The effect of acquired resistance on adult worms of Trichostrong ylus colubriformis in lambs

Dineen J. K. and Windon R. G. 1980. The effect of acquired resistance on adult worms of Trichostrongylus colubriformis in Lambs. International Journal for Parasitology10: 249–252. Wether lambs were classified as either responders or non-responders to vaccination and challenge with Trichostrongylus colubriformis. Worms recovered from these animals and unvaccinated controls were measured, eggs in utero counted and sex ratios (male/female) were determined for individual burdens. The results showed that all three parameters were reduced in responder lambs compared with non-responder and unvaccinated lambs and were positively correlated with worm counts in responder lambs whereas they tended to be negatively correlated with worm counts in non-responders and unvaccinated animals.These various effects of resistance on parasite development in responder lambs may be due to the action of endogenous inflammatory agents generated immunologically. On the other hand negative correlation between developmental parameters and worm burdens in non-responders and unvaccinated lambs suggests that development of parasites in heavy infections may be restricted by competition for a limiting physiological, spatial or nutritional resource of the host.     

The effect of sire selection on the response of lambs to vaccination with irradiated Trichostrongylus colubriformis larvae

Dineen, J. K. and Windon, R. G. (1980). The effect of sire selection on the response of lambs to vaccination with irradiated Trichostrongylus colubriformis larvae. International Journal for Parasitology10: 189–196. Rams selected for responsiveness and unresponsiveness to vaccination with irradiated T. colubriformis larvae at an early age were mated to unselected random bred ewes. Progeny were vaccinated with 20,000 irradiated larvae at 8 and 12 weeks of age, given anthelmintic treatment at 16 weeks and challenged with 20,000 normai larvae at 17 weeks. The results, based on wether worm counts and ewe faecal egg counts, showed significant differences between responder and non-responder progeny. There was a significant correlation between worm counts and faecal egg counts of half-sibs from the same sire group. The occurrence of globule leucocytes was inversely related to worm burdens of wether progeny, however, no clear relationship was found with eosinophils. In vitro lymphocyte stimulation using T. colubriformis L₃ antigen, concanavalin A and lipopolysaccharide showed that statistically defined responder progeny, pooled from both responder and non-responder sire groups, gave higher responses than non-responder lambs after vaccination. The results confirm that genetically-determined factors are involved in the response of lambs to vaccination at an early age, and indicate that rapid genetic progress may be achieved in the type of mating usually carried out under fleld conditions.     

The response of lambs to vaccination and challenge with Trichostrongylus colubriformis: Effect of plane of nutrition on,and the inter-relationship between,immunological responsiveness and resistance

Wagland B. M., Steel J. W., Windon R. G. and Dineen J. K. 1984. The response of lambs to vaccination and challenge with Trichostrongylus colubriformis: effect of plane of nutrition on, and the inter-relationship between, immunological responsiveness and resistance. International Journal for Parasitology14: 39–44. Merino lambs weaned at 8 weeks of age were fed either ground and pelleted (high plane, HP) or chopped (low plane, LP) lucerne hay ad libitum to achieve an approximate 2-fold difference in liveweight gain. When aged 17 and 21 weeks, 15 of the 20 lambs in each diet group were vaccinated with 80,000 irradiated T. colubriformis larvae. At 25 weeks, vaccinated and unvaccinated lambs were treated with anthelmintic and one week later challenged with 30,000 normal larvae. Four weeks after challenge the animals were killed for worm counts. After vaccination HP lambs had higher titres of antibodies to the parasite and after challenge had lower worm egg outputs, and lower worm burdens than LP lambs. Immunological responsiveness (serum titre of complement-fixing antibodies against worm antigen) and manifestations of resistance (eggs produced per female worm per day and percent protection calculated from worm counts) were significantly correlated within dietary groups. Percent protection and egg production per female worm were highly correlated (r = ?0.81) in individual animals pooled over dietary groups, suggesting that both manifestations of resistance respond to essentially the same immunological mechanism. Failure to obtain significant correlation between weight gain pre-vaccination and immunological and resistance parameters indicated that animal production and resistance to infection are not genetically linked. Negative correlation of weight gain during the vaccination period with serum antibody titre at challenge suggests that the developing immune response competes with weight gain for limited physiological resources of the animal.     

