Persistence of acquired immunity to Trichostrongylus colubriformis in sheep after termination of infection

Lambs were infected with 6000 Trichostrongylus colubriformis L3 per week for 18, 12 or 6 weeks, beginning at ages 14, 20 and 26 weeks, respectively. At the end of the primary infection subgroups had geometric mean adult worm burdens of 5000, 15,000 and 18,000, respectively. In the remaining sheep the worm population was removed with anthelmintic, and sheep had no further larval intake until challenged with T. colubriformis 1,6, 12, 18 or 24 weeks later. In the groups given 18 or 12 weeks primary infection, establishment of challenge doses was low (less than 25% of establishment in helminth naive controls) in most animals at all challenge times. However, for the groups given 6 weeks primary infection, establishment was low only at the first two challenge times. Thereafter it had similar mean to control groups, but much greater variance. Other subgroups were challenged with T. colubriformis, Ostertagia circumcincta and Haemonchus contortus 1 week after worm removal. In these animals T. colubriformis establishment was not different to animals challenged at the same time with T. colubriformis alone, however immunity to T. colubriformis afforded little protection against the other species. The results of this experiment were incorporated into a simulation model of the population dynamics of T. colubriformis.     

Infection with Haemonchus contortus in sheep and the role of adaptive immunity in selection of the parasite

1988. Infection with Haemonchus contortus in sheep and the role of adaptive immunity in selection of the parasite. International Journal for Parasitology 18: 1071–1075. A series of infections with Haemonchus contortus in immune and non-immune sheep gave no indication that successive generations of the parasite were selected for the enhanced ability to cope with host-protective immunity. Separate subpopulations of the parasite were passaged through individual immune sheep and were compared at each generation with infections by the pooled populations in an additional panel of immune sheep. The experiment ceased when infections became too low to allow the production of sufficient infective larvae for reinfection. Results showed that H. contortus is unable to make adjustments to the immune status of a given sheep. Attention is thus diverted from the process through which host identifies self and non-self and from the histocompatibility system as the targets for possible antigen-mimicry by H. contortus.     

Population dynamics of Trichostrongylus colubriformis and Ostertagia circumcincta in single and concurrent infections.

Twenty-one-week-old worm-free pen-reared lambs were infected weekly with either 10,000 T. colubriformis larvae, 5000 O. circumcincta larvae, or with both species (15,000 larvae per week). Larval establishment and total worm burdens were estimated after 4, 7, 10 and 13 weeks of infection. Faecal egg counts and lamb bodyweights were measured weekly, and numbers of eosinophils in blood were estimated before infection and at weeks 5, 8 and 14. For both species of worms, the dynamics of infection (establishment, worm burdens, egg counts) were not affected by concurrent or pre-existing infection with the other species. Infection with T. colubriformis alone did not protect against O. circumcincta, but infection with O. circumcincta alone provided slight protection against the T. colubriformis larvae. Blood eosinophils increased between 5 and 8 weeks of infection and were similar for the three infections. This corresponded to the reduction in establishment for both species.     

The interaction of Trypanosoma congolense and Haemonchus contortus in Djallonké sheep

The interaction between Trypanosoma congolense and Haemonchus contortus was studied in 5 groups of 8 Djallonké sheep. Two groups received a single infection with either H. contortus or T. congolense, and 2 groups were infected with T. congolense followed by H. contortus (TH) or vice versa (HT). One group was kept as uninfected controls. Mortality due to infection was observed only in the dual infection groups. In the TH group, the effects were more acute whereas in the HT group they were more chronic. No significant differences in weight gain could be demonstrated between infected and control groups. Djallonké sheep are able to withstand a single infection with either T. congolense or H. contortus, which confirms their trypanotolerant nature and provides preliminary indication of resistance against helminth infections. However, when exposed to successive infections with both parasites, some of the animals lose this tolerance.     

