The Caenorhabditis elegans DEG-3/DES-2 Channel Is a Betaine-Gated Receptor Insensitive to Monepantel.

Natural plant compounds, such as betaine, are described to have nematocidal properties. Betaine also acts as a neurotransmitter in the free-living model nematode Caenorhabditis elegans, where it is required for normal motility. Worm motility is mediated by nicotinic acetylcholine receptors (nAChRs), including subunits from the nematode-specific DEG-3 group. Not all types of nAChRs in this group are associated with motility, and one of these is the DEG-3/DES-2 channel from C. elegans, which is involved in nociception and possibly chemotaxis. Interestingly, the activity of DEG-3/DES-2 channel from the parasitic nematode of ruminants, Haemonchus contortus, is modulated by monepantel and its sulfone metabolite, which belong to the amino-acetonitrile derivative anthelmintic drug class. Here, our aim was to advance the pharmacological knowledge of the DEG-3/DES-2 channel from C. elegans by functionally expressing the DEG-3/DES-2 channel in Xenopus laevis oocytes and using two-electrode voltage-clamp electrophysiology. We found that the DEG-3/DES-2 channel was more sensitive to betaine than ACh and choline, but insensitive to monepantel and monepantel sulfone when used as direct agonists and as allosteric modulators in co-application with betaine. These findings provide important insight into the pharmacology of DEG-3/DES-2 from C. elegans and highlight the pharmacological differences between non-parasitic and parasitic nematode species.

Functional Characterization of the Oxantel-Sensitive Acetylcholine Receptor from Trichuris muris.

The human whipworm, Trichuris trichiura, is estimated to infect 289.6 million people globally. Control of human trichuriasis is a particular challenge, as most anthelmintics have a limited single-dose efficacy, with the striking exception of the narrow-spectrum anthelmintic, oxantel. We recently identified a novel ACR-16-like subunit from the pig whipworm, T. suis which gave rise to a functional acetylcholine receptor (nAChR) preferentially activated by oxantel. However, there is no ion channel described in the mouse model parasite T. muris so far. Here, we have identified the ACR-16-like and ACR-19 subunits from T. muris, and performed the functional characterization of the receptors in Xenopus laevis oocytes using two-electrode voltage-clamp electrophysiology. We found that the ACR-16-like subunit from T. muris formed a homomeric receptor gated by acetylcholine whereas the ACR-19 failed to create a functional channel. The subsequent pharmacological analysis of the Tmu-ACR-16-like receptor revealed that acetylcholine and oxantel were equally potent. The Tmu-ACR-16-like was more responsive to the toxic agonist epibatidine, but insensitive to pyrantel, in contrast to the Tsu-ACR-16-like receptor. These findings confirm that the ACR-16-like nAChR from Trichuris spp. is a preferential drug target for oxantel, and highlights the pharmacological difference between Trichuris species.

The narrow-spectrum anthelmintic oxantel is a potent agonist of a novel acetylcholine receptor subtype in whipworms.

In the absence of efficient alternative strategies, the control of parasitic nematodes, impacting human and animal health, mainly relies on the use of broad-spectrum anthelmintic compounds. Unfortunately, most of these drugs have a limited single-dose efficacy against infections caused by the whipworm, Trichuris. These infections are of both human and veterinary importance. However, in contrast to a wide range of parasitic nematode species, the narrow-spectrum anthelmintic oxantel has a high efficacy on Trichuris spp. Despite this knowledge, the molecular target(s) of oxantel within Trichuris is still unknown. In the distantly related pig roundworm, Ascaris suum, oxantel has a small, but significant effect on the recombinant homomeric Nicotine-sensitive ionotropic acetylcholine receptor (N-AChR) made up of five ACR-16 subunits. Therefore, we hypothesized that in whipworms, a putative homolog of an ACR-16 subunit, can form a functional oxantel-sensitive receptor. Using the pig whipworm T. suis as a model, we identified and cloned a novel ACR-16-like subunit and successfully expressed the corresponding homomeric channel in Xenopus laevis oocytes. Electrophysiological experiments revealed this receptor to have distinctive pharmacological properties with oxantel acting as a full agonist, hence we refer to the receptor as an O-AChR subtype. Pyrantel activated this novel O-AChR subtype moderately, whereas classic nicotinic agonists surprisingly resulted in only minor responses. We observed that the expression of the ACR-16-like subunit in the free-living nematode Caenorhabditis elegans conferred an increased sensitivity to oxantel of recombinant worms. We demonstrated that the novel Tsu-ACR-16-like receptor is indeed a target for oxantel, although other receptors may be involved. These finding brings new insight into the understanding of the high sensitivity of whipworms to oxantel, and highlights the importance of the discovery of additional distinct receptor subunit types within Trichuris that can be used as screening tools to evaluate the effect of new synthetic or natural anthelmintic compounds.

