Dietary seaweed (Saccharina latissima) supplementation in pigs induces localized immunomodulatory effects and minor gut microbiota changes during intestinal helminth infection.

Brown seaweeds have a rich bioactive content known to modulate biological processes, including the mucosal immune response and microbiota function, and may therefore have the potential to control enteric pathogens. Here, we tested if dietary seaweed (Saccharina latissima) supplementation could modulate pig gut health with a specific focus on parasitic helminth burdens, gut microbiota composition, and host immune response during a five week feeding period in pigs co-infected with the helminths Ascaris suum and Oesophagostomum dentatum. We found that inclusion of fermented S. latissima (Fer-SL) at 8% of the diet increased gut microbiota α-diversity with higher relative abundances of Firmicutes, Tenericutes, Verrucomicrobia, Spirochaetes and Elusimicrobia, and lower abundance of Prevotella copri. In the absence of helminth infection, transcription of immune-related genes in the intestine was only moderately influenced by dietary seaweed. However, Fer-SL modulated the transcriptional response to infection in a site-specific manner in the gut, with an attenuation of infection-induced gene expression in the jejunum and an amplification of gene expression in the colon. Effects on systemic immune parameters (e.g. blood lymphocyte populations) were limited, indicating the effects of Fer-SL were mainly localized to the intestinal tissues. Despite previously documented in vitro anti-parasitic activity against pig helminths, Fer-SL inclusion did not significantly affect parasite egg excretion or worm establishment. Collectively, our results show that although Fer-SL inclusion did not reduce parasite burdens, it may modify the gut environment during enteric parasite infection, which encourages continued investigations into the use of seaweeds or related products as novel tools to improve gut health.

Development of In Vitro Assays with the Canine Hookworm Uncinaria stenocephala and Assessment of Natural Plant Products for Anti-Parasitic Activity.

Enteric helminth infection is an increasing concern in companion animals due to reports of resistance to commonly used anthelmintic drugs. Thus, the assessment of new therapeutic options such as bioactive dietary additives is of high importance. Here, we adapted egg hatch, larval migration, and larval motility assays to screen extracts of several natural ingredients against the canine hookworm Uncinaria stenocephala, a prevalent parasite of dogs in northern Europe. Egg hatch and larval migration assays were established showing that the anthelmintic drugs levamisole and albendazole had strong anti-parasitic activity against U. stenocephala, validating the use of these assays for the assessment of novel anti-parasitic substances. Subsequently, we identified that extracts from the seaweed Saccharina latissima, but not extracts from grape seed or chicory, significantly inhibited both hatching and larval migration. Finally, we showed that α-linolenic acid, a putative anti-parasitic compound from S. latissima, also exhibited anti-parasitic activity. Collectively, our results established a platform for the screening for anthelmintic resistance or novel drug candidates against U. stenocephala and highlighted the potential use of seaweed extracts as a functional food component to help control hookworm infection in dogs.

Assessing welfare risks in unowned unsocialised domestic cats in Denmark based on associations with low body condition score.

BACKGROUND: Populations of unowned unsocialised cats are present worldwide. Generally, there is concern about their welfare. Low body condition score (BCS) is a potentially relevant indicator that is relatively easy to assess: emaciated cats are likely to have welfare problems while thin cats may be at risk of becoming emaciated. The objective of this study was to assess the association of low BSC with a selection of factors relating to the host, disease, and infection in unowned unsocialised domestic cats. We necropsied 598 euthanised unowned unsocialised cats from Denmark. We recorded each cat's age-group, sex, and neuter status, together with its pregnancy status, the season and location of trapping, as well as gross lesions at necropsy. We also tested for feline immunodeficiency virus and feline leukaemia virus, recorded presence of ectoparasites, and a subsample of the cats were also tested for endoparasites. Cats with no or sparse adipose deposits were categorised as having low BCS, and logistic regression was used to determine the factors associated with low BCS. RESULTS: Of the cats, 11.4% had low BCS. Season, age-group and sex were associated with low BCS and confounded potential associations with other variables. Intact adult males and females in spring and early summer were at highest risk of low BCS. When these factors were taken into account, cats with biting lice had 2.8 (95% confidence interval (CI) 1.4-5.4) times higher odds of low BCS, and cats with gastro-intestinal findings (i.e., enlarged mesenteric lymph nodes, abdominal hernia, diarrhoea, obstructive foreign bodies, or diaphragmatic hernia) had 50 (95% CI 10-417) times higher odds of low BCS, than cats with no such findings. Cats with low BCS were primarily adult intact cats with tooth lesions and skin lesions, ear mite infection, and positive test result for feline immunodeficiency virus. CONCLUSIONS: The results highlight associations that can be used to define a risk profile: low BCS, notably in summer-autumn, in an unowned unsocialised cat was associated with underlying, less visible problems. Thus, low BCS can be more than a step towards being emaciated; it can also be an indicator of other underlying welfare problems.

