Characterisation of seryl tRNA synthetase (srs-2) in Haemonchus contortus and Teladorsagia circumcincta.

The aim of the study was to purify and characterise recombinant proteins with the potential as an anti-parasite vaccine. Full-length cDNAs encoding seryl-tRNA synthetase (srs-2) were cloned from Haemonchus contortus (HcSRS-2) and Teladorsagia circumcincta (TcSRS-2). TcSRS-2 and HcSRS-2 cDNA (1458bp) encoded proteins of 486 amino acids, each of which was present as a single band of about 55 kDa on SDS-PAGE. Multiple alignments of the protein sequences showed homology of 94% between TcSRS-2 and HcSRS-2, 76-93% with SRS-2s of eight nematodes and 68% with Mus musculus SRS-2. The predicted three-dimensional structures revealed an overall structural homology of TcSRS-2 and HcSRS-2, highly conserved binding and catalytic sites, and minor differences in the tautomerase binding site residues in other nematode SRS-2 homologues. A phylogenetic tree was constructed using helminth and mammalian SRS-2 sequences. Soluble C-terminal SRS-2 proteins were expressed in Escherichia coli strain AY2.4 and purified. Recombinant HcSRS-2 assay shows that the recombinant enzyme was active and stable. The Km and Vmax for ATP were 3.9 ± 1.0 µM and 2.7 ± 0.1 µmol min-1 mg-1 protein, respectively. Antibodies in serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant HcSRS-2 and TcSRS-2 in enzyme-linked immunosorbent assays. Recognition of the recombinant proteins by antibodies generated by exposure of sheep to the native enzyme indicates similar antigenicity of the two proteins.

A single subcutaneous dose of eprinomectin (Eprecis®) is effective against common gastrointestinal nematodes and lungworms in experimentally infected lactating goats.

BACKGROUND: The health and productivity of dairy goats continue to be impacted by gastrointestinal nematodes (GIN) and lungworms (LW). Eprinomectin (EPN) is frequently selected for treatment because it is generally effective and does not require a milk withdrawal period. However, some factors, such as lactation, can have an impact on EPN pharmacokinetics and potentially its efficacy. To evaluate whether this can alter the efficacy of Eprecis® 2%, an eprinomectin injectable solution, a study was performed in lactating goats using the dose currently registered in cattle, sheep and goats (0.2 mg/kg). METHODS: This study was a blinded, randomized, controlled trial performed according to the VICH guidelines. Eighteen (18) worm-free lactating goats were included and experimentally challenged on day 28 with a mixed culture of infective gastrointestinal and lung nematode larvae (Haemonchus contortus, Trichostrongylus colubriformis, Teladorsagia circumcincta, Dictyocaulus filaria). At D-1, fecal samples were collected to confirm patent infection in all animals. On D0, the goats were randomly allocated into two groups of nine goats; group 1 was treated with Eprecis® 2% at 0.2 mg/kg BW by subcutaneous injection, while group 2 remained untreated. Fecal samples for egg counts were collected from all animals on days 3, 5, 7, 9, 11 and 14. On D14, all goats were killed, and the abomasum, small intestine and lungs were removed, processed and subsampled to record the number and species of worms. RESULTS: The treatment was well tolerated. After treatment, the arithmetic mean FEC decreased in the treated group and remained < 5 EPG until the end of the study, while the arithmetic mean FEC in the control group remained > 849.0 EPG. At D14, goats in the treated group had very limited or zero total worm counts, whereas all animals from the control group had a high worm burden. The measured efficacy was 100.0% against H. contortus and T. colubriformis, 99.9% against T. circumcincta and 98.0% against D. filaria. CONCLUSIONS: Eprinomectin (Eprecis®, 20 mg/ml), administered at the label dose (0.2 mg/kg), is highly effective against gastrointestinal nematodes and lungworms in lactating goats.

The feeding of heather (Calluna vulgaris) to Teladorsagia circumcincta infected lambs reduces parasitism but can detrimentally impact performance.