The effect of selection of both sire and dam on the response of F1 generation lambs to vaccination with irradiated Trichostrongylus colubriformis larvae

Windon R. G. and Dineen J. K. (1981). The effect of selection of both sire and dam on the response of F₁ generation lambs to vaccination with irradiated Trichostrongylus colubriformis larvae. International Journal for Parasitology11: 11–18. Rams and ewes, tested for responsiveness to vaccination with irradiated T. colubriformis larvae at an early age, were mated on the basis of responder × responder and non-responder × non-responder. Progeny were vaccinated at 8 and 12 weeks of age with 20,000 irradiated larvae, treated with anthelmintic at 16 weeks and challenged with 20,000 normal larvae at 17 weeks. Faecal egg counts of progeny from responder matings were significantly lower than progeny from non-responders, and within each mating type, ewe lambs had markedly lower egg counts than ram lambs. The level of circulating complement-fixing antibodies to T. colubriformis larval extract were inversely related to egg counts. Thus, ewe progeny from responder matings had the highest serum antibody levels, non-responder ram progeny had the lowest levels and responder rams and non-responder ewes had similar intermediate levels. In vitro responses of cells stimulated with T. colubriformis L₃ antigen were greater in progeny from responder matings, whereas responses to bacterial lipopolysaccharide were higher in progeny from non-responder matings. The results confirm that the response to vaccination at an early age is genetically determined, and show that the response of progeny is most vigorously expressed when both sires and dams have been selected.     

The segregation of lambs into ‘responders’ and ‘non-responders’: Response to vaccination with irradiated Trichostrongylus colubriformis larvae before weaning

Random bred Merino ram and ewe lambs were vaccinated at 1, 2 and/or 3 months of age with irradiated T. colubriformis larvae. An exponentially increasing challenge of normal larvae was given to all groups including unvaccinated controls commencing at 1 month of age. The results, based on faecal egg counts, showed a dissociation into animals which responded to vaccination (geometric mean egg count 441) and those which did not (geometric mean egg count 1567). The proportion of responders was greatest in groups first vaccinated at the earliest age (1 month). Wool growth and liveweight gains showed severe depression corresponding to peak egg counts, however, responders were less affected than non-responders. There was no correlation between haemoglobin type and resistance to challenge. Faecal egg counts after impulse challenge with 10,000 normal larvae given at about 6$\text{1}\text{2}$ months of age showed a significant ranked correlation with those obtained during the primary exponential challenge. These results confirm that a proportion of young lambs respond to vaccination with irradiated larvae, and that genetically-determined factors are implicated in the ability of animals to respond to vaccination at an early age.     

The role of thresholds in the response of lambs to vaccination with irradiated Trichostrongylus colubriformis larvae

A piecewise logarithmic model fitted to worm counts of ewe lambs vaccinated and challenged in pens with a range of doses of irradiated and normal Trichostrongylus colubriformis larvae respectively, indicated that the threshold for response to both vaccine (V₀ = 4400) and challenge dose is exceeded by 5000 larvae. Whereas response was vaccine dose dependent, it was independent of challenge dose.Ram lambs vaccinated at low dose levels were as resistant against challenge as ewe lambs, but by contrast, failed to show increased protection after vaccination with high doses of irradiated larvae.Serum titre of antiworm complement-fixing antibodies at the time of challenge also indicated that ram lambs were less responsive immunologically than ewe lambs following vaccination at the higher dose levels.A field study showed that response to vaccination was only apparent after transfer of the sheep to heavily contaminated pastures, suggesting that previous exposure of the vaccinated animals to the low dose of infective larvae available on a lightly contaminated pasture constituted a challenge which was below the threshold.     

