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Strategies to reduce dependence on synthetic drugs for the treatment of gastrointestinal nematodes (GIN) infections in ruminants include the search for novel anthelmintic scaffolds on plants, yet salt-tolerant plants remain overlooked. This study aims to evaluate the in vitro anthelmintic properties of selected salt-tolerant plants against GIN, and identify the potential bioactive secondary metabolites involved. For that purpose, 80% acetone/water extracts were prepared from dried biomass of aerial organs of nine salt-tolerant plant species and tested against Haemonchus contortus and Trichostrongylus colubriformis by the Larval Exsheathment Inhibition Assay (LEIA) and Egg Hatching Inhibition Assay (EHIA). Pistacia lentiscus, Limoniatrum monopetalum, Cladium mariscus and Helychrisum italicum picardi were the most active in both GIN and life stages. To investigate the role of polyphenols in the anthelmintic activity, four selected extracts were treated with polyvinylpolypyrrolidone (PVPP), and non-treated and treated samples were further characterized by high-performance liquid chromatography with electrospray ionization mass spectrometric detection (HPLC-ESI-MS n ). While polyphenols seem responsible for the EHIA properties, they are partially accountable to LEIA results. Several phenolics involved in the anthelmintic effects were identified and discussed. In sum, these species are rich sources of anthelmintic compounds and, therefore, are of major interest for nutraceutical and/or phytotherapeutic applications against GIN in ruminants., (© 2021. The Author(s).)

Worldwide, parasitic gastrointestinal nematodes continue to threaten animal health, welfare, and production in outdoor breeding systems of small ruminants. For more than 50 years, the control of these parasitic worms has relied on the use of commercial synthetic anthelmintics. However, anthelmintic resistance in worm populations is nowadays widespread and requires novel solutions. The use of tannin-rich plants has been suggested as an alternative to synthetic anthelmintics to control gastrointestinal nematodes. The majority of previous studies have focused on the activity of proanthocyanidins (syn condensed tannins), and less is known about ellagitannins. In this study, the effects of 30 structurally unique ellagitannins on the exsheathment of third-stage infective larvae were examined on Haemonchus contortus and Trichostrongylus colubriformis by the in vitro larval exsheathment inhibition assay. Ellagitannins were found to be promising natural anthelmintics as they showed direct inhibition on larval exsheathment for both nematode species. In general, ellagitannins were more efficient at inhibiting the exsheathment of H. contortus larvae than those of T. colubriformis . The efficiency of inhibition increased as the degree of oligomerization or the molecular weight of the ellagitannin increased. Otherwise, we found no other structural features of ellagitannins that significantly affected the anthelmintic activity on the third-stage infective larvae. The effective concentrations were physiologically relevant and should be achievable in the gastrointestinal tract also in in vivo conditions.

Plants containing condensed tannins (CTs) may hold promise as alternatives to synthetic anthelmintic (AH) drugs for controlling gastrointestinal nematodes (GINs). However, the structural features that contribute to the AH activities of CTs remain elusive. This study probed the relationships between CT structures and their AH activities. Eighteen plant resources were selected on the basis of their diverse CT structures. From each plant resource, two CT fractions were isolated and their in vitro AH activities were measured with the larval exsheathment inhibition assay, which was applied to Haemonchus contortus and Trichostrongylus colubriformis. Calculation of mean EC50 values indicated that H. contortus was more susceptible than T. colubriformis to the different fractions and that the F1 fractions were less efficient than the F2 ones, as indicated by the respective mean values for H. contortus, F1 = 136.9 ± 74.1 μg/mL and F2 = 108.1 ± 53.2 μg/mL, and for T. colubriformis, F1 = 233 ± 54.3 μg/mL and F2 = 166 ± 39.9 μg/mL. The results showed that the AH activity against H. contortus was associated with the monomeric subunits that give rise to prodelphinidins (P < 0.05) and with CT polymer size (P < 0.10). However, for T. colubriformis AH activity was correlated only with prodelphinidins (P < 0.05). These results suggest that CTs have different modes of action against different parasite species.