The Intestinal Expulsion of the Roundworm Ascaris suum Is Associated with Eosinophils,Intra-Epithelial T Cells and Decreased Intestinal Transit Time

Ascaris lumbricoides remains the most common endoparasite in humans, yet there is still very little information available about the immunological principles of protection, especially those directed against larval stages. Due to the natural host-parasite relationship, pigs infected with A. suum make an excellent model to study the mechanisms of protection against this nematode. In pigs, a self-cure reaction eliminates most larvae from the small intestine between 14 and 21 days post infection. In this study, we investigated the mucosal immune response leading to the expulsion of A. suum and the contribution of the hepato-tracheal migration. Self-cure was independent of previous passage through the liver or lungs, as infection with lung stage larvae did not impair self-cure. When animals were infected with 14-day-old intestinal larvae, the larvae were being driven distally in the small intestine around 7 days post infection but by 18 days post infection they re-inhabited the proximal part of the small intestine, indicating that more developed larvae can counter the expulsion mechanism. Self-cure was consistently associated with eosinophilia and intra-epithelial T cells in the jejunum. Furthermore, we identified increased gut movement as a possible mechanism of self-cure as the small intestinal transit time was markedly decreased at the time of expulsion of the worms. Taken together, these results shed new light on the mechanisms of self-cure that occur during A. suum infections.     

Population dynamics of Trichostrongylus colubriformis in sheep: the effect of host age on the establishment of infective larvae

Sheep, reared worm-free in pens and aged 12-36 weeks, were infected each weekday with 2000 infective T. colubriformis larvae (L3). Establishment was measured at various times during the course of infection and the rate of development of resistance to new infection was found to be faster in older than in younger hosts. In addition arrested development at the exsheathed L3 stage was found to be less marked in older hosts. Sheep which had experienced natural infection on pasture up to 20 weeks of age before exposure, in pens, to the same experimental infections as their worm-free counterparts showed similar immune responses. However, at 36 weeks of age, pasture-reared sheep had acquired a high level of resistance to infection with T. colubriformis.     

Cryopreservation of the first-stage larvae of trichostrongylid nematode parasites

First stage (L1) larvae of Haemonchus contortus, Trichostrongylus colubriformis and Ostertagia circumcincta can be cryopreserved in the presence of DMSO using a two-step freezing protocol involving an initial period at −80°C prior to transfer to liquid nitrogen. Thawed L1 larvae continue development in vitro producing third stage (L3) larvae that are infective to sheep when dosed per os. Establishment rates for L3 larvae grown from thawed L1 larvae were 40 and 80% for H. contortus and T. colubriformis, respectively. There was no difference in survival or infectivity between benzimidazole (BZ)-susceptible and BZ-resistant H. contortus isolates and cryopreservation caused no shift in their BZ-resistance status as indicated in an in vitro larval development assay. Cryopreservation also had no effect on the sensitivity of these isolates to the avermectins or levamisole in vitro. High survival rates (60–70%), good levels of establishment and the stability of anthelmintic resistance status of isolates indicate that little if any selection occurs during the cryopreservation process. L1 larvae of all 3 species have been successfully recovered after 16 months storage in liquid nitrogen, cultured to the L3 stage and established in sheep.     

Trichinella spiralis: expression of rapid expulsion in rats exposed to an abbreviated enteral infection.

Rats infected with Trichinella spiralis for the first week of the enteral infectious cycle displayed a strong rapid expulsion reaction during a challenge infection. The response was induced with equal facility in animals given low or high immunizing doses of infectious larvae (500 to 5000 larvae). Large challenge infections resulted in a 10–15% reduction in the efficiency of rejection as assessed 24 hr after challenge. Rats became primed to express rapid expulsion within the first week of primary infection whether the infection remained patent or not. However, maximum effectiveness was not realized until the second week after the initial infection. Once induced, the capacity to express rapid expulsion persisted for 6 weeks after the primary infection. Immunized hosts were capable of resisting two challenge infections spaced by periods of from 12 to 72 hr. This finding suggests that a mediator is not consumed by the initial response.     

Resistance of young lambs to Haemonchus contortus infection, and its loss following anthelmintic treatment

1988. Resistance of young lambs to Haemonchus contortus infection, and its loss following anthelmintic treatment. International Journal for Parasitology 18: 1107–1109. A comparison of worm burdens of grazing Merino lambs that were untreated, treated once and allowed to graze for 2 weeks, or given suppressive anthelmintic treatment until their final 2 weeks on pasture, showed that resistance to reinfection with Ostertagia circumcincta and Trichostrongylus colubriformis did not develop until the lambs were about 8 months old. By contrast, there was clear evidence that untreated lambs, but not lambs given one or more treatments, had acquired substantial resistance to infection with Haemonchus contortus by an age of 4 months.     