Gastrointestinal nematode (GIN) infections impact small ruminant health, welfare, and production across farming systems. Rising anthelmintic resistance and regulation of synthetic drug use in organic farming is driving research and development of sustainable alternatives for GIN control. One alternative is the feeding of plants that contain secondary metabolites (PSMs) e.g., proanthocyanidins (PA, syn. condensed tannins) that have shown anthelmintic potential. However, PSMs can potentially impair performance, arising from reduced palatability and thus intake, digestibility or even toxicity effects. In this study, we tested the trade-off between the antiparasitic and anti-nutritional effects of heather consumption by lambs. The impact of additional feeding of a nematophagous fungus (Duddingtonia flagrans) on larval development was also explored. Lambs infected with Teladorsagia circumcincta or uninfected controls, were offered ad libitum heather, or a control chopped hay for 22 days during the infection patent period. Eight days into the patent period, parasitised lambs were supplemented (or remained unsupplemented) with D. flagrans for a 5-day period. Performance and infection metrics were recorded, and polyphenol levels in the heather and control hay were measured to investigate their association with activity. The lambs consumed heather at approximately 20% of their dry matter intake, which was sufficient to exhibit significant anthelmintic effects via a reduction in total egg output (P = 0.007), compared to hay-fed lambs; the magnitude of the reduction over time in heather fed lambs was almost 10-fold compared to control lambs. Negative effects on production were shown, as heather-fed lambs weighed 6% less than hay-fed lambs (P < 0.001), even though dry matter intake (DMI) of heather increased over time. D. flagrans supplementation lowered larval recovery in the faeces of infected lambs by 31.8% (P = 0.003), although no interactions between feeding heather and D. flagrans were observed (P = 0.337). There was no significant correlation between PA, or other polyphenol subgroups in the diet and egg output, which suggests that any association between heather feeding and anthelmintic effect is not simply and directly attributable to the measured polyphenols. The level of heather intake in this study showed no antagonistic effects on D. flagrans, demonstrating the methods can be used in combination, but provide no additive effect on overall anthelmintic efficacies. In conclusion, heather feeding can assist to reduce egg outputs in infected sheep, but at 20% of DMI negative effects on lamb performance can be expected which may outweigh any antiparasitic benefits.

Exploring the transcriptomic changes underlying recombinant vaccine efficacy against Teladorsagia circumcincta in 3-month-old lambs.

Teladorsagia circumcincta is an abomasal parasitic nematode that can cause serious issues in small ruminant production, which are aggravated by drug resistance. Vaccines have been suggested as a feasible, long-lasting alternative for control since adaptation to the host's immune mechanisms by helminths develops at a much slower pace than anthelmintic resistance. Recently, a T. circumcincta recombinant subunit vaccine yielded over a 60% reduction in egg excretion and worm burden and induced strong humoral and cellular anti-helminth responses in vaccinated 3-month-old Canaria Hair Breed (CHB) lambs, but Canaria Sheep (CS) of a similar age were not protected by the vaccine. Here, we compared the transcriptomic profiles in the abomasal lymph nodes of such 3-month-old CHB and CS vaccinates 40 days after infection with T. circumcincta to understand differences in responsiveness at the molecular level. In the CS, differentially expressed genes (DEG) identified were related to general immunity processes such as antigen presentation or antimicrobial proteins and down-regulation of inflammation and immune response through regulatory T cell-associated genes. However, upregulated genes in CHB vaccinates were associated with type-2 oriented immune responses, i.e., immunoglobulin production, activation of eosinophils, as well as tissue structure and wound repair-related genes and protein metabolism pathways such as DNA and RNA processing. These results highlight potentially more optimal timing and orientation of immune responses in CHB sheep compared to CS associated with vaccine-induced protection. The data obtained in this study thus deepens our understanding of variations in responsiveness to vaccination in young lamb and provides insights for vaccine refinement strategies.

Refugia or reservoir? Feral goats and their role in the maintenance and circulation of benzimidazole-resistant gastrointestinal nematodes on shared pastures.