This study was carried out to evaluate the in vitro effects of different heather species on Trichostrongylus colubriformis eggs, larvae and adult worms, and obtain scientific evidence to attribute these effects to the action of their phenolic compounds and/or tannins. Total phenolic extracts of three heather species (Calluna vulgaris, Erica cinerea, and Erica umbellata) and an equal mixture of these three extracts were tested in vitro in the three development stages of T. colubriformis using an egg hatching assay (EHA), larval exsheathment inhibition assay (LEIA), and adult motility inhibition assay (AMIA). The egg hatching rate was measured after incubation with heather extracts for 48 h at 25°C. Infective third-stage larvae (L3) were incubated for 3h at 25°C with heather extracts. The evolution of artificial exsheathment over time was measured with repeated observations at 20-min intervals for 60 min. Adult worms were obtained from one donor goat and incubated with the extracts at 37°C for 5 days in 48-multiwell plates. Worm motility was measured at 0, 19, 24, 43, 48, 67, 72, 96 and 115 h after the beginning of the experiment. The extracts were tested at concentrations of 75, 150, 300, 600 and 1,200 μg/ml. All extracts significantly (P<0.001) inhibited egg hatching and the effect was dose dependent. All extracts inhibited or delayed the exsheathment of T. colubriformis L3, and the effect was dose dependent for C. vulgaris. Incubation with heather extracts induced a reduction in adult worm motility compared to control, although significant (P<0.05) differences were only found at the highest concentrations. Additional studies showed that purified tannins of the same heather species disturbed T. colubriformis larval exsheathment. All these results confirm the anthelmintic properties of heather against T. colubriformis, and suggest that not only tannins but also some other phenolic compounds might be involved., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The present study aimed at examining the possible role of tannins and flavonoids on the in vitro anthelmintic properties of the extracts of two plants from the southern area of Western Africa, i.e. Newbouldia laevis and Zanthoxylum zanthoxyloïdes. Extracts of the two plants were prepared by use of acetone/water (70/30) and their anthelmintic activity was measured by use of the larval exsheathment inhibition assay (LEIA) applied on the abomasal species, Haemonchus contortus and the intestinal species Trichostrongylus colubriformis. Three concentrations of extracts were evaluated to examine the possible dose effect. In addition, the possible involvement of tannins and flavonoids was examined by comparing the levels of inhibition of larval exsheathment obtained with the same extracts, after of not addition of PVPP which forms complexes with these compounds. The results indicate significant effects with both plants and both nematode species. In the range of concentrations examined, the results were dose-dependent for N. laevis extracts but not for Z. zanthoxyloïdes because the three doses applied provoked a similar highly significant inhibition whatever the tested dose. The use of PVPP indicated for both plant and nematode species, that tannins and flavonoids are involved partly in the effect but that some other biochemical compounds were also involved in both plants., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The anthelmintic (AH) effect of Acacia pennatula, Leucaena leucocephala, Lisyloma latisiliquum and Piscidia piscipula was evaluated in the infective larvae (L(3)) of Trichostrongylus colubriformis. Different concentrations of lyophilized extracts were tested using the larval migration inhibition (LMI) test. An inhibitor of tannins (the polyvinyl polypyrrolidone [PVPP]) was used to verify whether these compounds were responsible for the AH effects. Then, the effect of extracts on larval exsheathment was examined by observing the exsheathment process at 10-min intervals for 70 min. The LMI test showed a dose-dependant AH effect for A. pennatula, L. leucocephala and L. latisiliquum (P<0.01), but not for P. piscipula. The restoration of L(3) migration to values similar to those of controls after the addition of PVPP, indicates that tannins are involved in AH effects. Trichostrongylus colubriformis exsheathment was partially or totally blocked by the four plants extracts. Tropical tanniniferous plants evaluated in the current study may have potential as AH for the control of T. colubriformis if in vivo investigations indicate useful effects.

The current control of parasitic nematodes in small ruminants relies on the use of chemical anthelmintics, but the development of resistance requires research in alternatives. Bioactive plants represent one of those solutions. Previous results suggest that plants rich in condensed tannins (CTs) might have direct anthelmintic effects. However, the relationships between the biochemistry of active compounds and the mechanisms of action remain to be elucidated. Therefore, this study examined in vitro the effects of different CT monomers on the exsheathment of infective larvae for two nematode species. Monomers of prodelphinidins were more potent inhibitors of exsheathment than those of procyanidins. More severe inhibition was also found with galloyl derivatives. These results suggest that the number of free hydroxy groups of CT monomers is a key factor in interactions with parasite larvae. Comparison of effects between the two nematodes suggests different susceptibility to monomers depending on species, which might be related to the protein composition of sheaths.