Studies of stage-specific immunity against Trichostrongylus colubriformis in sheep: immunization by normal and truncated infections.

Sheep immunized with multiple normal infections of 30,000 Trichostrongylus colubriformis larvae (T.c. L3) suppressed the fecundity, establishment and survival of adoptively transferred adult worms, showing that these parasites were susceptible to the effects of host immunity. When sheep were immunized by four 'truncated' larval infections of 4, 7 or 10 days' (d) duration with 10(5) T.c. L3, animals given 4 x 4d infections were susceptible to challenge, whereas sheep given 4 x 7d and 4 x 10d infections were significantly protected. A serial analysis of the rejection of T. colubriformis from nine sheep given 5 x 7d infections revealed that the challenge larval infection given intraduodenally was expelled within 3 days after challenge (DAC). However, another five of these sheep only rejected around 50% of transferred adult worms by 21 DAC when compared with control animals. The results indicate that stage-specific antigens produced by early L3 and L4 stages of T. colubriformis effectively immunize sheep against a larval challenge but appear less reliably protective against adult worms.     

Response to helminth infection of sheep selected for resistance to Haemonchus contortus

Lines of Merino sheep selected for increased (IRH) and decreased (DRH) resistance to Haemonchus contortus were compared with an unselected (CH) line, after approximately four generations of selection. Measurements were recorded on 69 IRH, 47 DRH and 84 CH animals. Following artificial challenge with H. contortus, the IRH line had significantly (P < 0.001) lower faecal egg counts than the CH and DRH lines (2730,12,720 and 17,400 epg, respectively). Significant differences (P<0.05) were found between all lines in the minimum packed cell volumes during artificial infection (25.7, 22.0 and 20.3%) and in faecal egg counts after natural infection (140, 3590 and 8750 epg). Differences were also recorded (P<0.05) following artificial challenge with Trichostrongylus colubriformis (490, 840 and 1340 epg). On a percentage basis, faecal egg counts in the IRH line deviated less from the CH line following artificial infection with T. colubriformis (42%) than with H. contortus (79%). The reverse was true for the DRH line (60 and 37%, respectively). Differences in egg output of this magnitude should have a marked effect on requirements for anthelmintic treatment, rate of development of drug resistance and level of pasture contamination when the lines are grazed separately.     

Studies of stage-specific immunity against Trichostrongylus colubriformis in sheep: immunization with adult parasites.

Merino sheep immunized by the adoptive transfer of adult T. colubriformis for 8 weeks were significantly protected against a challenge infection of 20,000 larvae. Two additional groups of sheep received a primary infection of 9000 adult worms which were allowed to persist for 14 weeks before one group was drenched. When both groups were challenged 10 days later with 30,000 larvae, serial necropsies of these and naive sheep revealed that worm rejection did not occur until 7-10 days after challenge. By comparison with the rapid rejection of larval challenges from sheep immunized with normal primary infections, the results suggest that the antigens which elicited rejection in these experiments are stage-specific and were only present or synthesized in sufficient quantities when parasites had developed for 1 week.     

Studies on the role of mucus and mucosal hypersensitivity reactions during rejection of Trichostrongylus colubriformis from the intestine of immune sheep using an experimental challenge model.

Nematode-naive sheep and sheep immunised by truncated infections with Trichostrongylus colubriformis were fitted with intestinal cannulae to allow administration of challenge infection and collection of intestinal fluids. Sheep were slaughtered at various times after challenge and the distribution of larvae along the small intestine was determined. Results showed that immune sheep had significantly fewer larvae in their intestines and that some sheep could expel the challenge infection within 2 h. Mucus samples from immune sheep contained increased parasite-specific antibody, histamine and anti-parasite activity as measured by larval migration inhibition assay. Higher levels of antibody and histamine were seen in intestinal fluids of immune sheep after challenge. Immunisation of sheep by truncated infections stimulated serum IgE and resulted in significantly higher numbers of IgE-positive cells in gut tissue sections before challenge and at 2 h and 24 h after challenge. Immune sheep also had greater numbers of mucosal mast cells and globule leucocytes after challenge, compared with naive sheep. When challenge larvae were mixed with mucus from immune sheep and infused back into naive recipient sheep, there was a distinct displacement of the larval population towards the distal part of the intestine, compared with the profile of larval establishment after infusion with mucus from naive sheep. These results are further evidence for an immediate hypersensitivity reaction in the intestine of immune sheep, where challenge larvae are expelled within 2 h and confirm the direct anti-larval properties of mucus. The cannulated-sheep challenge model described here will be a useful tool to unravel the mechanism of larval rejection from immune sheep and could lead to novel therapies.     