Gastrointestinal nematodes threaten the productivity of grazing livestock and anthelmintic resistance has emerged globally. It is broadly understood that wild ruminants living in sympatry with livestock act as a positive source of refugia for anthelmintic-susceptible nematodes. However, they might also act as reservoirs of anthelmintic-resistant nematodes, contributing to the spread of anthelmintic resistance at a regional scale. Here, we sampled managed sheep and cattle together with feral goats within the same property in New South Wales, Australia. Internal transcribed spacer 2 (ITS-2) nemabiome metabarcoding identified 12 gastrointestinal nematodes (Cooperia oncophora, Cooperia punctata, Haemonchus contortus, Haemonchus placei, Nematodirus spathiger, Ostertagia ostertagi, Teladorsagia circumcincta, Oesophagostomum radiatum, Oesophagostomum venulosum, Trichostrongylus axei, Trichostrongylus colubriformis and Trichostrongylus rugatus). Isotype-1 ß-tubulin metabarcoding targeting benzimidazole resistance polymorphisms identified 6 of these nematode species (C. oncophora, C. punctata, H. contortus, H. placei, O. ostertagi and T. circumcincta), with the remaining 3 genera unable to be identified to the species level (Nematodirus, Oesophagostomum, Trichostrongylus). Both ITS-2 and ß-tubulin metabarcoding showed the presence of a cryptic species of T. circumcincta, known from domestic goats in France. Of the gastrointestinal nematodes detected via ß-tubulin metabarcoding, H. contortus, T. circumcincta, Nematodirus and Trichostrongylus exhibited the presence of at least one resistance genotype. We found that generalist gastrointestinal nematodes in untreated feral goats had a similarly high frequency of the benzimidazole-resistant F200Y polymorphism as those nematodes in sheep and cattle. This suggests cross-transmission and maintenance of the resistant genotype within the wild ruminant population, affirming that wild ruminants should be considered potential reservoirs of anthelmintic resistance.

Chromosome-length genome assembly of Teladorsagia circumcincta - a globally important helminth parasite in livestock.

BACKGROUND: Gastrointestinal (GIT) helminthiasis is a global problem that affects livestock health, especially in small ruminants. One of the major helminth parasites of sheep and goats, Teladorsagia circumcincta, infects the abomasum and causes production losses, reductions in weight gain, diarrhoea and, in some cases, death in young animals. Control strategies have relied heavily on the use of anthelmintic medication but, unfortunately, T. circumcincta has developed resistance, as have many helminths. Vaccination offers a sustainable and practical solution, but there is no commercially available vaccine to prevent Teladorsagiosis. The discovery of new strategies for controlling T. circumcincta, such as novel vaccine targets and drug candidates, would be greatly accelerated by the availability of better quality, chromosome-length, genome assembly because it would allow the identification of key genetic determinants of the pathophysiology of infection and host-parasite interaction. The available draft genome assembly of T. circumcincta (GCA_002352805.1) is highly fragmented and thus impedes large-scale investigations of population and functional genomics. RESULTS: We have constructed a high-quality reference genome, with chromosome-length scaffolds, by purging alternative haplotypes from the existing draft genome assembly and scaffolding the result using chromosome conformation, capture-based, in situ Hi-C technique. The improved (Hi-C) assembly resulted in six chromosome-length scaffolds with length ranging from 66.6 Mbp to 49.6 Mbp, 35% fewer sequences and reduction in size. Substantial improvements were also achieved in both the values for N50 (57.1 Mbp) and L50 (5 Mbp). A higher and comparable level of genome and proteome completeness was achieved for Hi-C assembly on BUSCO parameters. The Hi-C assembly had a greater synteny and number of orthologs with a closely related nematode, Haemonchus contortus. CONCLUSION: This improved genomic resource is suitable as a foundation for the identification of potential targets for vaccine and drug development.

Comparative analysis of the anthelmintic efficacy of European heather extracts on Teladorsagia circumcincta and Trichostrongylus colubriformis egg hatching and larval motility.

BACKGROUND: Gastrointestinal nematode (GIN) control is traditionally achieved with the use of anthelmintic drugs, however due to regulations in organic farming and the rise in anthelmintic resistance, alternatives are sought after. A promising alternative is the use of bioactive plant feeding due to the presence of plant secondary metabolites (PSMs) such as proanthocyanidins (PAs). This study focussed on the perennial shrub heather (Ericaceae family), a plant rich in PAs, highly abundant across Europe and with previously demonstrated anthelmintic potential. METHODS: In vitro assays were used to investigate heather's anthelmintic efficacy against egg hatching and larval motility. Heather samples were collected from five European countries across two seasons, and extracts were tested against two GIN species: Teladorsagia circumcincta and Trichostrongylus colubriformis. Polyphenol group-specific ultraperformance liquid chromatography-tandem mass spectrometry analysis was performed to identify relevant polyphenol subgroups present, including the PA concentration and size and ratio of the subunits. Partial least squares analysis was performed to associate efficacy with variation in PSM composition. RESULTS: Heather extracts reduced egg hatching of both GIN species in a dose-dependent manner by up to 100%, while three extracts at the highest concentration (10 mg/ml) reduced larval motility to levels that were not significantly different from dead larvae controls. PAs, particularly the procyanidin type, and flavonol derivatives were associated with anthelmintic activity, and the particular subgroup of polyphenols associated with the efficacy was dependent on the GIN species and life stage. CONCLUSIONS: Our results provide in vitro evidence that heather, a widely available plant often managed as a weed in grazing systems, has anthelmintic properties attributed to various groups of PSMs and could contribute to sustainable GIN control in ruminant production systems across Europe.