The purpose of this experiment was to determine if an earlier infection with Oestrus ovis would down regulate an infection with Trichostrongylus colubriformis when the larvae of O. ovis were expelled from the nasal cavities of sheep by a specific treatment. Three groups of five lambs were used: group 1 was artificially infected with O. ovis larvae and later with T. colubriformis, group 2 received O. ovis larvae and later was treated with ivermectin 14 days before being infected with T. colubriformis. Group 3 was infected with T. colubriformis only. The criteria examined were: the effects on nematode egg excretion, worm fecundity, nematode burdens and the kinetics of blood eosinophils. Significant decreases of nematode egg excretion, worm fecundity, nematode burdens were observed in group 1 compared to group 3. However, no changes were observed in either group 2 or 3. In group 2 it was noted that antiparasitic treatment induced a rapid decrease in blood eosinophils to a range close to the non-infected control group and this was associated with the removal of the down regulation effects of nematode burdens. This experiment showed that there is no cross immunity between O. ovis and T. colubriformis and that eosinophils may act against any parasite without specific priming.

The anthelmintic properties of tanniferous plants and of their secondary metabolites represent one possible alternative to chemotherapy that is currently being explored as a means of achieving sustainable control of gastrointestinal nematodes in ruminants. Previous in vivo and in vitro results suggest that tanniferous plants can have direct anti-parasitic effect against different stages of nematodes. However, the mode of action of the bioactive plant compounds remains obscure. The objectives of the current study were (1) to examine the hypothesis that extracts of tanniferous plants might interfere with the exsheathment of third-stage infective larvae (L3); (2) to assess the role of tannins in the process by examining the consequence of adding an inhibitor of tannins (polyethylene glycol: PEG) to extracts. The effects of 4 tanniferous plant extracts on exsheathment have been examined on L3 of Haemonchus contortus and Trichostrongylus colubriformis. Artificial exsheathment was induced in vitro by adding hypochloride solution to larval suspension. The evolution of exsheathment with time was measured by repeated observations at 10-min interval for 60 min. The selected plants were: genista (Sarothamnus scoparius), heather (Erica erigena), pine tree (Pinus sylvestris), and chestnut tree (Castanea sativa), with tannin contents ranging from 1.5 to 24.7% of DM. Extracts of a non-tanniferous plant (rye grass, tannin content: 0.3% of DM) were included in the assay as negative controls. The extracts were tested at the concentration of 600 microg/ml and the effects were compared to the rate of exsheathment of control larvae in PBS. No statistical differences in the pattern of exsheathment was observed after addition of rye grass or genista extracts for both nematode species and with heather extracts for T. colubriformis. In contrast, pine tree extracts on larvae of both species and heather extracts with H. contortus induced a significant delay in exsheathment. Last, contact with chest nut extracts led to a total inhibition of the process for both nematodes. These results suggest that extracts of tanniferous plants might affect a key process in the very early stages of larval invasion of the host. In most cases, the addition of PEG led to a total or partial restoration towards control values. This suggests that tannins are largely involved in the inhibitory process. However, other secondary metabolites may also interfere with the process that would help to explain some of the differences in response observed between the two nematode species.

The in vitro effects of extracts of four tropical plants (Zanthoxylum zanthoxyloides, Newbouldia laevis, Morinda lucida and Carica papaya) on the egg, infective larvae and adult worms of Trichostrongylus colubriformis were screened for potential anthelmintic properties. Significant effects were observed with the four plants on T. colubriformis but they differed depending on the stage of the parasite. Extracts of each plant induced a dose-dependent inhibition of egg hatching. Using a larval inhibition migration test, the effects on the infective larvae were also detected with the four plant extracts. In contrast, for adult worms, the effects were statistically significant only for N. laevis and C. papaya. No significant activity was shown for M. lucida and Z. zanthoxyloides. These in vitro results suggest the presence of some anthelmintic properties associated with these four plants, which are traditionally used by small farmers in western Africa. These effects need to be studied under in vivo conditions.