How Serious Are Health-Related Welfare Problems in Unowned Unsocialised Domestic Cats? A Study from Denmark Based on 598 Necropsies.

Free ranging unsocialised domestic cats are widely believed to suffer from a high load of welfare problems. We assessed the validity of this belief by performing necropsies on the corpses of 598 unsocialised cats, originating from all parts of Denmark, that had been euthanised by two Danish cat welfare organisations. We selected a number of variables for health-related cat welfare that could be assessed through necropsy (e.g., gross lesions, ectoparasites and body condition) or by laboratory analysis (e.g., infection with feline immunodeficiency virus (FIV), and by feline leukaemia virus (FeLV)). Each finding was classified as having either a major or minor welfare impact on the cat. More than 83% of the cats had no major finding, and 54% had no finding indicating a welfare issue at all. More than 83% of the cats had a body condition within normal range. Only 0.3% were emaciated. The most common finding was infestation with ectoparasites, with 15.9% infected with lice, 12.3% with fleas, 4.7% with ticks, and 6.7% with ear mites. FIV and FeLV were detected in 9.2% and 1.2% of the cases, respectively. The most common lesion related to the cats' teeth. Overall, unsocialised cats in Denmark have a moderate level of health-related welfare problems.

Dietary proanthocyanidins promote localized antioxidant responses in porcine pulmonary and gastrointestinal tissues during Ascaris suum-induced type 2 inflammation.

Proanthocyanidins (PAC) are dietary polyphenols with putative anti-inflammatory and immunomodulatory effects. However, whether dietary PAC can regulate type-2 immune function and inflammation at mucosal surfaces remains unclear. Here, we investigated if diets supplemented with purified PAC modulated pulmonary and intestinal mucosal immune responses during infection with the helminth parasite Ascaris suum in pigs. A. suum infection induced a type-2 biased immune response in lung and intestinal tissues, characterized by pulmonary granulocytosis, increased Th2/Th1 T cell ratios in tracheal-bronchial lymph nodes, intestinal eosinophilia, and modulation of genes involved in mucosal barrier function and immunity. Whilst PAC had only minor effects on pulmonary immune responses, RNA-sequencing of intestinal tissues revealed that dietary PAC significantly enhanced transcriptional responses related to immune function and antioxidant responses in the gut of both naïve and A. suum-infected animals. A. suum infection and dietary PAC induced distinct changes in gut microbiota composition, primarily in the jejunum and colon, respectively. Notably, PAC consumption substantially increased the abundance of Limosilactobacillus reuteri. In vitro experiments with porcine macrophages and intestinal epithelial cells supported a role for both PAC polymers and PAC-derived microbial metabolites in regulating oxidative stress responses in host tissues. Thus, dietary PAC may have distinct beneficial effects on intestinal health during infection with mucosal pathogens, while having a limited activity to modulate naturally-induced type-2 pulmonary inflammation. Our results shed further light on the mechanisms underlying the health-promoting properties of PAC-rich foods, and may aid in the design of novel dietary supplements to regulate mucosal inflammatory responses in the gastrointestinal tract.

Bio-Guided Fractionation and Molecular Networking Reveal Fatty Acids to Be Principal Anti-Parasitic Compounds in Nordic Seaweeds.