Excretory-secretory products from the brown stomach worm, Teladorsagia circumcincta, exert antimicrobial activity in in vitro growth assays.

BACKGROUND: Over the past decade, evidence has emerged of the ability of gastrointestinal (GI) helminth parasites to alter the composition of the host gut microbiome; however, the mechanism(s) underpinning such interactions remain unclear. In the current study, we (i) undertake proteomic analyses of the excretory-secretory products (ESPs), including secreted extracellular vesicles (EVs), of the 'brown stomach worm' Teladorsagia circumcincta, one of the major agents causing parasite gastroenteritis in temperate areas worldwide; (ii) conduct bioinformatic analyses to identify and characterise antimicrobial peptides (AMPs) with putative antimicrobial activity; and (iii) assess the bactericidal and/or bacteriostatic properties of T. circumcincta EVs, and whole and EV-depleted ESPs, using bacterial growth inhibition assays. METHODS: Size-exclusion chromatography was applied to the isolation of EVs from whole T. circumcincta ESPs, followed by EV characterisation via nanoparticle tracking analysis and transmission electron microscopy. Proteomic analysis of EVs and EV-depleted ESPs was conducted using liquid chromatography-tandem mass spectrometry, and prediction of putative AMPs was performed using available online tools. The antimicrobial activities of T. circumcincta EVs and of whole and EV-depleted ESPs against Escherichia coli were evaluated using bacterial growth inhibition assays. RESULTS: Several molecules with putative antimicrobial activity were identified in both EVs and EV-depleted ESPs from adult T. circumcincta. Whilst exposure of E. coli to whole ESPs resulted in a significant reduction of colony-forming units over 3 h, bacterial growth was not reduced following exposure to worm EVs or EV-depleted ESPs. CONCLUSIONS: Our data points towards a bactericidal and/or bacteriostatic function of T. circumcincta ESPs, likely mediated by molecules with antimicrobial activity.

Nematode galectin binds IgE and modulates mast cell activity.

Mast cell degranulation is the major mechanism influencing establishment and survival of the abomasal nematode Teladorsagia circumcincta and probably many other gastrointestinal nematodes. Host galectins-3 and -9 have been shown to bind IgE and positively and negatively influence mast cell degranulation. As incoming nematodes produce large amounts of galectin, we hypothesised that nematode galectin competes with host galectin and inhibits mast cell degranulation. ELISA was used to show that nematode galectin reduced total IgE activity. Galectin also reduced the binding of sheep IgE to the surface of a mast cell line and decreased the release of LCT-4 and Beta hexosaminidase but not MMP-9. These results indicate that nematode galectin influences mast cell degranulation and identify a potential immunomodulatory mechanism used by nematodes to enhance their establishment and survival.

Differences in the protection elicited by a recombinant Teladorsagia circumcincta vaccine in weaned lambs of two Canarian sheep breeds.