The relationship of abnormal mucosal microtopography with distribution of Trichostrongylus colubriformis in the small intestines of lambs

The relationship of abnormal mucosal microtopography with distribution of Trichostrongylus colubriformis in the small intestines of lambs. International Journal for Parasitology4: 153–163. The distribution of T. colubriformis was studied by counting worms in sequential 1-m segments from the guts of 14 infected lambs. Mucosal morphology was described at corresponding 1-m intervals and compared with similar samples from uninfected controls. A mean of 90 per cent of worms was recovered in the first 6 metres of gut. Maximum worm counts occurred in the first (eight lambs) second (three lambs) or third (three lambs) metre. Less than 0·8 per cent of worms were found in the abomasa of five lambs. Flat mucosae or abnormal surface patterns were seen frequently in the anterior small intestine of infected lambs. Degree of mucosal abnormality was positively associated with magnitude of worm numbers/m, and negatively correlated with distance of the sample from the pylorus, by analysis of partial correlation of worm numbers/m, mucosal type score, and distance from the pylorus. Mucosae from areas with > 4000 worms/m tended to have significantly shorter villi than intestine of control lambs. Factors influencing worm distribution and pattern of establishment are discussed, as is the association of extreme villus atrophy with poor performance by infected lambs.     

Investigations of 'transfer factor' activity in immunity to Ostertagia circumcincta and Trichostrongylus colubriformis infections in sheep.

Immunity was successfully transferred by ‘Transfer Factor’ prepared from leucocytes of two adult Scottish Blackface donor rams infected with O. circumcincta and T. colubriformis to 4-month-old susceptible Fin X Dorset lambs. The immunity was expressed by a significantly reduced faecal egg count and worm burden compared to challenged, untreated controls. The immunity was comparable to that produced in another group of lambs given an initial infection prior to challenge with both parasites.     

Studies of the responses of basophil and eosinophil leucocytes and mast cells to the nematode Trichostrongylus colubriformis: Comparison of cell populations in parasite resistant and susceptible guinea-pigs

Handlinger J. H. and Rothwell T. L. W. 1981. Studies of the responses of basophil and eosinophil leucocytes and mast cells to the nematode Trichostrongylus colubriformis: comparison of cell populations in parasite resistant and susceptible guinea-pigs. Internationaljournal for Parasitology11: 67–70. Basophil and eosinophil leucocytes and mast cells in T. colubriformis resistant and susceptible guinea-pigs were compared. There were significantly more circulating and small intestinal eosinophils in the resistant guinea-pigs. Intestinal eosinophils increased in both groups following infection with T. colubriformis but after 10 days the count in susceptible animals had only reached the pre-infection count in the resistant group. Pre-infection intestinal mast cell counts in the two groups were similar. Mast cell counts in susceptible guinea-pigs did not change during the period of observation but almost doubled within seven days of infection in the resistant animals.     

The effect of vaccinating infection during pregnancy and dietary protein supply on the peri-parturient immune response of sheep to infection with Teladorsagia circumcincta and Trichostrongylus colubriformis larvae