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.

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.

Pathway of oxfendazole from the host into the worm: Trichuris suis in pigs.

It is well known that the efficacy of a single oral dose of benzimidazoles against Trichuris spp. infections in humans and animals is poor, but is currently still used in control programmes against human trichuriasis. However, the route of the benzimidazoles from the treated host to Trichuris remains unknown. As parts of adult Trichuris are situated intracellularly in the caecum, they might be exposed to anthelmintic drugs in the intestinal content as well as the mucosa. In this study, the pathway of oxfendazole and its metabolites was explored using a T. suis-pig infection model, by simultaneously measuring drug concentrations within the worms and the caecal mucosa, caecal tissue, caecal content and plasma of pigs over time after a single oral dose of 5 mg/kg oxfendazole. Additionally, for comparison to the in vivo study, drug uptake and metabolism of oxfendazole by T. suis was examined after in vitro incubation. Oxfendazole and metabolites were quantified by High Performance Liquid Chromatography. Multivariate linear regression analysis showed a strong and highly significant association between OFZ concentrations within T. suis and in plasma, along with a weaker association between OFZ concentrations in caecal tissue/mucosa and T. suis, suggesting that oxfendazole reaches T. suis after absorption from the gastrointestinal tract and enters the worms by the blood-enterocyte pathway. The fenbendazole sulfone level in T. suis was highly affected by the concentrations in plasma. In addition, correlations between drug concentrations in the host compartments, were generally highest for this metabolite. In comparison to oxfendazole, the correlation between plasma and content was particularly high for this metabolite, suggesting a high level of drug movement between these compartments and the possible involvement of the enterohepatic circulation.

Dietary cinnamaldehyde enhances acquisition of specific antibodies following helminth infection in pigs.

Dietary phytonutrients such as cinnamaldehyde (CA) may contribute to immune function during pathogen infections, and CA has been reported to have positive effects on gut health when used as feed additive for livestock. Here, we investigated whether CA could enhance antibody production and specific immune responses during infection with an enteric pathogen. We examined the effect of dietary CA on plasma antibody levels in parasite-naïve pigs, and subsequently acquisition of humoral immune responses during infection with the parasitic nematode Ascaris suum. Parasite-naïve pigs fed diets supplemented with CA had higher levels of total IgA and IgG in plasma, and A. suum-infected pigs fed CA had higher levels of parasite-specific IgM and IgA in plasma 14days post-infection. Moreover, dietary CA increased expression of genes encoding the B-cell marker CD19, sodium/glucose co-transporter1 (SCA5L1) and glucose transporter 2 (SLC2A2) in the jejunal mucosa of A.suum-infected pigs. Dietary CA induced only limited changes in the composition of the prokaryotic gut microbiota of A. suum-infected pigs, and in vitro experiments showed that CA did not directly induce proliferation or increase secretion of IgG and IgA from lymphocytes. Our results demonstrate that dietary CA can significantly enhance acquisition of specific immune responses in pigs. The underlying mechanism remains obscure, but apparently does not derive simply from direct contact between CA and host lymphocytes and appears to be independent of the gut microbiota.

Glucose Absorption by the Bacillary Band of Trichuris muris.