Widespread use of antimicrobial drugs has led to high levels of drug-resistance in pathogen populations and a need for novel sources of anti-bacterial and anti-parasitic compounds. Macroalgae (seaweed) are potentially a rich source of bioactive compounds, and several species have traditionally been used as vermifuges. Here, we investigated the anti-parasitic properties of four common cold-water Nordic seaweeds; Palmaria palmata (Rhodophyta), Laminaria digitata, Saccharina latissima and Ascophyllum nodosum (Ochrophyta, Phaeophyceae). Screening of organic extracts against helminths of swine (Ascaris suum) and sheep (Teladorsagia circumcincta) revealed that S. latissima and L. digitata had particularly high biological activity. A combination of molecular networking and bio-guided fractionation led to the isolation of six compounds from extracts of these two species identified in both fermented and non-fermented samples. The six isolated compounds were tentatively identified by using MS-FINDER as five fatty acids and one monoglyceride: Stearidonic acid (1), Eicosapentaenoic acid (2), Alpha-Linolenic acid (3), Docosahexaenoic acid (4), Arachidonic acid (5), and Monoacylglycerol (MG 20:5) (6). Individual compounds showed only modest activity against A. suum, but a clear synergistic effect was apparent when selected compounds were tested in combination. Collectively, our data reveal that fatty acids may have a previously unappreciated role as natural anti-parasitic compounds, which suggests that seaweed products may represent a viable option for control of intestinal helminth infections.

Emerging interactions between diet, gastrointestinal helminth infection, and the gut microbiota in livestock.

Increasing evidence suggests that nutritional manipulation of the commensal gut microbiota (GM) may play a key role in maintaining animal health and production in an era of reduced antimicrobial usage. Gastrointestinal helminth infections impose a considerable burden on animal performance, and recent studies suggest that infection may substantially alter the composition and function of the GM. Here, we discuss the potential interactions between different bioactive dietary components (prebiotics, probiotics and phytonutrients) and helminth infection on the GM in livestock. A number of recent studies suggest that host diet can strongly influence the nature of the helminth-GM interaction. Nutritional manipulation of the GM may thus impact helminth infection, and conversely infection may also influence how the GM responds to dietary interventions. Moreover, a dynamic interaction exists between helminths, the GM, intestinal immune responses, and inflammation. Deciphering the mechanisms underlying the diet-GM-helminth axis will likely inform future helminth control strategies, as well as having implications for how health-promoting feed additives, such as probiotics, can play a role in sustainable animal production.

Parasite-Probiotic Interactions in the Gut: Bacillus sp. and Enterococcus faecium Regulate Type-2 Inflammatory Responses and Modify the Gut Microbiota of Pigs During Helminth Infection.

Dietary probiotics may enhance gut health by directly competing with pathogenic agents and through immunostimulatory effects. These properties are recognized in the context of bacterial and viral pathogens, but less is known about interactions with eukaryotic pathogens such as parasitic worms (helminths). In this study we investigated whether two probiotic mixtures (comprised of Bacillus amyloliquefaciens, B. subtilis, and Enterococcus faecium [BBE], or Lactobacillus rhamnosus LGG and Bifidobacterium animalis subspecies Lactis Bb12 [LB]) could modulate helminth infection kinetics as well as the gut microbiome and intestinal immune responses in pigs infected with the nodular worm Oesophagostomum dentatum. We observed that neither probiotic mixture influenced helminth infection levels. BBE, and to a lesser extent LB, changed the alpha- and beta-diversity indices of the colon and fecal microbiota, notably including an enrichment of fecal Bifidobacterium spp. by BBE. However, these effects were muted by concurrent O. dentatum infection. BBE (but not LB) significantly attenuated the O. dentatum-induced upregulation of genes involved in type-2 inflammation and restored normal lymphocyte ratios in the ileo-caecal lymph nodes that were altered by infection. Moreover, inflammatory cytokine release from blood mononuclear cells and intestinal lymphocytes was diminished by BBE. Collectively, our data suggest that selected probiotic mixtures can play a role in maintaining immune homeostasis during type 2-biased inflammation. In addition, potentially beneficial changes in the microbiome induced by dietary probiotics may be counteracted by helminths, highlighting the complex inter-relationships that potentially exist between probiotic bacteria and intestinal parasites.

Cardiopulmonary nematodes of wild carnivores from Denmark: Do they serve as reservoir hosts for infections in domestic animals?