Simultaneous infections of Trichostrongylus colubriformis and T. vitrinus in the small intestine of the sheep were examined by comparing the numbers of worms which established and their distribution within the intestine in both monospecific infections and mixed infections. The results differed depending upon the species and number of parasites. The establishment of T. colubriformis was reduced and the distribution of the nematode population was displaced posteriorly within the intestine when 30,000 larvae of both species were administered, compared with pure infections of T. colubriformis. The reduced establishment was less marked with infections of 15,000 larvae of both species and there was only a slight posterior displacement of T. colubriformis. Neither effect was evident with infections of 7,500 larvae of both species. The rate of establishment and distribution of T. vitrinus were unaffected by the presence of T. colubriformis at all three rates of infection. Atrophy of villi and hypertrophy of crypts occurred at the main site of infection in the anterior duodenum. The severity of villus atrophy was related to the number of infective larvae administered and/or the worm burden. In the ileum, beyond the main site of infection, hypertrophy of villi was only found in sheep receiving the greatest number of infective larvae.

Concurrent infections of sheep with Oestrus ovis and trichostrongyles of the digestive tract are common in the field. Previous results have shown that a previous infection with O. ovis adversely affects worm populations of either Trichostrongylus colubriformis or Haemonchus contortus. However, no information was available to determine the influence of the succession of infections on the expression of interactions between these parasites located in remote anatomical sites. In order to investigate the role of these modulating factors, an experimental study was conducted on four groups of naïve sheep, examining the consequences of a delayed infection with O. ovis on a pre-existing population of T. colubriformis. group T was infected four times with 4000 T. colubriformis larvae on days 0, 14, 28 and 42 of experiment; group O received multiple infections with O. ovis first instar larvae on days 42, 49, 56, 70 and 77; sheep from group TO received both infections and animals from group C remained as uninfected controls. Faecal egg counts and eosinophilia were measured weekly throughout the study. At necropsy (day 91), the mucosal cellular responses in the nasal cavities (septum, turbinates, ethmoid and sinus) and in the digestive tract (stomach and small intestine) from all animals were analysed from histological sections. Infection of the digestive tract with nematodes did not modify the biology of Oestrus populations, as measured by the number and weight of larvae. In contrast, infections with O. ovis after T. colubriformis infection was related to significant reductions (P < 0.01) in nematode egg excretion and worm burdens. These changes were associated with significant modifications in populations of mast cells, globule leucocytes and eosinophils in the respiratory and digestive tracts. These results indicate that an antagonistic interaction exists between the populations of O. ovis in the nasal cavities and T. colubriformis in the small intestine but that the order of succession of infections with the two parasites is not a major modulating factor for expression of interactions. They also confirm that parasitic infection in one particular anatomical site induces "at distance" inflammatory reactions of the whole mucosal system.

In an experiment lasting 4 years, changes in the Teladorsagia circumcincta and Trichostrongylus colubriformis populations were compared in lambs and adult sheep with differing resistance statuses. Two flocks of 30 rams (resistant R and susceptible S) grazed separate pastures and 8 rams were slaughtered in the middle and at the end of each grazing season. Five groups of tracer lambs were added each year to estimate the pasture infectivity and were killed for worm counts. The availability of animals with differing resistance statuses (rams and tracer lambs) and differing levels of infection made it possible to investigate the number, size and fecundity of worms of these two species. The inflammatory response was measured in the rams by counting the globule leukocytes, mast cells and eosinophils in the fundic, pyloric and intestinal mucosa. In the tracer lambs, the daily egg production by the female worms of both species was negatively correlated with the worm burden. Worm length accounted for 60 and 70% of the variation in the number of eggs in utero for T. circumcincta and T. colubriformis respectively. Worm length was closely associated with the resistance status of the host; there were greater differences between lambs, and S and R rams for T. colubriformis. T. circumcincta worm lengths were not affected by the worm number. Globule leukocyte counts were related to the worm burdens, and mast cell counts to worm length in the R and S rams. The number, size and fecundity of the worms may well be regulated by similar mechanisms in both species, but T. colubriformis seemed to be more intensively regulated than T. circumcincta. This finding could be useful in devising more effective methods of parasite control.

The chronology of the life cycle of Trichostrongylus retortaeformis (Zeder, 1800) (Nematoda, Trichostrongyloidea) is studied in its natural host Oryctolagus cuniculus. The free living period lasted 5 days at 24 degrees C. Worm-free rabbits were each infected per os with T. retortaeformis larvae. Rabbits were killed at 12 h post-infection (p.i.) and every day from one day to 13 days p.i. By 12 h p.i., all the larvae were exsheathed and in the small intestine. The third moult occurred between 3 and 5 days p.i. and the last moult between 4 and 7 days p.i. The prepatent period lasted 12 to 13 days. The patent period lasted five and a half months. The four known life cycles of species of Trichostrongylus in ruminants were compared with that of T. retortaeformis. No significant difference was found except in the duration of the prepatent period. These similarities in the life cycles confirm the previously formulated hypotheses on the relationship between the parasites of the two host groups (Durette-Desset, 1985).

