Contortus L3 Research Articles

Vaccination of goats with DNA vaccines encoding H11 and IL-2 induces partial protection against Haemonchus contortus infection

DNA vaccines expressing Haemonchus contortus H11 antigen with or without interleukin (IL)-2 were tested for protection against H. contortus infection in goats. Sixteen goats (8-10 months of age) were allocated into four trial groups. On days 0 and 14, group 1 was immunised with a DNA vaccine expressing H11 and IL-2 and group 2 was immunised with a DNA vaccine expressing H11 only. Group 3 was an unvaccinated positive control group challenged with H. contortus third stage larvae (L3). Group 4 was an unvaccinated negative control group that was not challenged with L3. Animals in groups 1-3 were challenged with 5000 infective H. contortus L3 14 days after the second immunisation. Transcription of H11 and IL-2 was demonstrated in muscle by reverse transcriptase-PCR 10 days after primary immunisation and translation of H11 was detected by Western blot analysis 7 days after the second immunisation. Following immunisation with a DNA vaccine expressing H11 and IL-2, high levels of specific serum immunoglobulin (Ig) G, non-specific serum IgA, mucosal IgA, CD4(+) T lymphocytes, CD8(+) T lymphocytes and B lymphocytes were produced. Following challenge with L3, cumulative mean faecal worm egg counts and worm burdens in group 1 were reduced by 56.6% and 46.7%, respectively, while corresponding reductions in group 2 were 44.8% and 38.0%. There was a small but significant difference in abomasal worm burdens in goats in groups 1 (395.3±37.6) and 2 (459.5±101.6) compared to group 3 (741.5±241.5; P<0.05). Use of a DNA vaccine expressing H11 and IL-2 conferred partial protection against Haemonchus contortus infection in goats.

Effect of feeding sericea lespedeza leaf meal in goats experimentally infected with Haemonchus contortus

Effect of sericea lespedeza [SL; Lespedeza cuneata (Dum-Cours.) G. Don.] leaf meal feeding was evaluated in two experiments in indoor reared goats with experimental infection of Haemonchus contortus larvae. In the first experiment, ten 8–10 month old male Spanish and Alpine cross kids pair matched for body weight and age were fed SL or bermudagrass [BG; Cynodon dactylon (L.) Pers.] hay one week before infection and were infected with 5000 H. contortus L3. The animals were maintained on the same diet for the remaining period and were slaughtered 28 days post-infection (DPI) to determine the establishment of incoming infective larvae. Goats fed SL had lower establishment (P<0.05) of H. contortus larvae than that of the control goats fed BG hay. In the second experiment, twenty-five 8–10 months old male Alpine cross, Saanen, Nubian×Saanen and Spanish kids reared in confinement on BG were experimentally infected with 5000 H. contortus L3. On 35 DPI, the animals were allocated to two groups after blocking by fecal egg count (FEC), and one group was fed SL leaf meal (n=13), and another control group remained on BG (n=12). Four goats/group were slaughtered successively on days 7, 14, and 28 days post SL feeding, except on day 7, when five SL fed goats were slaughtered. Fecal egg counts and blood packed cell volume (PCV) were measured at weekly intervals and worm count, female worm fecundity, worm length and mucosal eosinophils, mast cells and globule leucocytes were measured after slaughter. Goats fed SL had a lower FEC (P<0.05) one week after feeding, as compared to those fed on BG, and the values remained at low level thereafter. Similarly, PCV was also significantly affected by feeding (P<0.01), and feeding and time interaction (P<0.05). However, worm burden, female worm fecundity, parasite length, and mucosal inflammatory cell count were similar between the groups. Feeding SL reduced the establishment of infective larvae and FEC of H. contortus in experimental studies and this plant could be used for biological control of parasite infection under field conditions to limit the harmful effects of the parasites in goats.