The cardiopulmonary nematodes Angiostrongylus vasorum, Crenosoma vulpis, Capillaria aerophila and Aelurostrongylus abstrusus, are a cause of concern in the scientific and veterinary community, potentially causing significant disease in domestic animals. To investigate the potential of wild carnivores as reservoir hosts to these parasites, a total of 1041 animals from seven regions of Denmark were sampled: 476 raccoon dogs (Nyctereutes procyonoides), 367 red foxes (Vulpes vulpes), 123 American mink (Neovison vison), 31 beech martens (Martes foina), 30 Eurasian otters (Lutra lutra) and 14 polecats (Mustela putorius). Hearts and lungs were collected and examined for cardiopulmonary parasites. Capillaria aerophila was identified using morphology, whereas A. vasorum and C. vulpis were identified by a duplex real-time PCR, and A. abstrusus by conventional PCR. This is the first Danish report of A. vasorum and C. vulpis infections in raccoon dogs, mink and polecats, and of C. aerophila in raccoon dogs and beech martens. In addition, this is the first time A. vasorum and C. vulpis have been identified in wild animals from the island of Bornholm, just as it is the first report of C. vulpis in American mink, and C. vulpis and A. vasorum in polecats in Europe. The prevalence of A. vasorum appears to have increased in red foxes in Denmark compared to previous studies, while C. vulpis and C. aerophila prevalences are lower. Our data show that several wild carnivores can serve as reservoir hosts for A. vasorum, C. vulpis and C. aerophila in Denmark, and that A. vasorum appears more abundant than previously reported. It is speculated that the A. vasorum increase might relate to increased snail abundance, which may be due to a rise in mean yearly temperatures in Denmark.

Fermentable Dietary Fiber Promotes Helminth Infection and Exacerbates Host Inflammatory Responses.

Fermentable dietary fibers promote the growth of beneficial bacteria, can enhance mucosal barrier integrity, and reduce chronic inflammation. However, effects on intestinal type 2 immune function remain unclear. In this study, we used the murine whipworm Trichuris muris to investigate the effect of the fermentable fiber inulin on host responses to infection regimes that promote distinct Th1 and Th2 responses in C57BL/6 mice. In uninfected mice, dietary inulin stimulated the growth of beneficial bacteria, such as Bifidobacterium (Actinobacteria) and Akkermansia (Verrucomicrobia). Despite this, inulin prevented worm expulsion in normally resistant mice, instead resulting in chronic infection, whereas mice fed an equivalent amount of nonfermentable fiber (cellulose) expelled worms normally. Lack of expulsion in the mice fed inulin was accompanied by a significantly Th1-skewed immune profile characterized by increased T-bet+ T cells and IFN-γ production in mesenteric lymph nodes, increased expression of Ido1 in the cecum, and a complete absence of mast cell and IgE production. Furthermore, the combination of dietary inulin and high-dose T. muris infection caused marked dysbiosis, with expansion of the Firmicutes and Proteobacteria phyla, near elimination of Bacteroidetes, and marked reductions in cecal short-chain fatty acids. Neutralization of IFN-γ during infection abrogated Ido1 expression and was sufficient to restore IgE production and worm expulsion in inulin-fed mice. Our results indicate that, whereas inulin promoted gut health in otherwise healthy mice, during T. muris infection, it exacerbated inflammatory responses and dysbiosis. Thus, the positive effects of fermentable fiber on gut inflammation appear to be context dependent, revealing a novel interaction between diet and infection.

Dietary Inulin and Trichuris suis Infection Promote Beneficial Bacteria Throughout the Porcine Gut.

The gut microbiota (GM) displays a profound ability to adapt to extrinsic factors, such as gastrointestinal pathogens and/or dietary alterations. Parasitic worms (helminths) and host-associated GM share a long co-evolutionary relationship, exerting mutually modulatory effects which may impact the health of the host. Moreover, dietary components such as prebiotic fibers (e.g. inulin) are capable of modulating microbiota toward a composition often associated with a healthier gut function. The effect of helminth infection on the host microbiota is still equivocal, and it is also unclear how parasites and prebiotic dietary components interact to influence the microbiota and host health status. Some helminths, such as Trichuris suis (porcine whipworm), also exhibit strong immunomodulatory and anti-inflammatory effects. We therefore explored the effects of T. suis, alone and in interaction with inulin, both in fecal microbiota during the infection period and luminal microbiota across four intestinal segments at the end of a 4-week infection period. We observed that T. suis generally had minimal, but mainly positive, effects on the microbiota. T. suis increased the relative abundance of bacterial genera putatively associated with gut health such as Prevotella, and decreased bacteria such as Proteobacteria that have been associated with dysbiosis. Interestingly, dietary inulin interacted with T. suis to enhance these effects, thereby modulating the microbiota toward a composition associated with reduced inflammation. Our results show that administration of T. suis together with the consumption of prebiotic inulin may have the potential to positively affect gut health.