It is well established that dietary protein supply can influence the peri-parturient breakdown of immunity to nematode parasites but there is no information on the importance of exposure to nematode larvae during pregnancy for this response. We investigated this by exposing housed pregnant sheep, scanned as carrying two lambs, to a vaccinating infection with a trickle mixed infection of Teladorsagia circumcincta and Trichostrongylus colubriformis larvae (L3) or to no infection during weeks − 9 to − 4 relative to parturition. At the beginning of week − 3 all sheep were treated with anthelmintic to remove any vaccinating worm burden and from week − 2 to week +6 received a trickle challenge infection with the same nematodes. Within each vaccinating treatment there were two nutritional treatments (no. = 20 per subgroup) designed to provide 1.5 or 1.0 and 1.3 or 0.8 of metabolisable protein (MP) requirement during pregnancy and lactation, respectively. Five ewes were necropsied during weeks +1 and +3 to measure worm burdens and mucosal inflammatory cells and the remainder maintained until week +6. Serum levels of total, IgA and IgE antibodies against L3 antigen of each nematode were measured.Scanning errors and lamb losses resulted in some ewes carrying and/or rearing only one lamb. Numbers of lambs reared was therefore introduced as a treatment effect. Vaccinating infection delayed the peri-parturient rise in faecal egg count (FEC) by an average of 2 weeks but its effect on FEC during the first 6 weeks of lactation was smaller and less persistent than that of dietary MP supply and single- v. twin-suckling.Populations of both nematodes were lower in association with high MP supply, vaccination and single suckling. These changes were associated with increases in numbers of mucosal mast cells (MMC) as a result of both increased MP supply and vaccination. Evidence for a more rapid return of host ability to limit populations of the abdominal nematode T. circumcincta than of the intestinal nematode T. colubriformis was associated with fewer eosinophils and more globule leucocytes (GL) in abomasal than in intestinal tissue.None of the serum antibody isotypes was affected by dietary protein supply. Total and IgA antibodies were maintained by a current larval (vaccinating) intake. IgA titres, however, increased progressively during pregnancy, especially in twin-bearing ewes. IgE titres appeared to be sensitive primarily to the reproductive cycle itself, peaking around parturition.This work supports the conclusion that availability of MP supply influences the recruitment and activity of cells of the immune armoury of the gastro-intestinal tract to nematode parasites. The precise outcome may differ with site and/or nematode species.     

Studies on the kinetics of expulsion by guinea-pigs of primary infections with the intestinal nematode parasite Trichostrongylus colubriformis

Heston strain guinea-pigs were infected with 1000 Trichostrongylus colubriformis infective larvae and the kinetics of parasite expulsion from the host studied. Expulsion began approximately 15 days after infection, after which log worm count declined from an initial level of approximately 2·77 at a rate of approximately 0·1 per day. There was no significant difference in worm counts in each sex.     

Worm burden and immunological responses in Corriedale and Crioula Lanada sheep following natural infection with Haemonchus contortus

The effects of natural infection with Haemonchus contortus were compared in 15 Corriedale lambs and 18 Crioula Lanada lambs kept in the grazing conditions after weaning. The following parameters were assessed weekly for 9 consecutive weeks: number of eggs per gram of feces (EPG), packed cell volume (PCV), total serum proteins (TSP) and albumin (ALB). At 10 weeks, the animals were slaughtered and autopsied to evaluate to the worm burden and eosinophil, mast cell and globule leukocyte counts in histological sections of the abomasum. EPG counts and worm burden were significantly lower in Crioula lambs, which also showed higher levels of PCV, TSP and ALB, as well as higher eosinophil and globule leukocyte counts. Mast cell counts were similar for the two breeds. These results indicate that, under intensive pasture conditions, the Crioula Lanada breed shows a better response to natural infection with H. contortus, suggesting greater resistance to parasitism.     

An association between a lymphocyte antigen in sheep and the response to vaccination against the parasite Trichostrongylus colubriformis

Lymphocyte antigens were tested in sheep which had been selected for responsiveness to vaccination against the intestinal nematode Trichostrongylus colubriformis. These sheep had been bred in an assortative mating programme which produced offspring designated as either “high responders” or “low responders”, with highly heritable resistance or susceptibility.Ovine lymphocyte antigen (OLA) typing antisera were obtained from parous ewes in the course of matings which produced the high and low responder flocks. A particular antigen (SY1) was found to be present in high frequency on the lymphocytes of high responder (72·2%) and in lower frequency (21·9%) on the lymphocytes of low responder rams. In ewes, the frequency for high responders was 65·7% and for low responders it was 33·5%. A similar association between the SY1 antigen and low faecal egg count was found in random-bred sheep which had been vaccinated with irradiated larvae and challenged with normal larvae. The conclusion was drawn that this lymphocyte antigen was likely to be part of the sheep major histocompatibility complex which influenced the immune response of sheep to vaccination against the parasite.     