Factors affecting susceptibility to RNA interference in Haemonchuscontortus and in vivo silencing of an H11 aminopeptidase gene

Gene silencing by RNA interference (RNAi) has been applied very successfully to Caenorhabditis elegans to study gene function but has proven less effective in parasitic nematodes. In the sheep gastrointestinal nematode Haemonchus contortus, previous studies demonstrated reproducible silencing of β-tubulin but not of other genes targeted. Here we aimed to examine whether the level of target transcript or site of gene expression influence susceptibility to RNAi by soaking. Target genes represented by a high number of expressed sequence tags (ESTs) in the H. contortus L3 stage were not reproducibly silenced. In contrast, four out of six genes putatively expressed in the intestine, excretory cell or amphids were consistently silenced by RNAi. This suggests that genes expressed in sites accessible to the environment are more likely to be susceptible to RNAi by soaking. Silenced genes included those encoding the highly protective gut aminopeptidase H11, secretory protein Hc-ASP-1, β-tubulin and homologues of aquaporin and RNA helicase. To determine whether RNAi silencing of H11 could mimic H11 vaccination in reducing worm and egg counts, we examined the in vivo effects of H11 RNAi. This is the first, to our knowledge, in vivo study of RNAi in an animal parasitic nematode. RNAi of the H11 gene in infective larvae prior to infection resulted in a 57% reduction in faecal egg count (FEC), 40% reduction in worm burden and 64% decrease in aminopeptidase activity compared with pre-soaking in control dsRNA. Thus, in this study we have established that RNAi is a valid and feasible approach to identify essential gene function. However, using current methods, this may be limited to genes expressed in accessible sites.

Activity in vitro of fungal conidia of Duddingtonia flagrans and Monacrosporium thaumasium on Haemonchus contortus infective larvae

The objective of this work was to evaluate the predatory activity of the fungi Duddingtonia flagrans (AC001) and Monacrosporium thaumasium (NF34a) on Haemonchus contortus infective larvae (L3) in two experimental assays (A and B). In assay A, two treatments and one control were formed and kept for 7 days in Petri dishes with 2% water-agar. Each treatment consisted of 1000 H. contortus L3 and 1000 conidia of only one fungal isolate, and the control group consisted of 1000 L3, without fungus, with 10 repetitions per group. In assay B, 1000 conidia of one of the fungal isolates, AC001 or NF34a, were added to coprocultures made from 20 g of faeces collected from sheep naturally infected with H. contortus. At the end of the experiment, the Baermann method was used to count the non-predated larvae of all Petri dishes from treatment and control groups. In assay A, no difference was observed (P>0.05) between the groups treated with AC001 and NF34a fungi. A difference was observed (P < 0.05) between the treated and control groups. The L3 reduction percentages at the end of the experiment were 87.75 and 85.57%, respectively, for the fungal isolates compared to the control group. In assay B, the reduction percentages for conidia of these isolates were 85.82 and 87.32%, respectively. The results obtained show that D. flagrans (AC001) and M. thaumasium (NF34a) were effective in the in vitro control of sheep H. contortus L3 and could be used in the biological control of this nematode.

Comparative analysis of destruction of the infective forms of Trichuris trichiura and Haemonchus contortus by nematophagous fungi Pochonia chlamydosporia; Duddingtonia flagrans and Monacrosporium thaumasium by scanning electron microscopy

The present study aimed to demonstrate by scanning electron microscopy (SEM) the in vitro predatory activity of nematophagous fungi Pochonia chlamydosporia (VC1 and VC4 isolates) Duddingtonia flagrans (AC001 isolate) and Monacrosporium thaumasium (NF34a isolate) on eggs of Trichuristrichiura and infective larvae (L3) of Haemonchus contortus. The work was divided into two experimental tests (A and B). In tests A and B, the predatory activity of nematophagous fungi P. chlamydosporia, D. flagrans and M. thaumasium on eggs of T. trichiura and H. contortus L3 was observed. After 6h, in test A, isolates P. chlamydosporia (VC1 and VC4) had a role in destroying eggs of T.trichiura. For fungi D. flagrans and M. thaumasium the ovicidal activity on T. trichiura eggs was not observed. Test B showed that D. flagrans (AC001) and M. thaumasium (NF34a) were capable of predating H. contortus L3, but no predation by the fungus P. chlamydosporia was seen. These fungi can offer potential for the biological control of nematodes.