Gastrointestinal nematode (GIN) infections are a serious drawback on small ruminant production. Since anthelmintic resistance has extended, optimisation of alternative non-chemical control strategies has attracted interest. Recently, a prototype recombinant vaccine protected immunologically mature sheep from Texel-cross and Canaria Sheep breeds against Teladorsagia circumcincta. The level of protective immunity stimulated by the vaccine varied between individuals and with age. Previous studies suggest that Canaria Hair Breed (CHB) sheep is naturally resistant to GIN infection, with some evidence suggesting that this protection is present in young lambs. Here, we sought to enhance this resistance by immunising three-month-old CHB lambs with a T. circumcincta prototype recombinant vaccine. Following vaccination and a larval challenge period, levels of protection against T. circumcincta infection were compared in CHB lambs with Canaria Sheep (CS) lambs (a breed considered less resistant to GIN). Lambs from the resistant CHB breed appeared to respond more favourably to vaccination, shedding 63% fewer eggs over the sampling period than unvaccinated CHB lambs. No protection was evident in CS vaccinated lambs. At post-mortem, CHB vaccine recipients had a 68% reduction in mean total worm burden, and female worms were significantly shorter and contained fewer eggs in utero compared to unvaccinated CHB lambs. A higher anti-parasite IgG2 level was detected in immunised CHB lambs compared to unvaccinated control CHB animals, with data suggesting that IgA, globular leucocytes, CD45RA+, CD4+ and CD8+ T cells are implicated in this protective response. The development of effective immunity in vaccinated CHB lambs did not reduce lamb growth rate as immunised CHB lambs had a significantly higher average daily weight gain after challenge than their unvaccinated counterparts. Therefore, the protection of CHB lambs was enhanced by immunisation at weaning, suggesting a synergistic effect when combining vaccination with presumed genetic resistance.

Local immune response of Canarian Majorera goats infected with Teladorsagia circumcincta.

BACKGROUND: Due to increased anthelmintic resistance, alternative methods to drugs are necessary to control gastrointestinal nematodes (GINs). Some of the most promising alternatives are based on the immune response of the host, such as the selection of genetically resistant breeds or the use of vaccines against these parasites. Given the limited information available on the immune response against GINs in goats, this study investigated the local immune response of goat kids of an indigenous Canary Islands breed (Majorera breed) experimentally infected with Teladorsagia circumcincta, one of the most pathogenic and prevalent GIN species. METHODS: For this purpose, the relationship between different parasitological (number of mature and immature worms, worm length, and number of intrauterine eggs) and immunological parameters at the local level (related to both the humoral and cellular immune response) was analyzed at early (1 week post-infection [wpi]) and late (8 wpi) stages of infection. RESULTS: Primary infection of goat kids with T. circumcincta infective larvae (L3) generated a complex immune response that could be defined as Th2 type, characterized by increased infiltration in abomasal tissues of several effector cells as well as a progressive presence of specific antibodies against parasitic antigens in the gastric mucus. Cellular responses were evidenced from 1 wpi onward, showing an increase in antigen-presenting cells and various lymphocyte subsets in the gastric mucosa. CONCLUSIONS: The complexity of the host response was evidenced by statistically significant changes in the number of all these subpopulations (MHCII+, CD4+, CD8+, γδ+, CD45R+, IgA+, and IgG+), as well as in the evolution of the relative cytokine gene expression. From a functional point of view, negative associations were observed between the number of most of the immune cells (CD4, IgA, IgG, and CD45R cells) and parameters that could be related to the fecundity of worms, a phenomenon that was especially evident when the number of IgG and CD45R cells or the specific IgA levels of the gastric mucus were compared with parasitological parameters such as the female worm length or fecal egg counts at 8 wpi.

Quantifying the sources of variation in eosinophilia among Scottish blackface lambs with mixed, predominantly Teladorsagia circumcincta nematode infection.

Eosinophils play a key role in defence against gastrointestinal nematodes. There is considerable variation among animals in the intensity of eosinophilia following nematode infection. However, the statistical distribution of eosinophils among animals has still to be determined. A better description of the variation among animals could provide biological insight and determine the most appropriate way to analyse the effect of eosinophils. We estimated blood eosinophil numbers in a flock of Scottish Blackface sheep that were naturally exposed to mixed, predominantly Teladorsagia circumcincta infection. Three of the four eosinophil counts were better described by a gamma distribution than by a lognormal distribution. The scale and shape parameters of the gamma distribution varied over time. Eosinophil counts differed among animals kept on separate fields before weaning and between singletons and twins but were not significantly different between years and genders. Eosinophil counts also differed among offspring from different sires and dams. The parameters of the gamma distribution were used to enable a power analysis. Large numbers of animals were required to reliably detect even large differences between two groups. These results indicate that methods appropriate for gamma distributions, such as generalized linear mixed models, will provide more reliable inferences than traditional methods of analysis and experimental design.