The response of immune sheep to challenge with Trichostrongylus colubriformis: enteric plasma loss and secretion of biogenic amines

Sheep were challenged with a single large dose of larvae after vaccination with irradiated Trichostrongylus colubriformis larvae. Worm counts performed on vaccinated sheep 6 or 7 days after challenge (DAC) showed that they were solidly immune and only retarded L3 larvae were recovered at this time. Enteric plasma loss (EPL) in vaccinated animals increased immediately after challenge to peak 4–6 DAC and then decreased to pre-challenge levels. In contrast, a substantial rise in EPL did not occur in unvaccinated sheep until 10 DAC. Secretion of histamine into the duodenum of immunized sheep increased significantly from 2 to 7 DAC with the highest value at 6 DAC which corresponded with a lower duodenal tissue histamine level at this time. Histamine and 5HT secretion into the small intestine of previously uninfected sheep gradually increased during 12 weeks of a trickle infection of 3000 normal T. colubriformis larvae per week. The results indicate that rejection of incoming larvae by immune sheep is accompanied by an intestinal inflammatory response involving secretion of biogenic amines and a concurrent plasma loss.     

Studies on the immune response of sheep to Chabertia ovina (Fabricius, 1788)

The immunological basis of host resistance to C. ovina was established by the demonstration of a marked anamnestic response by 6 sheep to a second infection of C. ovina after previous sensitization. Reaginic antibody was detected in the circulation of 20 sheep tested during both primary and secondary infection. It was first detected 5 weeks after infection with 2000 larvae, 8 weeks after infection with 1000 larvae and 12 weeks after infection with 400 larvae. In contrast, precipitating antibody was detected in only 10 per cent of sheep tested, while in vivo and in vitro tests for delayed hypersensitivity were negative. Studies on the effect of antilymphocytic serum on immunity to C. ovina suggested that the development of resistance to C. ovina requires the participation of the thymus-derived lymphocyte.     

Population dynamics of Haemonchus contortus and Trichostrongylus spp. in sheep

Twenty-five lambs grazing a parasite-contaminated pasture were divided into four groups. One group of seven lambs were left untreated and three groups of six lambs each were treated with levamisole at 2 week, 3 week or 1 month intervals respectively. Body weights, fecal egg counts and packed cell volumes were determined every 2 weeks for 8 weeks. Lambs were then necropsied on the day they would have received another treatment, thus the maximum accumulation of parasites at each treatment frequency was measured. Treatment at 2 and 3 week intervals markedly suppressed fecal egg counts, but egg counts of sheep treated every month rose between treatments and untreated animals continually shed eggs. Treatment at all three frequencies provided only slight protection from blood loss, but it lessened weight loss. There was little difference between treatment groups in burdens of adult H. contortus, but burdens of T. axei and T. colubriformis and arrested larvae of H. contortus increased with decreasing frequency of treatment. The results support the findings of other workers that Trichostrongylus spp. have a long adult life span and continue to accumulate with time, whereas H. contortus may have a short adult life span and a rapid population turnover. In the face of constant reinfection, repeated anthelmintic treatment gives more protection from the effects of the long-lived parasites than from those with a rapid population turnover.     