Immune cell kinetics in the ovine abomasal mucosa following hyperimmunization and challenge withHaemonchus contortus

Sheep were sensitized by repeated infection with Haemonchus contortus L3, followed by a 12 week rest period, and an abomasal cannula was surgically implanted in all sheep. Seven of the sensitized sheep were subsequently challenged with 50 000 H. contortus L3 while 4 control sheep were challenged with saline. Biopsy samples were taken using a fibreoptic endoscope on days 0, 1, 2, 3, 5, 7 and 28 after challenge and leukocyte subpopulations quantified by (immuno)histology. Differential blood cell counts were performed on the same days. At the end of the trial, sheep showed significantly reduced worm burdens compared to unsensitized control sheep, confirming their resistance status. Both blood and tissue eosinophils, as well as tissue γδ TCR+ cells were rapidly elevated by day 1 post L3 challenge (pc), peaking at day 3 pc. There was a slight increase in tissue CD4 T cells at day 2 pc, peaking at day 3 pc while no significant changes in CD8 T cells were observed. B cells (CD45R+) increased later into challenged tissues with a peak at 5 days pc. All tissue lymphocyte subpopulations as well as tissue and blood eosinophils were reduced by day 7 pc before increasing again at day 28 pc, suggesting separate responses to larval and adult antigens. In contrast, globule leukocytes and mucosal mast cells only showed one peak at day 5 pc and 28 pc, respectively. Unexpectedly, globule leukocytes correlated significantly with tissue eosinophils but not mucosal mast cells. The results are consistent with an early eosinophil-mediated killing of L3, possibly recruited by IL-5 produced by γδ T cells. In contrast to post-mortem studies, abomasal cannulation allowed sequential analysis of both early and late time points in the same animal, providing a more complete picture of cellular interactions at both peripheral and local sites, and their correlation with the different stages of parasite development.

Effect of plant trichomes on the vertical migration of Haemonchus contortus infective larvae on five tropical forages

The influence of trichomes on vertical migration and survival of Haemonchus contortus infective larvae (L3) on different forages was investigated. Four different forages showing different distributions of trichomes (Brachiaria brizantha cv. Marandu, Brachiaria brizantha cv. Xaraes, Andropogon gayanus, and Stylosanthes spp.), and one forage species without trichomes (Panicum maximum cv. Tanzania), were used. Forages cut at the post-grazing height were contaminated with faeces containing L3. Samples of different grass strata (0-10, 10-20, >20 cm) and faeces were collected for L3 quantification once per week over four weeks. In all forages studied, the highest L3 recovery occurred seven days after contamination, with the lowest recovery on A. gayanus. In general, larvae were found on all forages' strata. However, most of the larvae were at the lower stratum. There was no influence of trichomes on migration and survival of H. contortus L3 on the forages.

Serum antibody responses in Creole kids experimentally infected with Haemonchus contortus