Teladorsagia Circumcincta Galectin-Mucosal Interactome in Sheep.

Teladorsagia circumcincta is the most important gastrointestinal parasite in the livestock industry in temperate regions around the world, causing great economic losses. The infective third-stage larvae (L3) of Teladorsagia circumcincta secrete a large number of excretory-secretory (E/S) molecules, some of which are likely to play critical roles in modulating the host immune response. One of the most abundant E/S molecules is a protein termed Tci-gal-1, which has similarity to mammalian galectins. Galectins are a family of carbohydrate-binding molecules, with characteristic domain organisation and affinity for ß-galactosids that mediate a variety of important cellular functions including inflammation and immune responses. To understand the role of Tci-gal-1 at the host-parasite interface, we used a proteomics pull-down approach to identify Tc-gal-1 interacting proteins from sheep abomasal scrapes and whole tissue. A total of 135 unique proteins were identified from whole abomasal tissue samples, while 89 proteins were isolated from abomasal scrape samples. Of these proteins, 63 were present in both samples. Many of the host proteins identified, such as trefoil factors and mucin-like proteins, play critical roles in the host response. The identification of Tci-gal-1 binding partners provides new insights on host-parasite interactions and could lead to the development of new control strategies.

Development and evaluation of an indirect ELISA for detection of Teladorsagia circumcincta infection in sheep.

BACKGROUND: The gastrointestinal helminth, Teladorsagia circumcincta, is one of the major health risks and production-limiting diseases in small ruminant populations, particularly in temperate regions. With the increasing importance of disease management and recruited anthelmintic resistant types, accurate approaches are needed for the diagnosis of the infection in the host. Due to uncertain results using faecal examinations, the ELISA method was indicated for the detection of nematode antigenic materials. Despite some promising results, problems were described in terms of test specificity and cross-reactions. Therefore, this study aimed to evaluate the IgG response to worm somatic and excretory/secretory (ES) products using western blot analysis and an indirect ELISA for the detection of T. circumcincta infection in sheep. RESULTS: Based on the immuno-reactivity analysis, immunogenic fractions with molecular weights (MWs) of approximately 60, 75 and 100 kDa were detected in somatic content and two antigens of about 63 and 75 kDa in ES material. Accordingly, a specific product at 75 kDa had the strongest reaction and appeared as the most common antigenic protein. In ELISA, all the sera from the infected sheep revealed the OD rates above the calculated cut-off value with about two-fold greater average. Negative control samples were also specifically recognized with the mean OD rate of about 1/3 of the estimated cut-off value. The cross-reaction test, using rabbit anti-T. circumcincta IgG, did not show reactivity with the ES antigens of other prevalent nematodes including Haemonchus contortus, Protostrongylus rufescens and Marshallagia marshalli. In contrast, a strong positive reaction was observed with the somatic antigens of M. marshalli. CONCLUSIONS: The results of this study indicated that the indirect ELISA method using the ES content enables distinguishing the T. circumcincta infected sheep with high specificity. Those antigenic ES peptides with 63 and particularly 75 kDa MWs should be further investigated due to the potential for serological diagnostic methods and immunoprotective targets in the host.

Integrating applied parasitological and molecular epidemiological methodologies to investigate the capacity of Haemonchus contortus to over-winter on pasture in Scotland.

BACKGROUND: The Barber's Pole worm, Haemonchus contortus is of major concern to sheep producers, particularly in the southern hemisphere. This nematode is also commonly found in many sheep flocks in Northern hemisphere countries but is generally not associated with acute clinical pathology. As with other nematode species, the pattern of disease is changing in the United Kingdom. Changes in management practices, climate, anthelmintic resistance prevalence and parasite adaptation are possible factors thought to be responsible for this. METHODS: In the present study, a combination of traditional applied parasitological and molecular species identification techniques were used to assess the capability of H. contortus infective larvae to over-winter on pasture and infect lambs in early spring. RESULTS: Adult and inhibited H. contortus worms were identified in previously worm-free tracer lambs that had grazed contaminated pasture in late winter/early spring (February/March). CONCLUSION: The study illustrated the benefit of using classical applied parasitology techniques in conjunction with molecular species identification methods to explore the epidemiology of gastro-intestinal nematodes of livestock. This study also demonstrated that larvae were able to survive over-winter, albeit in small numbers, and potentially contaminate pastures earlier than previously considered in northern regions of the UK.