Immunity acquired by sheep from an experimental infection with haemonchus contortus

Adams D.B. and Beh K.J. 1981. Immunity acquired by sheep from an experimental infection with Haemonchus contortus. International Journal for Parasitology11: 381–386. A primary infection of sheep with a single dose of Haemonchus contortus larvae was traced by faecal egg counts until it had substantially declined after 55 weeks. These primed sheep were then given a sequence of two reinfections with the parasite. Comparison of faecal egg counts in primed sheep and in two separate groups of previously worm-free sheep showed that primary infection conferred significant immunity. This, however, was not sufficiently protective to prevent the development of further anaemia and faecal egg counts indicative of clinical haemonchosis. It is suggested that an adaptation in the host-parasite relationship which promotes the longevity of primary infection with H. contortus may also moderate the induction of acquired immunity.The titre of haemagglutinating antibody specific for H. contortus rose in serum during the course of primary infection, but the two reinfections did not stimulate a rise in titre. Titres of haemagglutinating antibody before reinfection did not correlate with subsequent faecal egg counts.     

Thymus-dependency of the immune response of mice to a primary infection with the nematode Trichuris muris

Adult mice which had been thymectomized, irradiated and given stem-cell protection were incapable of making a self-cure response to a primary infection with the nematode Trichuris muris. The capacity to mount a self-cure response was restored by the injection of 2·5 × 10⁶ mesenteric lymph node lymphocytes or by 2·5 × 10⁸, but not 2·5 × 10⁷, thymocytes. Restoration of the ability to respond to sheep red blood cells was achieved with all three cell injections. Suppression of the immune response was also achieved by injection of ALS during the second week of infection and at intervals thereafter. The results of thymectomy and ALS treatment show that immunity to T. muris is dependent upon the presence of an intact thymus and thymus-dependent cell population.     

Regulation of Haemonchus contortus populations in sheep exposed to continuous infection

Groups of sheep were infected three times weekly for 15 weeks with infective larvae of Haemonchus contortus at four rates ranging from 600 to 4800 larvae per week. At 3-weekly intervals, sheep from each group were killed for total worm counts after receiving a dose of radiolabelled larvae which enabled a direct measurement of establishment of incoming larvae during the continuing infection. Peak H. contortus burdens were reached between 6 and 9 weeks of infection, and were related to infection rate. In the groups receiving 2400 and 4800 larvae per week, worm numbers then declined rapidly and by the end of the experiment were lower than those in the groups given 600 or 1200 larvae per week. Establishment of incoming larvae was not influenced by infection rate, and declined from 45% in the first 4 weeks of infection to insignificant levels during the final 6 weeks. The proportion of incoming larvae arrested in their development increased as the infection progressed. It was concluded that H. contortus numbers were regulated by development of resistance to infection and by a loss of established worms which was related to the current rate of larval intake, and to the host's previous experience of infection.     

Effects of sainfoin (Onobrychis viciifolia) extract and monomers of condensed tannins on the association of abomasal nematode larvae with fundic explants

Tannin-rich forages offer an alternative to anthelmintic chemicals to control gastrointestinal nematodes. However, the mode of action of such bioactive plants still needs to be assessed. Previous studies have shown that extracts of tannin-rich plants interfere with the first phase of host invasion, i.e., the exsheathment of infective larvae (L3s). In the current study, we examined the hypothesis that exposure to tannins could also affect the second phase of larval establishment, i.e., the tissue association/penetration of the exsheathed L3s into the digestive mucosae. An in vitro direct challenge technique using fundic explants was applied in this study. The main parasite model was Haemonchus contortus. The objectives were to verify: (i) whether a modification of the association/penetration of L3s with the mucosae occurred after contact with sainfoin extract; (ii) whether this is a dose-dependent phenomenon; (iii) whether tannins were responsible for these effects; (iv) whether these effects were dependent on the parasite species; and (v) how the biochemical structure of tannins might influence these effects. Following 3 h contact with sainfoin extract at 1,200 μg/ml, the penetration of exsheathed L3s of H. contortus and Teladorsagia circumcincta into fundic explants was significantly reduced. Moreover, a dose–response relationship was found for H. contortus. For both nematodes, the changes were totally alleviated after addition of polyvinyl polypyrrolidone, an inhibitor of tannins, to the sainfoin extract, suggesting that tannins play a major role in the observed effects. Comparison of results obtained with different monomers of condensed tannins confirms a relationship between structure and activity, the prodelphinidin monomers and galloyl-derivatives being more effective than the procyanidin monomers. Combined with the delay or the inhibition of larval exsheathment previously shown, these effects could explain how tanniniferous plants reduce the establishment of infective larvae in small ruminants.