The objective of this study was to evaluate the relationship of parasite-specific serum antibodies with the resistance status of Creole kids. The average breeding values on egg output predicted in a context of natural infection at 11 months of age were distant of 1.07 genetic standard deviation between resistant and susceptible animals. After drenching the animals were maintained worm-free during 1 month until experimental infection with 10,000 Haemonchus contortus infective larvae (L3). Enzyme-linked immunosorbent assay was carried out in serum samples to determine the level of IgG, IgA and IgE anti- H. contortus L3 crude extracts and adult excretion/secretion products (ESP). Parasitological and blood immunological parameters were measured on the 2 extreme groups. Despite the absence of any typical signs of haemonchosis, susceptible kids had more than 11 times higher faecal egg counts (FEC) at 35 days post-infection (d.p.i.) than resistant kids had. Levels of immunoglobulin against H. contortus L3 and ESP increased significantly after infection in both groups. However, no difference in the host immune response mediated by immunoglobulin against H. contortus was evidenced between groups. This finding suggests that, in goats previously infected by H. contortus, a degree of protection occurred and the phenotypic and genetic segregation in resistant and susceptible animals were not related to the humoral immune response. The correlation coefficients between FEC and IgE anti-ESP ( r = 0.593; P < 0.05 was significant in both resistant and susceptible animals. Such correlation suggesting a hypersensitivity reaction dependent on worm prolificacy has never been described. This result needs further studies to understand the mechanisms underlying this observation.

Vertical migration of Haemonchus contortus third stage larvae on Brachiaria decumbens grass

The present study aimed at evaluating the vertical migration of Haemonchus contortus third stage larvae (L3) on Brachiaria decumbens grass, as well as at verifying whether larval numbers on pasture varies over the day due to climatic conditions. Feces containing H. contortus L3 were deposited on the soil in the middle of herbage which was initially 30 cm high. Seven days later, samples of different herbage strata (0–10, 10–20 and >20 cm), remaining feces and a layer of approximately 1 cm soil were collected. Tests were carried out in four periods: September 2006, December 2006, March 2007, and June 2007. Samples were collected at sunrise, mid-day, sunset, and mid-night. The humidity and temperature conditions observed in different months influenced larval migration from the feces to the grass. In September, December and March, it rained after fecal deposition on pasture, which favored migration of larvae from the feces to the herbage. Conversely, in June 2007, when there was no rainfall after fecal deposition and temperatures were lower, L3 were mainly recovered from feces. As regards the vertical migration of larvae, the numbers of H. contortus L3 in the forage strata remained relatively constant over the day. This indicates there is not a determined period in which sheep on pasture are at higher risk of infection. Finally, in all collection periods a considerable amount of third stage larvae was observed on the herbage top, which is the first plant part consumed by sheep.

Experimental infection of Haemonchus contortus strains resistant and susceptible to benzimidazoles and the effect on mast cells distribution in the stomach of Mongolian gerbils (Meriones unguiculatus)

Establishment rate of Haemonchus contortus in non-suppressed and immunosuppressed gerbils within 14 days post-infection was compared after inoculation with 1,000 third-stage larvae (L3), exsheathed BZ-susceptible larvae. Based on significantly higher number of larvae in gerbils receiving low doses of immunosuppressant agent hydrocortisone, development of benzimidazole (BZ)-susceptible and BZ-resistant strain of nematode in the stomach was studied on days 4, 7, 10, and 14 p.i. Sections of stomach from both groups of animals were examined for overall histopathological response and dynamics of mucosal mast cells (MMC) and connective tissue mast cells (CTMC). In the immunosuppressed gerbils, H. contortus L3 stage larvae developed to the L4 stage on days 10 and 14 p.i., and their sex ratio was higher toward female worms. Significantly higher ratios of establishment rate were recorded for BZ-susceptible than BZ-resistant strain. Infection elicited strong inflammation mainly in the lamina propria mucosae, where MMC numbers peaked on day 7 p.i., being present in a significantly higher numbers in gerbils infected with BZ-susceptible strain. Infection with BZ-susceptible strain of nematode also resulted in a higher number of CTMC in comparison with the effect of BZ-resistant strain, which were observed in the tela submucosa only. Thus, H. contortus infection in gerbils seems to be a suitable model to study host-parasite interactions. Our results indicate that BZ-resistant strain of H. contortus have a decreased capacity to establish infection in direct relation with lower mucosal and connective tissue MCs counts in the stomach.