BACKGROUND: A common characteristic of Trichuris spp. infections in humans and animals is the variable but low efficacy of single-dose benzimidazoles currently used in mass drug administration programmes against human trichuriasis. The bacillary band, a specialised morphological structure of Trichuris spp., as well as the unique partly intracellular habitat of adult Trichuris spp. may affect drug absorption and perhaps contribute to the low drug accumulation in the worm. However, the exact function of the bacillary band is still unknown. METHODOLOGY: We studied the dependency of adult Trichuris muris on glucose and/or amino acids for survival in vitro and the absorptive function of the bacillary band. The viability of the worms was evaluated using a motility scale from 0 to 3, and the colorimetric assay Alamar Blue was utilised to measure the metabolic activity. The absorptive function of the bacillary band in living worms was explored using a fluorescent glucose analogue (6-NBDG) and confocal microscopy. To study the absorptive function of the bacillary band in relation to 6-NBDG, the oral uptake was minimised or excluded by sealing the oral cavity with glue and agarose. PRINCIPAL FINDINGS: Glucose had a positive effect on both the motility (p < 0.001) and metabolic activity (p < 0.001) of T. muris in vitro, whereas this was not the case for amino acids. The 6-NBDG was observed in the pores of the bacillary band and within the stichocytes of the living worms, independent of oral sealing. CONCLUSIONS/SIGNIFICANCE: Trichuris muris is dependent on glucose for viability in vitro, and the bacillary band has an absorptive function in relation to 6-NBDG, which accumulates within the stichocytes. The absorptive function of the bacillary band calls for an exploration of its possible role in the uptake of anthelmintics, and as a potential anthelmintic target relevant for future drug development.

Anti-parasitic activity of pelleted sainfoin (Onobrychis viciifolia) against Ostertagia ostertagi and Cooperia oncophora in calves.

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

Anthelmintic effects of forage chicory (Cichorium intybus) against gastrointestinal nematode parasites in experimentally infected cattle.

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

Proanthocyanidins inhibit Ascaris suum glutathione-S-transferase activity and increase susceptibility of larvae to levamisole in vitro.

Proanthocyanidins (PAC) are a class of plant secondary metabolites commonly found in the diet that have shown potential to control gastrointestinal nematode infections. The anti-parasitic mechanism(s) of PAC remain obscure, however the protein-binding properties of PAC suggest that disturbance of key enzyme functions may be a potential mode of action. Glutathione-S-transferases (GSTs) are essential for parasite detoxification and have been investigated as drug and vaccine targets. Here, we show that purified PAC strongly inhibit the activity of both recombinant and native GSTs from the parasitic nematode Ascaris suum. As GSTs are involved in detoxifying xenobiotic substances within the parasite, we hypothesised that this inhibition may render parasites hyper-susceptible to anthelmintic drugs. Migration inhibition assays with A. suum larvae demonstrated that the potency of levamisole (LEV) and ivermectin (IVM) were significantly increased in the presence of PAC purified from pine bark (4.6-fold and 3.2-fold reduction in IC50 value for LEV and IVM, respectively). Synergy analysis revealed that the relationship between PAC and LEV appeared to be synergistic in nature, suggesting a specific enhancement of LEV activity, whilst the relationship between PAC and IVM was additive rather than synergistic, suggesting independent actions. Our results demonstrate that these common dietary compounds may increase the efficacy of synthetic anthelmintic drugs in vitro, and also suggest one possible mechanism for their well-known anti-parasitic activity.

Anthelmintic activity of trans-cinnamaldehyde and A- and B-type proanthocyanidins derived from cinnamon (Cinnamomum verum).

Cinnamon (Cinnamomum verum) has been shown to have anti-inflammatory and antimicrobial properties, but effects on parasitic worms of the intestine have not been investigated. Here, extracts of cinnamon bark were shown to have potent in vitro anthelmintic properties against the swine nematode Ascaris suum. Analysis of the extract revealed high concentrations of proanthocyanidins (PAC) and trans-cinnamaldehyde (CA). The PAC were subjected to thiolysis and HPLC-MS analysis which demonstrated that they were exclusively procyanidins, had a mean degree of polymerization of 5.2 and 21% of their inter-flavan-3-ol links were A-type linkages. Purification of the PAC revealed that whilst they had activity against A. suum, most of the potency of the extract derived from CA. Trichuris suis and Oesophagostomum dentatum larvae were similarly susceptible to CA. To test whether CA could reduce A. suum infection in pigs in vivo, CA was administered daily in the diet or as a targeted, encapsulated dose. However, infection was not significantly reduced. It is proposed that the rapid absorption or metabolism of CA in vivo may prevent it from being present in sufficient concentrations in situ to exert efficacy. Therefore, further work should focus on whether formulation of CA can enhance its activity against internal parasites.

Uptake of benzimidazoles by Trichuris suis in vivo in pigs.