The aims of this study were to characterise trichostrongyle egg excretion in dairy goat flocks under various epidemiological conditions, and to assess the relative importance of animals in their first lactation and goats with high levels of milk production on the total egg output of the flock. The repeatability of egg excretion was estimated on 4 different dates in 14 dairy goat farms in France. Egg outputs were positively skewed on most farms suggesting an aggregated distribution of parasites. Within-year repeatability coefficients ranged from 0.29 to 0.82 suggesting that, on most farms, animals with high egg excretion on one occasion are likely to show high rates of excretion on other occasions. Comparisons of egg outputs from the selected subgroups showed that the high producing goats contributed prominently to egg excretion, particularly at times of peak parasitism. Results from goats in their first lactation were more equivocal. These results provide a rationale for anthelmintic strategies in dairy goats based on phenotypic characters., (Copyright 2002 Published by Elsevier Science Ltd.)

Concurrent infections of sheep with Oestrus ovis and trichostrongyles of the digestive tract are common in the field. In order to examine the possible occurrence of interactions between these two parasites and the consequences on parasite biology and the associated pathophysiological changes, an experimental study was conducted on four groups of naïve sheep. Group O was infected repeatedly with O. ovis larvae from D0 to D42 of experiment; group T received a single infection of Trichostrongylus colubriformis on D49; sheep from group OT received both infections, and animals from group C remained uninfected as controls. Parasitological and pathophysiological parameters were measured regularly. At necropsy (D94), the mucosal cellular responses in the nasal cavities and digestive tract from all animals were analysed from histological sections. Infection of the digestive tract with nematodes did not modify the biology of Oestrus populations. In contrast, the presence of O. ovis was related to significant reductions in nematode egg excretion, worm fecundity and worm burdens. These changes were associated with significant modifications in tissular populations of mast cells, globule leucocytes and eosinophils in the respiratory and digestive tracts. These results indicate that parasitic infection in one particular anatomical site induces "at distance" inflammatory reactions of the whole mucosal system. The relationships between the cellular response and the changes in worm biology are discussed.

Culled dry dairy goats, which differed in their level of production in previous lactations, received a single infection with Trichostrongylus colubriformis. The objectives of the study were twofold. First, the study aimed at examining the ability of dairy goats to develop an immune response to trichostrongyle infection and the associated cellular changes developing within the intestinal mucosa. Second, a comparison between animals differing in their level of production was assessed, in order to determine whether these differed in their susceptibility to infection. No difference occurred in egg excretion, worm burden and local inflammatory cell responses between high (HP) and low (LP) producer dairy goats, in contrast to observations in previous studies. Because their nutrition was controlled and milk production absent in the goats employed in the present study, these results suggest that any genetic component associated with the selection of HP and LP goats had little influence on the development of acquired resistance to T. colubriformis. The analysis of the relationship between different cell types in the mucosa and some characteristics of the worm population show that eosinophils are negatively related to worm burden. Also, a role is suggested for mast cells and globule leukocytes in the modulation of egg excretion.

The morphogenesis and the distribution along the digestive tract of Trichostrongylus retortaeformis(Zeder, 1800) (Nematoda) were studied in detail in one of its natural hosts, Oryctolagus cuniculus. Worm-free rabbits were each infected with T. retortaeformis larvae and were killed at 12 h post-infection (HPI) and on each day from 1 to 15 days post-infection (DPI). The distribution of worm populations along the small intestine was assessed. At the different dates of infection, more than 80% of the population was recovered from the first third of the intestine with more than 50% occurring in the first 30 cm. For each date, morphological descriptions of the different stages of the life cycle were also provided. In addition, adult worms collected from naturally infected rabbits from France were redescribed.