Correlation of parasitological and immunological parameters in sheep with high and low resistance to haemonchosis

Parasitological and immunological parameters of Blackbelly (Bb), a haemonchosis high-resistance sheep breed, and Columbia (Cb) a haemonchosis low-resistance breed were compared. Two experiments were performed: Bb and Cb lambs were infected weekly with 1000 third-stage larvae (L3) of Haemonchus contortus for 6 wk. In the first experiment, faecal egg counts (FEC) and blood eosinophils (BE) were determined for 15 wk. In the second experiment, the following parameters were measured weekly: FEC, plasma proteins, serum IgG to H. contortus L3 levels by ELISA and L3 antigens recognized by Western blot; animals were euthanized after 15 weeks and autopsies were performed to evaluate the total adult phases (AP), eosinophils and mast cells in the fundic abomasal region (FAR), pyloric abomasal region (PAR) and abomasal lymph node (ALN). Fecal egg counts and AP were significantly higher (P &lt; 0.05) in Cb lambs. BE showed a significant increase (P &lt; 0.05) in both breeds at week 9 and, subsequently, Cb lambs presented a sudden decrease in these levels. The Bb lambs revealed a higher amount (P &lt; 0.05) of antibodies between weeks 9 and 15 of the experiment, and Cb lambs showed a reduction in antibody levels at week 9. The Western blot revealed 11 bands of H. contortus L3 antigens; no differences were observed between breeds. The number of tissue eosinophils per cm2 in PAR and ALN was higher (P &lt; 0.05) in the Bb lambs. No differences were found (P &gt; 0.05) in the number of mast cells per cm2 in the sampled regions of both infected groups. The increase and maintenance of serum antibodies and circulating and local eosinophils in resistant animals agrees with the development and maintenance of a Th2 type immune response. The initial increase and further reduction of serum antibodies and circulating and local eosinophils in the low-resistance lamb breed suggest an initial Th2 type response and a subsequent change to a Th1 type response. Key words: Haemonchus, immune response, breed resistence, eosinophils, antibodies, sheep

Haemonchus contortus(Nematoda: Trichostrongylidae) infection in lambs elicits an unequivocal Th2 immune response

Selection of resistant animals and host immunization have been proposed as alternative methods for the control of gastrointestinal nematode parasites. However, a better knowledge of the mechanisms involved in protective immunity against these parasites is required for the development of optimal strategies. In this study, 3 month old INRA 401 lambs (n = 81) were allocated into three groups: uninfected control, challenged either once (primary-infected animals) or twice (previously-infected animals) with 10,000 Haemonchus contortus L3. Uninfected control and challenged animals were sequentially sacrificed at 3, 7, 15 and 28 days post challenge. In both challenged groups, a clear Th2-oriented immune response was observed in the abomasal lymph node and mucosa. IL-4 and IL-13 mRNA over-expression, recruitment of eosinophils, mast cells and globule leukocytes and production of specific systemic IgG and mucosal IgA were observed earlier in previously-infected animals than in primary-infected ones. At 28 days post infection, no differences between intensities of these responses were observed between the challenged groups. Worm establishment rates were similar in previously-infected and primary-infected lambs. However, reductions of worm development, female fecundity and fecal egg output were observed in previously-infected sheep. We conclude that H. contortus infection in young INRA 401 lambs induced an unequivocal Th2 immune response resulting in the regulation of worm egg production without affecting their establishment.