Variation in resistance to haemonchosis: selection of female sheep resistant to Haemonchus contortus.

Seventy female lambs (6-7 months old) which were exposed to natural infections of Haemonchus contortus were designated as responders or non-responders on the basis of 10 weekly cumulative faecal egg counts. Selected responder and non-responder lambs were treated with ivermectin, housed separately and 6 weeks post-housing, seven lambs from each group were given a trickle infection of Haemonchus contortus at 1000 L3 daily for 5 days per week up to 2 weeks and examined weekly for 10 weeks after first infection. Analysis of data revealed significantly lower mean faecal egg counts and non-significantly less weight loss in responder than non-responder lambs. Mean values of haemoglobin, packed cell volume, total serum protein and peripheral eosinophil counts were significantly higher in responders than non-responders. In contrast, serum pepsinogen concentration was significantly less in responders than in non-responders. At 10 weeks post-infection, there were fewer pathological lesions and significantly lower worm burdens in responders than in non-responders. These results demonstrate a distinct resistance in responders to Haemonchus contortus infection.     

Expression of genes in gastrointestinal and lymphatic tissues during parasite infection in sheep genetically resistant or susceptible to Trichostrongylus colubriformis and Haemonchus contortus

Resistance to an acute gastrointestinal nematode (GIN) infection is dependent on the ability of the host to recognise the parasite and mount a protective Th2 response. It is hypothesised that lambs which are genetically susceptible to GIN will differentially up-regulate Th1-type genes and therefore remain susceptible to chronic parasitism compared with genetically resistant lambs which will differentially up-regulate Th2-type genes and clear the parasite infection. Two selection flocks, in which lines of Merino sheep produced lambs genetically resistant or susceptible to GIN, were acutely challenged once or thrice with either Haemonchus contortus or Trichostrongylus colubriformis. Faecal-egg counts (FECs), and plasma and tissue anti-parasite (H. contortus or T. colubriformis) antibody isotype responses showed that resistant animals challenged three times with T. colubriformis established a protective Th2 response (negligible FEC, IgG1 and IgE) whereas susceptible animals required multiple challenges to establish a significant IgG1 response despite FECs remaining high. Trichostrongylus colubriformis elicited a more pronounced host response than H. contortus. RNA extracted from tissues at the site of each parasite infection and associated lymph nodes were interrogated by microarray and quantitative PCR analyses to correlate host gene expression to FECs and antibody responses. The IFN-γ inducible gene cxcl10 was up-regulated in the susceptible line of the Trichostrongylus selection flock sheep after a tertiary challenge with the parasites H. contortus and T. colubriformis. However, a uniform pattern of genes was not up-regulated in resistant animals from both selection flocks during both parasite infections, suggesting that the mode of host resistance to these parasites is different, although some similarities in host susceptibility were apparent.     

Parasitological and immunological responses of genetically resistant Merino sheep on pastures contaminated with parasitic nematodes.

One hundred and twenty lambs were grazed continuously from weaning until 9 months of age on 12 plots contaminated with larvae of three nematode species (Haemonchus contortus, Trichostrongylus colubriformis and Ostertagia circumcincta). The lambs were sired by either a genetically resistant ram or susceptible rams (determined by the response of previous progeny to artificial H. contortus infection). Half the resistant and half the susceptible lambs were given strategic anthelmintic treatment and the remainder remained untreated. Faecal egg counts and blood packed cell volume were measured frequently in all animals. One and 5 months after weaning, two lambs from each plot were slaughtered, and worm burdens and larval establishment rates of the three species of nematode were estimated. At the second slaughter, leukotriene levels and larval migration inhibitory (LMI) activity were measured in mucus collected from the small intestine. The dominant species in all faecal samples and the gastrointestinal tract was T. colubriformis. Lambs of the resistant genotype had lower faecal worm egg counts, lower worm burdens and higher levels of resistance to larval establishment. There were no differences in larval migration inhibition (LMI) activity, but resistant lambs had higher levels of the leukotriene LTC4/D4/E4. Further, the resistant genotype, identified on responsiveness to artificial infections with H. contortus, was more resistant to infections of three important species acquired naturally from contaminated pastures. All these genetic differences were maintained while the lambs were subject to strategic anthelmintic treatment.     