Numerous studies have examined the interactions between protein nutrition and the response to nematode parasitism in sheep, but very few in goats. Compared with other ruminants, goats are less resistant to nematode infection. In addition, in dairy goats, high producing animals have been shown to be less resistant and less resilient to infection compared to low producing ones. The objective of the present study was to examine the consequences of protein supplementation on both resistance and resilience of dairy goats to nematode trickle infection, taking into account the initial level of milk production of the animals. During a 14-week period, 40 milking goats received a high protein (HP) diet supplying 130% of the protein requirements, and 38 goats were fed a intermediate protein (IP) diet (120% of the protein requirements). In addition, half of each group was given a weekly trickle infection with Trichostrongylus colubriformis larvae, the other part of the flock remained non-infected. Faecal egg counts (FEC), eosinophil counts and pathophysiological data (urea, albumin and inorganic phosphate concentrations in the serum) were measured twice a month. Milk production data (milk yield, protein and fat contents) were also recorded every 15 days. The results showed that FECs were lower (p < 0.05) and eosinophil counts higher (p < 0.05) in the animals receiving the HP diet suggesting that resistance was enhanced by protein supplementation. Meanwhile, milk parameters (related to resilience) were not affected by the level of protein in the diet when considering the whole groups. In contrast, in the high producing goats, the milk production and milk composition parameters were improved with the HP diet. To conclude, we have seen that the expression of both resistance and resilience did not appear when the coverage of the protein requirements was insufficient. Because the milk production is dependent on the protein supply, we suggest that there is a competition in the use of the protein between the development of resistance and the milk production.

The effects of the excretory/secretory (ES) products of the parasitic nematode Trichostrongylus colubriformis were examined on the proliferation of seven cell lines derived from a digestive or non-digestive origin. The excretory/secretory products of T. colubriformis were incorporated in the culture medium of the different cell lines and cell proliferation was measured by means of the 5-bromo-2'-deoxy-uridine (Brdu) assay. An increase in cell numbers was found with the three epithelial intestinal cells (RIC, IEC-6, IRD-98) and with epithelial kidney cells (MDCK). In contrast, an inhibition in the proliferation of epithelial ovarian cells (CHO) and fibroblasts (3T3) was observed with the addition of the excretory/secretory products and no effect was detected on the cell growth of hepatocytes (HepG2). These data are discussed with respect to the tissue specificity of the existing mitogenic effect of the worms on the intestinal crypt cells during parasitism.

The excretory-secretory (E-S) products of the parasitic nematodes Trichostrongylus colubriformis and Nematodirus battus were found to modify the in vitro proliferation of the tumorous colic HT29-D4 cell line of epithelial origin. A characteristic feature of these E-S products is the presence of a high level of acetylcholinesterase (AChE) activity, the biological significance of which remains unclear. To determine a possible role of AChE on cell growth, the enzyme was purified from E-S products using edrophonium chloride. Purity was confirmed by polyacrylamide gel electrophoresis, using silver and Karnovsky stains, before assessing its effects on cell proliferation. The purified AChE was incorporated at different concentrations in a culture medium of HT29-D4 cells. A mitogenic effect was shown for low concentrations (0.1-14 units). By contrast, an inhibitory effect was noted at high concentrations (35-1400 units). Furthermore, polyclonal antibodies were prepared and depletion of AChE in E-S products by immunoprecipitation or affinity chromatography resulted in a partial or total disappearance of the stimulatory effect of cell growth. Thus, the results form this in vitro study suggest a modulatory role for AChE secreted by nematode parasites on the proliferation of epithelial cells of the host.

Sequences of the second internal transcribed spacer ribosomal DNA for the parasitic trichostrongylid nematodes Trichostrongylus probolurus, Trichostrongylus rugatus and Camelostrongylus mentulatus were compared with previously published sequences for five other species within the genus Trichostrongylus. The secondary structures of the second internal transcribed spacer pre-rRNA for these nematodes were predicted using an energy minimisation method. The results indicate that a common secondary structure of the second internal transcribed spacer of these nematodes is maintained despite distinct differences in primary sequence between species. Sequence differences among Trichostrongylus species ranged from 1.3 to 7.6%, but each species differed by 22-26% in sequence when compared with C. mentulatus which belongs to a different subfamily.

The first internal transcribed spacer (ITS-1) of the ribosomal DNA of seven species of Trichostrongylus was sequenced. The length of ITS-1 in the different species varied from 387 to 390 bases. The G + C content of the ITS-1 sequences were approximately 42%. Little or no intraspecific variation was detected in the three species. Trichostrongylus axei, Trichostrongylus colubriformis and Trichostrongylus vitrinus, for which multiple isolates from different geographical regions were sequenced. In contrast, the level of ITS-1 sequence differences between species ranged from 1.3% to 5.7%. The greatest sequence differences were detected between T. tenuis, the parasite species which infects birds and the six species found in mammals. Some of the nucleotide differences occurred at sites corresponding to recognition sites for restriction endonucleases. These results are compared with previous data obtained for the second internal transcribed spacer (ITS-2). The ITS-1 data indicate that this region of rDNA may also be useful for systematic studies in trichostrongylid nematodes.