Lack of effects of quebracho and sainfoin hay on incoming third-stage larvae of Haemonchus contortus in goats

The effects of tannins on adult populations of Haemonchus contortus, Trichostrongylus colubriformis and Teladorsagia circumcincta in goats are characterised mainly by a decrease in egg excretion without any significant changes in worm number. In contrast, the impact of tannins on T. colubriformis or T. circumcincta third-stage larvae (L3) is associated with a significant reduction in worm establishment. The objective of the present study was to examine the effects of quebracho extract tannins on H. contortus L3. The consequences of consumption of sainfoin hay were also examined. Twenty-one naïve kids were divided into three experimental groups. Group Q received quebracho extract and group S received sainfoin hay from days D3 to D5. Group C remained as an infected control group. All kids received 1500 L3 H. contortus on D0, D1 and D2. On D18, post-infection, the kids were slaughtered and the worm populations compared in the different groups. Compared to the control values, the worm counts decreased, respectively, by 33% and 38% in groups Q and S but the differences were not significant. No differences were found in pathophysiological measurements between the three groups. The results confirm differences in tannin effect according to nematode species but not parasitic stage.

Relative resistance to gastrointestinal nematode parasites in Dorper, Katahdin, and St. Croix lambs under conditions encountered in the southeastern region of the United States

A study was conducted to evaluate the relative resistance to a natural or induced gastrointestinal (GI) nematode infection in hair and wool breeds of lambs. In Experiment 1, natural GI nematode infection was evaluated among fall born Dorper crossbred (DO; n=17) or St. Croix (SC; n=17) lambs. Ewe and wether lambs were weaned at 84±1.1 days of age and dewormed. Fecal egg counts (FECs) and blood packed cell volume (PCV) were determined between weaning and 56 days later. In Experiment 2, natural GI nematode infection was evaluated among spring born DO (7/8 or 3/4 Dorper; n=24), Katahdin (KA; n=26), SC ( n=8), and Suffolk (SU; n=10; wethers only) ewe and wether lambs from weaning (60±1.3 days of age; April) to 115±1.3 days of age (June). In both studies, lambs grazed bermudagrass overseeded with ryegrass, previously grazed with sheep, and were supplemented with 225–500 g of corn/soybean meal supplement. In Experiment 3, the relative resistance was examined among DO ( n=8), KA ( n=8), and SC ( n=9) ewe lambs to induced GI nematode infection (20,000 Haemonchus contortus L3). Lambs were weaned at 62±2.3 days of age, dewormed 22 days later and inoculated 6 days later (Day 0). In Experiment 1, FEC was greater ( P<0.04) at weaning in DO lambs and PCV was similar between DO and SC lambs. In Experiment 2, FEC was greatest and PCV least in DO lambs when compared with other breeds (breed×time, P<0.003). In Experiment 3, FEC was greater in DO and KA lambs compared with SC lambs ( P<0.02). By Day 42, four DO, six KA, and three SC lambs had been dewormed. With a low to moderate challenge, resistance to infection was similar among the three hair breeds examined and greater than the wool breed lambs. When the challenge escalated, St. Croix lambs were most resistant and Dorper crossbred lambs were similar or less resistant than Katahdin lambs.

Experimental haemonchosis in goats: effects of single and multiple infections in the host response.

Histopathological changes and the distribution of T lymphocytes (CD3), B cells (CD79alpha) and IgG secreting plasma cells were recorded in the abomasum and abomasal lymph nodes of goats during early and late post-infection stages with one to four doses of Haemonchus contortus L3. The infiltration of eosinophils, mast cells, CD3(+) T lymphocytes, CD79alpha(+)B cells and IgG(+) plasma cells in the abomasal mucosa increased dramatically from 10dpi onwards, whereas globule leukocytes were observed only during chronic infection. In late post-infection stages abomasal infiltration of globule leukocytes, CD3(+) T lymphocytes, CD79alpha(+)B cells and IgG(+) plasma cells was significantly higher (P<0.05) in reinfected (groups 6-8) than in primarily infected goats (group 5). In the abomasal lymph nodes, marked hyperplasia of lymphoid follicles and medullary cords, with increase of CD3(+) T lymphocytes, CD79alpha(+)B cells and IgG(+) plasma cells was recorded from 10dpi (group 3) onwards. Worm burdens and the severe abomasal response during the late post-infection stages suggests that a rapid expulsion of nematodes did not occur. The prolonged time required for generating globule leukocytes suggested that immune mechanisms dependent of this cell type are of crucial importance in the protective immunity against H. contortus in goats.