Effects of the dietary fibre inulin and Trichuris suis products on inflammatory responses in lipopolysaccharide-stimulated macrophages.

Consumption of fermentable dietary fibres, such as inulin, or administration of helminth products (e.g. Trichuris suis ova) have independently been shown to alleviate inflammation in vivo. We recently found that dietary inulin and T. suis infection in pigs co-operatively suppressed type-1 inflammatory responses in the gut, suggesting the potential of dietary components to augment anti-inflammatory responses induced by certain helminths. Here, we explored whether T. suis antigens and inulin could directly suppress inflammatory responses in vitro in a cooperative manner. T. suis soluble products (TsSP) strongly suppressed lipopolysaccharide (LPS)-induced IL-6 and TNF-α secretion from murine macrophages and induced an anti-inflammatory phenotype as evidenced by transcriptomic and gene pathway analyses. Inulin regulated the expression of a small number of genes and transcriptional pathways in macrophages after exposure to LPS, but did not enhance the suppressive activity of TsSP, either directly or in co-culture experiments with intestinal epithelial cells. Culture of macrophages with short-chain fatty acids, the products of microbial fermentation of inulin, did however appear to enhance TsSP-mediated inhibition of TNF-α production. Our results confirm a direct role for helminth products in suppressing inflammatory responses in macrophages. In contrast, inulin had little capacity to directly modulate LPS-induced responses. Our results suggest distinct mode-of-actions of T. suis and inulin in regulating inflammatory responses, and that the role of inulin in modulating the response to helminth infection may be dependent on other factors such as production of metabolites by the gut microbiota.

Environmental DNA for improved detection and environmental surveillance of schistosomiasis.

Schistosomiasis is a water-based, infectious disease with high morbidity and significant economic burdens affecting >250 million people globally. Disease control has, with notable success, for decades focused on drug treatment of infected human populations, but a recent paradigm shift now entails moving from control to elimination. To achieve this ambitious goal, more sensitive diagnostic tools are needed to monitor progress toward transmission interruption in the environment, especially in low-intensity infection areas. We report on the development of an environmental DNA (eDNA)-based tool to efficiently detect DNA traces of the parasite Schistosoma mansoni directly in the aquatic environment, where the nonhuman part of the parasite life cycle occurs. This is a report of the successful detection of S. mansoni in freshwater samples by using aquatic eDNA. True eDNA was detected in as few as 10 cercariae per liter of water in laboratory experiments. The field applicability of the method was tested at known transmission sites in Kenya, where comparison of schistosome detection by conventional snail surveys (snail collection and cercariae shedding) with eDNA (water samples) showed 71% agreement between the methods. The eDNA method furthermore detected schistosome presence at two additional sites where snail shedding failed, demonstrating a higher sensitivity of eDNA sampling. We conclude that eDNA provides a promising tool to substantially improve the environmental surveillance of S. mansoni Given the proper method and guideline development, eDNA could become an essential future component of the schistosomiasis control tool box needed to achieve the goal of elimination.

Mucosal Barrier and Th2 Immune Responses Are Enhanced by Dietary Inulin in Pigs Infected With Trichuris suis.