A total of 70 strongyle-free French Alpine dairy goats were exposed to a combination of sequential and challenge infections with Haemonchus contortus and Trichostrongylus colubriformis third-stage larvae. The sequential infection consisted of three inoculations at 50-day intervals, each infection being abbreviated by anthelmintic treatment at 40 days postinoculation. The challenge infection, composed of the same nematode strains, was undertaken 2 months later, when goats were at their 1st month of lactation. Fecal egg counts (FECs), packed cell volumes (PCVs), pepsinogen concentrations, inorganic phosphate concentrations, and peripheral eosinophil numbers were measured at 30-40 days after each inoculation. Goats were defined as being resistant or susceptible according to their level of nematode egg output following the first inoculation. Significant differences in FECs were recorded between the two groups throughout the further inoculations and the challenge infection. The reliability of FECs was supported by the high repeatability values found within and between infections. With regard to blood constituents, only PCVs related to H. contortus infection showed values that differed significantly between the two groups, resistant goats having higher PCVs after the first and the third inoculations than did susceptible animals. However, this difference was not detectable after the challenge infection. The milk production yield for the current lactation was significantly lower in the resistant goats. Moreover, resistant animals exhibited constantly greater body condition scores as compared with susceptible animals. These results indicate that the individual responsiveness of dairy goats to experimental nematode infection can be estimated on the basis of FECs and PCVs (for H. contortus) and is negatively related to the level of milk production of the animals.

To investigate the consequences of subclinical Trichostrongylus colubriformis infection on the intestinal mucosa and the associated changes in entero-glucagon gene expression, sheep were infected with 30000 larvae and killed 5, 10, 15 or 20 days after infection. Histological and cytological changes were examined. In the main site of infection, the upper duodenum, villous atrophy associated with crypt hyperplasia developed gradually. Cytological changes in the enterocytes appeared concurrently, characterized by a progressive reduction in brush border and in the number of ribosomes in the cytoplasm, changes in the internal structure of mitochondria, and enlargement of the intercellular spaces. Neither histological nor cytological modifications were found before day 15. At the same time, villous hypertrophy developed distally, beyond the main site of infection; this was interpreted as an adaptive response to parasitism. Enteroglucagon gene expression in the ileum was measured in parallel with the mucosal changes but did not reveal any difference between infected and control sheep. The results indicate that this gastrointestinal hormone does not have a major role in the response to nematode parasitism.

Seven species of closely-related nematode parasite (Trichostrongylus axei, T. colubriformis, T. probolurus, T. retortaeformis, T. rugatus, T. vitrinus and T. tenuis) were characterized using a polymerase chain reaction-linked restriction fragment length polymorphism technique (PCR-RFLP). The rDNA region spanning the first and second internal transcribed spacers as well as the 5.8S rDNA gene (ITS+) was amplified from isolates of each of the seven species, digested separately with six restriction endonucleases (Dra I, Hinf I, Rsa I, Vsp I, Nla III and Tsp 509 I) and the fragments separated by agarose gel electrophoresis. PCR-RFLP of ITS+ produced characteristic patterns for each Trichostrongylus species examined. No variation in RFLP patterns was observed among different isolates for species where multiple isolates were examined. The present study demonstrates that the ITS+ provides genetic markers for the species identification of closely-related parasitic nematodes, and indicates the usefulness of these markers for diagnostic purposes, and epidemiologic and molecular-systematic studies on parasites and other eukaryotic organisms.

The second internal transcribed spacer (ITS-2) of the ribosomal DNA of 5 species of Trichostrongylus has been sequenced. The ITS-2 of the 5 species was 237 or 238 bases in length, and had a GC content of approximately 30%. No evidence of intraspecific variation was detected in the ITS-2 sequence of T. colubriformis, T. vitrinus or T. retortaeformis, irrespective of the life cycle stage examined. There was evidence, however, of variation at five positions in the ITS-2 sequence of T. vitrinus samples and at one position in T. axei, indicating intra-individual variation in the sequence of different copies of the ribosomal DNA. Nonetheless, there were consistent sequence differences between the five Trichostrongylus species examined. The level of interspecific differences in nucleotide sequence was low (1.3-7.6%), with the species infecting birds (T. tenuis) being genetically more different to the four species found in mammals. Some of the nucleotide differences between species occurred at the recognition sites of endonucleases, which makes them of important diagnostic value for species identification. Also of significance are the recognition sites for several enzymes located within the regions of sequence homology for the five species of Trichostrongylus. These may prove useful in distinguishing between genera of trichostrongyle nematodes.