It is recognized that the clinical efficacy of single dose benzimidazoles (BZs) against the nematode, Trichuris suis of pigs and the closely related Trichuris trichiura in humans is only poor to moderate. Recent in vitro studies have indicated that a low uptake of fenbendazole (FBZ) in T. suis may be responsible for its poor efficacy. The aim of this study was to investigate this hypothesis by measuring the concentrations of FBZ and its metabolites, oxfendazole (OXF) and FBZ sulphone (FBZSO2), in T. suis isolated from FBZ treated pigs and in plasma of the pigs. The highest concentration of FBZ measured in T. suis was 66.6 pmol/mg dry worm tissue which was approximately half of what was measured in a previous in vitro study. The correlation between drug concentrations in plasma and in T. suis worms was highly positive for OXF (r = 0.93, P = 0.0007) and FBZSO2 (r = 0.85, P = 0.007), but no correlation was found for FBZ. This study shows that the low uptake of FBZ observed for T. suis in vitro, also takes place in vivo. The high and significant correlations between OXF and FBZSO2 concentrations in plasma of the pigs and T. suis (and the lack of this correlation for FBZ) suggests that the metabolites reach the worms via the blood-enterocyte interface while FBZ primarily reaches the worms via the intestinal lumen of the host.

Trichuris suis and Oesophagostomum dentatum show different sensitivity and accumulation of fenbendazole, albendazole and levamisole in vitro.

BACKGROUND: The single-dose benzimidazoles used against Trichuris trichiura infections in humans are not satisfactory. Likewise, the benzimidazole, fenbendazole, has varied efficacy against Trichuris suis whereas Oesophagostomum dentatum is highly sensitive to the drug. The reasons for low treatment efficacy of Trichuris spp. infections are not known. METHODOLOGY: We studied the effect of fenbendazole, albendazole and levamisole on the motility of T. suis and O. dentatum and measured concentrations of the parent drug compounds and metabolites of the benzimidazoles within worms in vitro. The motility and concentrations of drug compounds within worms were compared between species and the maximum specific binding capacity (Bmax) of T. suis and O. dentatum towards the benzimidazoles was estimated. Comparisons of drug uptake in living and killed worms were made for both species. PRINCIPAL FINDINGS: The motility of T. suis was generally less decreased than the motility of O. dentatum when incubated in benzimidazoles, but was more decreased when incubated in levamisole. The Bmax were significantly lower for T. suis (106.6, and 612.7 pmol/mg dry worm tissue) than O. dentatum (395.2, 958.1 pmol/mg dry worm tissue) when incubated for 72 hours in fenbendazole and albendazole respectively. The total drug concentrations (pmol/mg dry worm tissue) were significantly lower within T. suis than O. dentatum whether killed or alive when incubated in all tested drugs (except in living worms exposed to fenbendazole). Relatively high proportions of the anthelmintic inactive metabolite fenbendazole sulphone was measured within T. suis (6-17.2%) as compared to O. dentatum (0.8-0.9%). CONCLUSION/SIGNIFICANCE: The general lower sensitivity of T. suis towards BZs in vitro seems to be related to a lower drug uptake. Furthermore, the relatively high occurrence of fenbendazole sulphone suggests a higher detoxifying capacity of T. suis as compared to O. dentatum.

Genetic variations in the beta-tubulin gene and the internal transcribed spacer 2 region of Trichuris species from man and baboons.

BACKGROUND: The whipworm Trichuris trichiura has been estimated to infect 604 - 795 million people worldwide. The current control strategy against trichuriasis using the benzimidazoles (BZs) albendazole (400 mg) or mebendazole (500 mg) as single-dose treatment is not satisfactory. The occurrence of single nucleotide polymorphisms (SNPs) in codons 167, 198 or 200 of the beta-tubulin gene has been reported to convey BZ-resistance in intestinal nematodes of veterinary importance. It was hypothesised that the low susceptibility of T. trichiura to BZ could be due to a natural occurrence of such SNPs. The aim of this study was to investigate whether these SNPs were present in the beta-tubulin gene of Trichuris spp. from humans and baboons. As a secondary objective, the degree of identity between T. trichiura from humans and Trichuris spp. from baboons was evaluated based on the beta-tubulin gene and the internal transcribed spacer 2 region (ITS2). METHODS: Nucleotide sequences of the beta-tubulin gene were generated by PCR using degenerate primers, specific primers and DNA from worms and eggs of T. trichiura and worms of Trichuris spp. from baboons. The ITS2 region was amplified using adult Trichuris spp. from baboons. PCR products were sequenced and analysed. The beta-tubulin fragments were studied for SNPs in codons 167, 198 or 200 and the ITS2 amplicons were compared with GenBank records of T. trichiura. RESULTS: No SNPs in codons 167, 198 or 200 were identified in any of the analysed Trichuris spp. from humans and baboons. Based on the ITS2 region, the similarity between Trichuris spp. from baboons and GenBank records of T. trichiura was found to be 98 - 99%. CONCLUSIONS: Single nucleotide polymorphisms in codon 167, 198 and 200, known to confer BZ-resistance in other nematodes, were absent in the studied material. This study does not provide data that could explain previous reports of poor BZ treatment efficacy in terms of polymorphism in these codons of beta-tubulin. Based on a fragment of the beta-tubulin gene and the ITS2 region sequenced, it was found that T. trichiura from humans and Trichuris spp. isolated from baboons are closely related and may be the same species.