Diet composition may play a crucial role in shaping host immune responses and commensal gut microbiota populations. Bioactive dietary components, such as inulin, have been extensively studied for their bioactive properties, particularly in modulating gut immune function and reducing inflammation. It has been shown that colonization with gastrointestinal parasitic worms (helminths) may alleviate chronic inflammation through promotion of T-helper cell type (Th) 2 and T-regulatory immune responses and alterations in the gut microbiome. In this study, we investigated if dietary inulin could modulate mucosal immune function in pigs during colonization with the porcine whipworm Trichuris suis. T. suis infection induced a typical Th2-biased immune response characterized by transcriptional changes in Th2- and barrier function-related genes, accompanied by intestinal remodeling through increased epithelial goblet and tuft cell proliferation. We observed that inulin also up-regulated Th2-related immune genes (IL13, IL5), and suppressed Th1-related pro-inflammatory genes (IFNG, IL1A, IL8) in the colon. Notably, inulin augmented the T. suis-induced responses with increased transcription of key Th2 and mucosal barrier genes (e.g., IL13, TFF3), and synergistically suppressed pro-inflammatory genes, such as IFNG and CXCL9. 16S rRNA sequencing of proximal colon digesta samples revealed that inulin supplementation reduced the abundance of bacterial phyla linked to inflammation, such as Proteobacteria and Firmicutes, and simultaneously increased Actinobacteria and Bacteroidetes. Interestingly, pigs treated with both inulin and T. suis displayed the highest Bacteroidetes: Firmicutes ratio and the lowest gut pH, suggesting an interaction of diet and helminth infection that stimulates the growth of beneficial bacterial species. Overall, our data demonstrate that T. suis infection and inulin co-operatively enhance anti-inflammatory immune responses, which is potentially mediated by changes in microbiota composition. Our results highlight the intricate interactions between diet, immune function and microbiota composition in a porcine helminth infection model. This porcine model should facilitate further investigations into the use of bioactive diets as immunomodulatory mediators against inflammatory conditions, and how diet and parasites may influence gut health.

Effect of the nematophagous fungus Pochonia chlamydosporia on soil content of ascarid eggs and infection levels in exposed hens.

BACKGROUND: The nematophagous fungus Pochonia chlamydosporia can degrade ascarid (e.g. Ascaridia galli) eggs in agar and soil in vitro. However, it has not been investigated how this translates to reduced infection levels in naturally exposed chickens. We thus tested the infectivity of soil artificially contaminated with A. galli (and a few Heterakis gallinarum) eggs and treated with P. chlamydosporia. Sterilised and non-sterilised soils were used to examine any influence of natural soil biota. METHODS: Unembryonated eggs were mixed with sterilised (S)/non-sterilised (N) soil, either treated with the fungus (F) or left as untreated controls (C) and incubated (22 °C, 35 days) to allow eggs to embryonate and fungus to grow. Egg number in soil was estimated on days 0 and 35 post-incubation. Hens were exposed to the soil (SC/SF/NC/NF) four times over 12 days by mixing soil into the feed. On day 42 post-first-exposure (p.f.e.), the hens were euthanized and parasites were recovered. Serum A. galli IgY level and ascarid eggs per gram of faeces (EPG) were examined on days -1 and 36 (IgY) or 40 p.f.e. (EPG). RESULTS: Egg recovery in SF soil was substantially lower than in SC soil, but recovery was not significantly different between NF and NC soils. SF hens had a mean worm count of 76 whereas the other groups had means of 355-453. Early mature/mature A. galli were recovered from SF hens whereas hens in the other groups harboured mainly immature A. galli. Heterakis gallinarum counts were low overall, especially in SF. The SF post-exposure IgY response was significantly lower while EPG was significantly higher compared to the other groups. CONCLUSIONS: Pochonia chlamydosporia was very effective in reducing ascarid egg numbers in sterilised soil and thus worm burdens in the exposed hens. However, reduced exposure of hens shifted A. galli populations toward a higher proportion of mature worms and resulted in a higher faecal egg excretion within the study period. This highlights a fundamental problem in ascarid control: if not all eggs in the farm environment are inactivated, the resulting low level infections may result in higher contamination levels with associated negative long-term consequences.

Balancing knowledge and basic principles in veterinary parasitology - Competencies for future Danish veterinary graduates.