Anthelmintic activity of aminoalcohol and diamine derivatives against the gastrointestinal nematode Teladorsagia circumcincta.

Gastrointestinal nematodes (GIN) infections are a serious problem in livestock production due to the great economic losses they cause. Their control is increasingly difficult because of the rapid development of drug resistance and the limited number of available drugs. Therefore, this study evaluated 18 aminoalcohol and 16 diamine derivatives against eggs, first and third stage larvae from a susceptible and a resistant isolate of Teladorsagia circumcincta collected from sheep. The effectiveness of the in vitro anthelmintic activity of the compounds was evaluated using three different procedures: Egg Hatch Test (EHT), Larval Mortality Test (LMT) and Larval Migration Inhibition Test (LMIT). Those compounds with activities higher than 90 % in the initial screening at 50 µM were selected to determine their half maximal effective concentration (EC50). In parallel, cytotoxicity assays were conducted on Caco2 and HepG2 cell lines to calculate Selectivity Indexes (SI) for each compound. The diamine 30 presented the best results in preventing egg hatching, displaying the lowest EC50 value (1.01 ±â€¯0.04 µM) of all compounds tested and the highest SI (21.21 vs. Caco-2 cells). For the LMIT, the diamine 34 showed the highest efficacy, with EC50 values of 2.67 ±â€¯0.08 and 3.02 ±â€¯0.09 µM on the susceptible and resistant isolate of the parasite, respectively.

Cellular and humoral immune responses associated with protection in sheep vaccinated against Teladorsagia circumcincta.

Due to increased anthelmintic resistance, complementary methods to drugs are necessary to control gastrointestinal nematodes (GIN). Vaccines are an environmentally-friendly and promising option. In a previous study, a Teladorsagia circumcincta recombinant sub-unit vaccine was administered to two sheep breeds with different levels of resistance against GIN. In the susceptible Canaria Sheep (CS) breed, vaccinates harboured smaller worms with fewer eggs in utero than the control group. Here, we extend this work, by investigating the cellular and humoral immune responses of these two sheep breeds following vaccination and experimental infection with T. circumcincta. In the vaccinated CS group, negative associations between antigen-specific IgA, IgG2 and Globule Leukocytes (GLs) with several parasitological parameters were established as well as a higher CD4+/CD8+ ratio than in control CS animals, suggesting a key role in the protection induced by the vaccine. In the more resistant Canaria Hair Breed (CHB) sheep the vaccine did not significantly impact on the parasitological parameters studied and none of these humoral associations were observed in vaccinated CHB lambs, although CHB had higher proportions of CD4+ and CD8+ T cells within the abomasal lymph nodes, suggesting higher mucosal T cell activation. Each of the component proteins in the vaccine induced an increase in immunoglobulin levels in vaccinated groups of each breed. However, levels of immunoglobulins to only three of the antigens (Tci-MEP-1, Tci-SAA-1, Tci-ASP-1) were negatively correlated with parasitological parameters in the CS breed and they may be, at least partially, responsible for the protective effect of the vaccine in this breed. These data could be useful for improving the current vaccine prototype.

Comparative evaluation of different molecular methods for DNA extraction from individual Teladorsagia circumcincta nematodes.

BACKGROUND: The purpose of this study was to develop a reliable DNA extraction protocol to use on individual Teladorsagia circumcincta nematode specimens to produce high quality DNA for genome sequencing and phylogenetic analysis. Pooled samples have been critical in providing the groundwork for T. circumcincta genome construction, but there is currently no standard method for extracting high-quality DNA from individual nematodes. 11 extraction kits were compared based on DNA quality, yield, and processing time. RESULTS: 11 extraction protocols were compared, and the concentration and purity of the extracted DNA was quantified. Median DNA concentration among all methods measured on NanoDrop 2000™ ranged between 0.45-11.5 ng/µL, and on Qubit™ ranged between undetectable - 0.962 ng/µL. Median A260/280 ranged between 0.505-3.925, and median A260/230 ranged - 0.005 - 1.545. Larval exsheathment to remove the nematode cuticle negatively impacted DNA concentration and purity. CONCLUSIONS: A Schistosoma sp. DNA extraction method was determined as most suitable for individual T. circumcincta nematode specimens due to its resulting DNA concentration, purity, and relatively fast processing time.