Genetic variation in codons 167, 198 and 200 of the beta-tubulin gene in whipworms (Trichuris spp.) from a range of domestic animals and wildlife.

A recurrent problem in the control of whipworm (Trichuris spp.) infections in many animal species and man is the relatively low efficacy of treatment with a single application of benzimidazoles (BZs). The presence of single nucleotide polymorphisms (SNPs) in codons 167, 198 and 200 in the beta-tubulin gene has been associated with BZ anthelmintic resistance in intestinal nematodes of veterinary importance. We hypothesized that the low susceptibility to BZ could be related to a natural tolerance or induced resistance caused by BZ-resistant associated SNPs. The aim of the present study was therefore to investigate the presence of these SNPs in the beta-tubulin gene of Trichuris spp. obtained from a range of animals. DNA was extracted from a total of 121 Trichuris spp. adult whipworm specimens obtained from 6 different host species. The number of worms from each host was pig: 31, deer: 21, sheep: 18, mouse: 17, dog: 19 and Arabian camels: 14. A pooled sample of Trichuris eggs from 3 moose was also used. In order to amplify the beta-tubulin fragments which covered codons 167, 198 and 200 of the gene, degenerate primers were designed. The sequences obtained were used to design species specific primers and used to amplify a ~476 bp fragment of the beta-tubulin gene. The PCR products were sequenced, analysed and evaluated. We did not identify SNPs in codons 167, 198 or 200 that led to amino acid substitutions in any of the studied Trichuris spp., but genetic variation expected to be related to species differences was observed. The cluster analysis showed close evolutionary relationship between Trichuris spp. from ruminants and between mouse and dog whereas the pig-derived worms, T. suis, clustered with T. trichiura obtained from Genbank.

Genetic analysis of Trichuris suis and Trichuris trichiura recovered from humans and pigs in a sympatric setting in Uganda.

The whipworms Trichuris trichiura and Trichuris suis in humans and pigs, respectively, are believed to be two different species yet closely related. Morphologically, adult worms, eggs and larvae of the two species are indistinguishable. The aim of this study was to examine the genetic variation of Trichuris sp. mainly recovered from natural infected pigs and humans. Worm material isolated from humans and pigs living in the same geographical region in Uganda were analyzed by PCR, cloning and sequencing. Measurements of morphometric characters were also performed. The analysis of the ITS-2 (internal transcribed spacer) region showed a high genetic variation in the human-derived worms with two sequence types, designated type 1 and type 2, differing with up to 45%, the type 2 being identical to the sequence found in pig-derived worms. A single human-derived worm showed exclusively the type 2-genotype (T. suis-type) and three cases of 'heterozygote' worms in humans were identified. However, the analysis showed that sympatric Trichuris primarily assorted with host origin. Sequence analysis of a part of the genetically conserved ß-tubulin gene confirmed two separate populations/species but also showed that the 'heterozygote' worms had a T. suis-like ß-tubulin gene. A PCR-RFLP on the ITS-2 region was developed, that could distinguish between worms of the pig, human and 'heterozygote' type. The data suggest that Trichuris in pigs and humans belong to two different populations (i.e. are two different species). However, the data presented also suggest that cross-infections of humans with T. suis takes place. Further studies on sympatric Trichuris populations are highly warranted in order to explore transmission dynamics and unravel the zoonotic potential of T. suis.