Vertical migration by the infective larvae of three species of parasitic nematodes: is the behaviour really a response to gravity?

Vertical migration by infective larvae (L3) of 3 species of nematodes was investigated. Upright truncated agarose cones were used to test upward migration, and comparable truncated cone-shaped agarose hollows were used to test downward migration. Flat agarose plates were control surfaces. When placed at the bases of upright cones, 74% of Ancylostoma caninum L3 migrated up, whereas only 16.5% migrated down the indented cones; this latter value was not significantly different from larval migration on flat plates. Strongyloides stercoralis L3 also migrated upward in significant numbers (80%). These larvae also failed to migrate downward under normal conditions. However, when the bottoms of the indented cones were 3-5 degrees C warmer than the tops, 75.5% of S. stercoralis L3 migrated down. In contrast, Haemonchus contortus L3 showed no tendency to crawl up or down cones, when compared with normal crawling behaviour on flat plates. Thus, L3 of A. caninum and S. stercoralis exhibited negative geotaxis, crawling against the pull of gravity, while H. contortus did not. The biology of these parasites may explain these behavioural differences.

Mechanisms of immunity to Haemonchus contortus infection in sheep.

In two separate experiments, sheep were immunized by nine to 12 weekly immunizing infections with 4000 Haemonchus contortus third stage larva (L3), drenched with anthelminthics and maintained free of H. contortus infection for a further 12 weeks. The anamnestic cellular immune responses in both the abomasal lymph node (ALN) and mucosa of the immunized sheep were examined 3 and 5 days post challenge with 50 000 H. contortus L3. Sheep in the two experiments clearly segregated out in two distinct groups, one in which challenge larvae were obviously present in the tissues of all 12 sheep at 3 and 5 days post challenge while no challenge larvae were detected in tissues of seven of the eight sheep in the other group. In sheep in which no tissue larvae were detected, very few changes were noted in either the ALN or mucosa. In contrast, dramatic changes were observed in the cellular profiles of the ALN and mucosa after challenge infection in sheep in which larvae were observed in the abomasal tissues. In the ALN, these changes were characterized by an increase in the relative percentage of gammadelta-TCR+ T cells and B cells and an increase in the proportion of CD4+ T cells coexpressing the activation markers MHC class II and CD25. In the abomasal mucosa, an increase in the number of infiltrating CD4+ and gammadelta-TCR+ T cells and B cells was observed by 3 days postinfection and these levels were further increased at 5 days postinfection. This infiltration of the abomasal mucosa by lymphocytes was accompanied by a dramatic increase in the number of infiltrating eosinophils, which were often in intimate association with the surface of H. contortus larvae. None of these changes occurred in the mucosa of the sheep that showed no sign of challenge larvae in the tissues; however, a transient increase in gammadelta T cells in the ALN and a drop in intraepithelial globule leucocytes were uniquely observed in these sheep at 5 days post challenge. These results suggest that two different types of immune responses can be generated after challenge infection of immunized sheep, one where tissue larvae are excluded from their tissue niche as observed previously and which is associated with changes in globular leucocyte population but no mobilization of the local immune system. In contrast, when challenge larvae reach their tissue niche, dramatic changes in the local immune system occur, including a pronounced infiltration of eosinophils. These two immune mechanisms may be associated with the rapid and delayed rejection of parasite infections in immune sheep.