Veterinary parasitology has always been considered to be relevant and interesting by the Danish veterinary students. Students have to acquaint themselves with many new, small creatures with complicated and varied life cycles and with intricate Latin names that are difficult to pronounce, as only a few parasites have Danish names. In our veterinary curriculum, zoology has disappeared as a discipline, and parasitology has gradually moved from the third year to the beginning of the second year, which implies that, for example, pathology and pharmacology are "unknown fields". The number of contact hours in veterinary parasitology has been gradually cut to 24 lectures (35 min each) and practical exercises (24 h), including 9 h on coprology. The course is taught and examined jointly with bacteriology and virology in a 8-week course. As a comprehensive course, it has become increasingly difficult to get students to acquire enough active knowledge of the most common parasites and an understanding of the basic principles in relation to, for example, transmission and control. Even though information is readily accessible through books and on-line resources, we still believe that a competent clinician should know a range of parasites by heart as an active resource for their work. The dilemma has been tackled (partly) by introducing a veterinary paraclinical refresher course of 18 h (half practicals and half lectures) in the fourth study year. The focus here is on host(herd)-oriented clinical and diagnostic parasitology. The students can also now select a One Health track for six months in which zoonotic parasites are obviously a relevant topic.

Novel approach to study gastropod-mediated innate immune reactions against metastrongyloid parasites.

The anthropozoonotic metastrongyloid nematodes Angiostrongylus cantonensis and Angiostrongylus costaricensis, as well as Angiostrongylus vasorum, Crenosoma vulpis, Aelurostrongylus abstrusus and Troglostrongylus brevior are currently considered as emerging gastropod-borne parasites and have gained growing scientific attention in the last years. However, the knowledge on invertebrate immune responses and on how metastrongyloid larvae are attacked by gastropod immune cells is still limited. This work aims to describe an in vitro system to investigate haemocyte-derived innate immune responses of terrestrial gastropods induced by vital axenic metastrongyloid larvae. We also provide protocols on slug/snail management and breeding under standardized climate conditions (circadian cycle, temperature and humidity) for the generation of parasite-free F0 stages which are essential for immune-related investigations. Adult slug species (Arion lusitanicus, Limax maximus) and giant snails (Achatina fulica) were maintained in fully automated climate chambers until mating and production of fertilized eggs. Newly hatched F0 juvenile specimens were kept under parasite-free conditions before experimental use. An improved protocol for gastropod haemolymph collection and haemocyte isolation was established. Giemsa-stained haemolymph preparations showed adequate haemocyte isolation in all three gastropod species. Additionally, a protocol for the production of axenic first and third stage larvae (L1, L3) was established. Haemocyte functionality was tested in haemocyte-nematode-co-cultures. Scanning electron microscopy (SEM) and light microscopy analyses revealed that gastropod-derived haemocytes formed clusters as well as DNA-rich extracellular aggregates catching larvae and decreasing their motility. These data confirm the usefulness of the presented methods to study haemocyte-mediated gastropod immune responses to better understand the complex biology of gastropod-borne diseases.

Survival and development of chicken ascarid eggs in temperate pastures.

Eggs of chicken ascarids (Ascaridia galli and Heterakis spp.) are believed to be hardy and survive for long periods. However, this has not been evaluated quantitatively and our study therefore aimed to determine development and recovery of chicken ascarid eggs after burying in pasture soil. Unembryonated eggs were mixed with soil, placed in sealed nylon bags and buried at 7 cm depth in pasture plots April (spring, n = 72) and December 2014 (winter, n = 72). Eight randomly selected bags per season were used to estimate pre-burial egg recovery [0 week post-burial (wpb)]. Eight random bags were removed at 5, 12, 23, 38, 52, 71 wpb per season and additionally at 104 wpb for spring burial. The content of each bag was analysed for numbers and development stages of eggs. Eggs buried in spring were fully embryonated within 12 wpb. In contrast, eggs buried in winter were developing between 23 and 38 wpb, so that all viable eggs seemed to be fully developed by 38 wpb. About 90% eggs disappeared within 23 wpb (spring) and 38 wpb (winter). Small proportions (2-3%) of seemingly viable and infective eggs were still recovered up to 2 years after deposition. In conclusion, most eggs buried in temperate pasture soil seem to experience a heavy mortality within a few months after the deposition, especially during warm periods. However, a small proportion of eggs may survive and remain infective for at least 2 years.

Gastropod-derived haemocyte extracellular traps entrap metastrongyloid larval stages of Angiostrongylus vasorum, Aelurostrongylus abstrusus and Troglostrongylus brevior.