Triple lectin staining of trichostrongyle eggs from naturally infected small ruminants.

Trichostrongyle nematodes can be a major threat to the profitability of small ruminant producers depending of the species and intensity of trichostrongyles parasitizing their herd. Haemonchus contortus, Teladorsagia circumcincta, and Trichostrongylus colubriformis are typically the most common and clinically important species. Three lectins (PNA, LCA and AAL) have been reported to bind specifically to eggs from these three genera and therefore could be used to quantify the intensity of each species in individual animals. Peanut agglutinin (PNA) has been the most commonly tested lectin because it selectively binds intensely to eggs of the most pathogenic species, H. contortus. Lens culinaris agglutinin (LCA) and Aleuria aurantia agglutinin (AAL) have shown specificity to T. circumcincta and Trichostrongylus spp. respectively, however, these lectins have only been evaluated using eggs harvested directly from adult females, and not from fecal samples. The purpose of the present study is to describe a method to sequentially stain H. contortus, T. circumcincta and Trichostrongylus spp. fecal eggs with PNA, LCA and AAL, and then evaluate the resultant staining patterns seen with eggs collected from a naturally infected goat shown with PCR to contain H. contortus, T. circumcincta, Ostertagia leptospicularis, Trichostrongylus colubriformis and Trichostrongylus axei eggs. These results were also compared with patterns observed with eggs stained with single lectins and double combinations of lectins. The various patterns were then compared to those seen with egg samples collected from an ewe shown to only contain H. contortus. PNA bound intensely and uniformly to all eggs from samples containing only H. contortus eggs; however, some eggs additionally bound LCA and AAL in localized patches of varying size, and a few eggs exhibited intense and uniform binding of all three lectins. Single PNA-staining of goat samples containing the five trichostrongyles species identified most eggs as H. contortus, and triple-staining showed patterns consistent with those seen for H. contortus. Binding of AAL to non-Haemonchus eggs was uniform but showed significant variations in intensity. Lesser staining eggs tended to also stain intensely with LCA, which is consistent with published binding pattern for T. circumcincta. Most eggs that AAL bound intensely to did not bind with LCA, which is consistent with published binding pattern for Trichostrongylus spp. Autofluorescence was observed with the DAPI filter-cube among most non-Haemonchus eggs. This study demonstrates the need for additional field studies to further validate the specificity of these three lectins for use in identifying eggs from the three species of trichostrongyles.

Vaccination against the brown stomach worm, Teladorsagia circumcincta, followed by parasite challenge, induces inconsistent modifications in gut microbiota composition of lambs.

BACKGROUND: Growing evidence points towards a role of gastrointestinal (GI) helminth parasites of ruminants in modifying the composition of the host gut flora, with likely repercussions on the pathophysiology of worm infection and disease, and on animal growth and productivity. However, a thorough understanding of the mechanisms governing helminth-microbiota interactions and of their impact on host health and welfare relies on reproducibility and replicability of findings. To this aim, in this study, we analysed quantitative and qualitative fluctuations in the faecal microbiota composition of lambs vaccinated against, and experimentally infected with, the parasitic GI nematode Teladorsagia circumcincta over the course of two separate trials performed over two consecutive years. METHODS: Two trials were conducted under similar experimental conditions in 2017 and 2018, respectively. In each trial, lambs were randomly assigned to one of the following experimental groups: (i) vaccinated/infected, (ii) unvaccinated/infected and (iii) unvaccinated/uninfected. Faecal samples collected from individual animals were subjected to DNA extraction followed by high-throughput sequencing of the V3-V4 region of the bacterial 16S rRNA gene and bioinformatics and biostatistical analyses of sequence data. RESULTS: Substantial differences in the populations of bacteria affected by immunisation against and infection by T. circumcincta were detected when comparing data from the two trials. Nevertheless, the abundance of Prevotella spp. was significantly linked to helminth infection in both trials. CONCLUSIONS: Despite the largely conflicting findings between the two trials, our data revealed that selected gut microbial populations are consistently affected by T. circumcincta infection and/or vaccination. Nevertheless, our study calls for caution when interpreting data generated from in vivo helminth-microbiome interaction studies that may be influenced by several intrinsic and extrinsic host-, parasite- and environment-related factors.