Two strains of Trichostrongylus colubriformis of ovine origin, one resistant and one susceptible to thiabendazole, were compared during experimental infection in rabbits. Groups of rabbits were infected with 5000 infective larvae (L3) of either the resistant or the susceptible strain. On days 21 and 42 post-infection (p.i.), 5 rabbits of each group were killed and the small intestine divided into three sections for worm counts and histological or biochemical analysis. Faecal egg counts were performed twice a week from day 15 to day 42 p.i. The physiology of the worms was characterised by in vitro acetylcholinesterase secretion of adult worms. The host inflammatory response was determined by peroxidase activity in mucosal homogenates and by histological counts of mast cells and eosinophils. Infectivity was not significantly different between the two strains. However, egg production was delayed and was significantly lower for the resistant strain. Conversely, in vitro secretion of resistant worms was significantly higher than the susceptible ones. The mucosal homogenetates of rabbits infected with the resistant strain had significantly higher levels of peroxidase activity in the duodenum on days 21 and 42 p.i. Mast cell counts were also significantly higher in the duodenum on day 42 p.i., in rabbits infected with the resistant strain. No difference was observed in the eosinophil counts. These results suggest that genetic variation in the nematode, such as anthelminthic resistance, is associated with variations in worm biology and physiology as well as differences in the inflammatory response of the host.

The presence of the nematode parasite Trichostrongylus colubriformis in the small intestine is associated with an increase in epithelial renewal. To assess the possible role of excretory/secretory products from the worm on cell proliferation, adult Trichostrongylus colubriformis were incubated in vitro in Dulbecco's Modified Eagle's Medium for 24 h and the conditioned medium was added to the culture medium of the transformed epithelial cell line HT29-D4. A stimulation of the HT29-D4 cell growth was ascertained at concentrations of 0.25-1.0 micrograms protein/ml using counts of cell numbers, the MTT method and incorporation of tritiated thymidine. An increased incorporation of tritiated thymidine was also observed with the excretory/secretory products from T. colubriformis fourth stage larvae at 1.0 microgram/ml. Dialysis of the medium conditioned by the worms indicated that the molecular weight of the factor is greater than 8000 Daltons in size. Heat treatment, acid hydrolysis and precipitation by trichloracetic acid of the conditioned medium resulted in the disappearance of the proliferative effect while treatment with trypsin partially depleted the stimulative activity. These results suggest that T. colubriformis produce some protein factor which could increase the epithelial regeneration in the host small intestine.

The nucleotide sequence of the second internal transcribed spacer (ITS-2) was determined for three populations of the parasitic nematode Trichostrongylus colubriformis which differed in their susceptibility to benzimidazole anthelmintics and/or in their geographical origin. No intraspecific variation was found in the ITS-2 sequence, indicating that this region of rDNA is inadequate to discriminate between resistant and susceptible populations of T. colubriformis, but it may prove useful for distinguishing between species of Trichostrongylus.

The influence of population size of Trichostrongylus colubriformis on the structures of the small intestine, especially with regard to the development and origin of an intestinal adaptive response, was examined in experimentally infected rabbits. The effects of low (500 L3) and high (50,000 L3) infection on histological (villous length, mucosa to serosa ratio, crypt surface) and biochemical (protein content, alkaline phosphatase and leucine aminopeptidase activities) aspects of the mucosa were assessed along the whole small intestine. The presence of a small number of worms induced only minor mucosal changes, indicating a regenerative response of the intestinal epithelium. The role of a local small population of T. colubriformis in the development of a previously described adaptive response appeared thus to be limited. On the other hand, the 50,000 L3 inoculum was associated with severe lesions of villi, marked crypt hyperplasia and with a major reduction of enzyme activities. The changes were found along the whole length of the small intestine. These results suggest that the generally recognized dose-dependent pathogenicity of the intestinal nematode infections could be ascribed to two different processes: firstly, a greater severity of the lesions; secondly, more extensive damage leading to the disappearance of any adaptive intestinal region.