10 wpi (48,056±52,574 S.D.). It has been reported that glucocorticoid (GC) profile can affect E. multilocularis establishment. This was assessed by measuring corticosterone in rodent hair to determine if parasite establishment or fertility was related to this stress hormone. No significant differences were found. Data presented here provides, for the first time, a protoscolex development window in this species that has the potential to shed light on the epizootiology of this parasite.

An insect-tapeworm model as a proxy for anthelminthic effects in the mammalian host.

Invertebrate models provide several important advantages over their vertebrate counterparts including fewer legislative stipulations and faster, more cost-effective experimental procedures. Furthermore, various similarities between insect and mammalian systems have been highlighted. To obtain maximum use of invertebrate models in pharmacology, their fidelity as analogues of vertebrate systems requires verification. We utilised a flour beetle (Tenebrio molitor)-tapeworm (Hymenolepis diminuta) model to evaluate the efficacy of known anthelmintic compounds, praziquantel, mebendazole and levamisole against H. diminuta cysticercoid larvae in vitro. Inhibition of cysticercoid activity during the excystation procedure was used as a proxy for worm removal. The effects of the three compounds mirrored their relative efficacy in treatment against adult worms in mammalian systems; however, further study is required to determine the fidelity of this model in relation to dose administered. The model precludes comparison of consecutive daily administration of pharmaceuticals in mammals due to cysticercoids not surviving outside of the host for multiple days. Treatment of beetles in vivo, followed by excystation of cysticercoids postdissection could potentially allow for such comparisons. Further model validation will include analysis of pharmaceutical efficacy in varying H. diminuta isolates and pharmaceutical dilution in solvents other than water. Notwithstanding, our results demonstrate that this model holds promise as a method to efficiently identify promising new cestocidal candidates.

Echinococcus multilocularis infection in the field vole (Microtus agrestis): an ecological model for studies on transmission dynamics.

We propose a model involving the oral inoculation of Echinococcus multilocularis eggs in a vole species and examine the infection dynamics in a dose-response experiment. Defined doses, 100 (n = 8), 500 (n = 5) and 1000 (n = 5) of E. multilocularis eggs were used to inoculate Microtus agrestis. Four female C57BL/6j mice were inoculated with 1000 eggs as positive controls. The groups inoculated with 100 and 500 eggs exhibited significantly higher lesion numbers, and relatively smaller lesion size was observed in the 1000 dose group. Undetectable abortive lesions may be responsible for some form of resource limitation early in the infection, resulting in lower lesion counts and size in the 1000 dose group. The C57BL/6j mice exhibited significantly fewer lesions than M. agrestis. The feasibility of measuring corticosterone (which has been shown to downregulate Th1 cytokines) in rodent hair and tumour necrosis factor (TNF) production in spleen cells was demonstrated by a positive correlation between corticosterone levels and higher lesion counts and TNF production in C57BL/6j, respectively. These results suggest that M. agrestis is more prone to a Th2 immune response than C57BL/6j, which is associated with E. multilocularis susceptibility and may explain why the parasite develops more slowly in murine models. This is the first data to suggest that M. agrestis is capable of supporting E. multilocularis transmission and thus may be suited as a model to describe the infection dynamics in an intermediate host that affects transmission under natural conditions.

Predictors of Echinococcus multilocularis Prevalence in Definitive and Intermediate Hosts: A Meta-Analysis Approach.

Echinococcus multilocularis (EM) is a pathogenic and potentially fatal cestode causing human alveolar echinococcosis (AE). A meta-analysis was conducted using a generalized estimation equation approach (GEE) to assess the effect of taxonomic, environmental, and diagnostic variables on EM prevalence in different hosts. Red foxes ( Vulpes vulpes ) had significantly higher prevalence of EM than domestic dogs ( Canis lupus familiaris), with the diagnostic method playing an important factor in assessing prevalence. For intermediate hosts genera was significantly associated with EM prevalence, although there was some indication of publication bias in this dataset. This study also highlights the possible importance of temperature and precipitation to EM transmission. This implies the possibility of a changing climate affecting the future distribution of the parasite.