Strategies for nematode transmission: selective migration of Trichostrongylus tenuis infective larvae

Successful transmission of macroparasites is dependent on exposure of susceptible hosts to free-living infective stages. When these hosts are herbivores that feed mostly on a single food plant then natural selection should favour those infective larvae that selectively ascend this main food plant. Red grouse feed predominantly on heather, Calluna vulgaris, so we predict that the infective larvae (L3) of the caecal nematode Trichostrongylus tenuis selectively locate and ascend heather plants. To determine whether the presence of heather influences the horizontal dispersal of T. tenuis L3 across soil, the movement of L3 across trays of soil with and without heather was investigated in the laboratory. More T. tenuis L3 were recovered from soil when heather was present, implying that larval migration may be influenced by chemical cues produced by heather plants. This was investigated in a second experiment, in which the horizontal dispersal of T. tenuis larvae was examined in the presence of heather and grass vegetation. This trial was repeated with larvae of a second species, Haemonchus contortus, a nematode whose hosts feed on a wide range of grass and shrub species. Significantly more larvae of both nematode species were recovered in the region of the heather than the grass or controls. This implies that T. tenuis and H. contortus L3 exhibit selective migration towards heather, perhaps reflecting a general response to plant cues which may be stronger for heather than for grass.

High-affinity ivermectin binding to recombinant subunits of the Haemonchus contortus glutamate-gated chloride channel

Glutamate-gated chloride channels (GluCls) are targets for the avermectin anthelmintics. A family of five GluCl subunit genes encoding seven subunits has been identified in Caenorhabditis elegans. We have previously shown that two orthologous genes in the parasite, Haemonchus contortus, encode three GluCl subunits (HcGluClβ, Hcgbr-2A and Hcgbr-2B) with high amino-acid identity (>80%) to their C. elegans counterparts. We amplified and cloned a further subunit cDNA, HcGluClα, from H. contortus eggs. Sequence comparisons suggested that this subunit was closely related to, but not orthologous with, the C. elegans GluClα1, α2 or α3/GBR-2 subunits (∼55% amino-acid identity). The HcGluClα cDNA from an ivermectin-resistant isolate contained no coding changes from the wild-type. All of the known H. contortus GluCl cDNA clones were subcloned into the expression vector pcDNA3.1 and transiently expressed in COS-7 cells. As predicted by functional data from the C. elegans orthologues, the Hcgbr-2A and HcGluClβ subunits failed to bind [ 3H]ivermectin. The Hcgbr-2B and HcGluClα subunits bound [ 3H]ivermectin with high affinity; the K d values were 70±16 and 26±12 pM, respectively. This binding was inhibited by a variety of avermectins, though cold ivermectin was the most potent inhibitor of [ 3H] ivermectin binding. Picrotoxin, fipronil, glutamate and GABA all failed to compete for ivermectin binding to either subunit. The affinity of [ 3H]ivermectin binding to H. contortus L3 P2 larval membrane preparations was re-examined and found to be 70±7 pM. The properties of orthologous GluCl subunits are likely to be conserved across species, but the repertoire and relative importance of those subunits may vary.

Identification and characterization of excreted-secreted products and surface coat antigens of animal and plant-parasitic nematodes.

Nematode surface coat (SC) proteins and excreted-secreted products (E-S) are likely to play important roles in the host-parasite interaction and considerable similarities can be found in SC proteins and E-S products from certain plant and animal parasitic nematodes. Monoclonal antibodies raised to E-S products of plant-parasitic nematodes were shown to cross-react with E-S products and the surface coats of the animal parasites Trichinella spiralis and Haemonchus contortus. Most of the antibodies recognized carbohydrate epitopes but the activity of 2 MAbs (IACR-CCNj.2a.15 and IACR-Misec.8D.3) which recognized proteic epitopes in these nematodes were further characterized. Antibody 2a.15 recognized the SC and oral exudate of Meloidogyne incognita, T. spiralis and H. contortus. This antigen was also immunolocalized in the lining of the oesophagus and gut and in the exudate present during ecdysis of H. contortus L3. Antibody 8D.3 reacted with the SC of these nematodes on cryosections but on live nematodes the immunofluorescence was very patchy and was shed from the nematode SC.