BACKGROUND: Phagocyte-derived extracellular traps (ETs) were recently demonstrated mainly in vertebrate hosts as an important effector mechanism against invading parasites. In the present study we aimed to characterize gastropod-derived invertebrate extracellular phagocyte trap (InEPT) formation in response to larval stages of important canine and feline metastrongyloid lungworms. Gastropod haemocytes were isolated from the slug species Arion lusitanicus and Limax maximus, and the snail Achatina fulica, and exposed to larval stages of Angiostrongylus vasorum, Aelurostrongylus abstrusus and Troglostrongylus brevior and investigated for gastropod-derived InEPT formation. RESULTS: Phase contrast as well as scanning electron microscopy (SEM) analyses of lungworm larvae-exposed haemocytes revealed ET-like structures to be extruded by haemocytes thereby contacting and ensnaring the parasites. Co-localization studies of haemocyte-derived extracellular DNA with histones and myeloperoxidase in larvae-entrapping structures confirmed classical characteristics of ETs. In vivo exposure of slugs to A. vasorum larvae resulted in InEPTs being extruded from haemocytes in the slug mucous extrapallial space emphasizing the pivotal role of this effector mechanism against invasive larvae. Functional larval entrapment assays demonstrated that almost half of the haemocyte-exposed larvae were contacted or even immobilized by released InEPTs. Overall, as reported for mammalian-derived ETs, different types of InEPTs were here observed, i.e. aggregated, spread and diffused InEPTs. CONCLUSIONS: To our knowledge, this study represents the first report on metastrongyloid lungworm-triggered ETosis in gastropods thereby providing evidence of early mollusc host innate immune reactions against invading larvae. These findings will contribute to the better understanding on complex parasite-intermediate host interactions since different gastropod species bear different transmitting capacities for metastrongyloid infections.

Occurrence and clinical significance of Aelurostrongylus abstrusus and other endoparasites in Danish cats.

Feline endoparasites are highly prevalent worldwide and may cause a variety of clinical signs in infected cats. Prevalence rates are dynamic and there is limited knowledge of the current prevalence in Denmark and the clinical manifestation and significance of especially the lungworm Aelurostrongylus abstrusus. This study investigated the total and local prevalence of Aelurostrongylus abstrusus and other endoparasites in Danish cats. The clinical significance of feline aelurostrongylosis was also examined through identification of frequency and severity of selected clinical signs. Faecal samples (n=327) and clinical data (n=312) were collected from August to October 2015, primarily from outdoor cats located at shelters distributed across Denmark. A modified Baermann method and a concentration McMaster technique was used to diagnose A. abstrusus first stage larvae and eggs/oocysts of other endoparasites. The total A. abstrusus prevalence was 8.3% [95% CI: 5.6-11.9] but local prevalence rates varied from 0% [95% CI: 0.0-8.8] to 31.4% [95% CI: 16.9-49.3]. A rural habitat appeared to increase the risk of A. abstrusus and this accounted for most of the local variation. Furthermore, the risk of infection was lower in kittens younger than 11 weeks compared to older cats (p=0.002). The cats were also infected with Toxocara cati (44.4% [95% CI: 38.3-50.7]), taeniid species (8.9% [95% CI: 5.7-13.0]), Capillaria aerophila (3.1% [95% CI: 1.3-6.0]), Aonchotheca putorii (3.9% [95% CI: 1.9-7.0]), Cystoisospora felis (3.1% [95% CI: 1.3-6.0]) and Cystoisospora rivolta (2.3% [95% CI: 0.9-5.0]), but there was no difference in local distribution. Co-infection was common, as 66.7% of A. abstrusus infected cats were also infected with one or more other parasites, the most common being T. cati. However, none of these parasites were significantly associated with A. abstrusus. The vast majority of the A. abstrusus infected cats displayed mild to moderate clinical signs. The main symptoms associated with the infection were increased sound on auscultation of the lungs (p=0.002), increased respiratory rate (p=0.02), coughing (p=0.007) and enlarged mandibular lymph nodes (p=0.002). None of these symptoms were associated with T. cati or C. aerophila which may also affect the lungs. This supports that the symptoms may be related to A. abstrusus and that aelurostrongylosis should be considered an important differential diagnosis in any feline respiratory patient.