Eosinophil responses in sheep selected for high and low responsiveness to Trichostrongylus colubriformis

A breeding programme, based on selection for faecal egg counts, has produced lines of sheep which demonstrate either increased resistance (high responder) or susceptibility (low responder) to challenge infection with T. colubriformis after vaccination with irradiated larvae. Circulating blood eosinophilia, a hallmark of helminth infections, was examined in third generation lambs from two separate selective matings and random bred control lambs. Numbers of eosinophils were higher in high responder lambs when compared to low responders after vaccination and challenge infections. Analysis of eosinophil counts confirmed a strong line effect and there was no evidence of a sex effect. Random bred lambs showed wide individual variations in eosinophil numbers and their response to infection. It was concluded that peripheral eosinophilia was more a measure of host responsiveness to infection than an indicator of helminthiasis. As such the eosinophil may serve as an indicator of the hosts ability to respond to T. colubriformis vaccination and infection.     

Intraspecific Epitopic Variation in a Carbohydrate Antigen Exposed on the Surface of Trichostrongylus colubriformis Infective L3 Larvae

The carbohydrate larval antigen, CarLA, is present on the exposed surface of all strongylid nematode infective L3 larvae tested, and antibodies against CarLA can promote rapid immune rejection of incoming Trichostrongylus colubriformis larvae in sheep. A library of ovine recombinant single chain Fv (scFv) antibody fragments, displayed on phage, was prepared from B cell mRNA of field-immune sheep. Phage displaying scFvs that bind to the surface of living exsheathed T. colubriformis L3 larvae were identified, and the majority of worm-binding scFvs recognized CarLA. Characterization of greater than 500 worm surface binding phage resulted in the identification of nine different anti-CarLA scFvs that recognized three distinct T. colubriformis CarLA epitopes based on blocking and additive ELISA. All anti-CarLA scFvs were specific to the T. colubriformis species of nematode. Each of the three scFv epitope classes displayed identical Western blot recognition patterns and recognized the exposed surface of living T. colubriformis exsheathed L3 larvae. Surprisingly, each of the anti-CarLA scFvs was able to bind to only a subset of worms. Double-labelling indirect immunofluorescence revealed that the three classes of anti-CarLA scFvs recognize distinct, non-overlapping, T. colubriformis sub-populations. These results demonstrate that individual T. colubriformis L3 larvae display only one of at least three distinct antigenic forms of CarLA on their surface at any given time, and suggest that antigenic variation within CarLA is likely a mechanism of immune evasion in strongylid nematodes.     

Effects of lactation on Trichostrongylus colubriformis infections in the guinea pig

Effects of lactation on Trichostrongylus colubriformis infections in the guinea pig. International journal for Parasitology, 4: 177–181. An increase in parasite egg output occurred in lactating guinea pigs infected with Trichostrongylus colubriformis, compared with that of either non-lactating or nulliparous females similarly infected. The lactating animals carried more parasites than either of the other two groups both as fourth stage larvae 9 days after infection and as adults at the peak of egg output 19 days after infection. Both the nulliparous and the non-lactating female guinea pigs had more mast cells and eosinophils in the small intestinal mucosa than did the lactating females.The behaviour of T. colubriformis in the lactating guinea pig is considered to be substantially the same as that of the various nematodes which have been studied in the lactating ewe.     

Studies on the inhibition of rejection of Trichostrongylus colubriformis larvae from immune sheep

A total of 8 drugs were examined for their ability to suppress the rejection of Trichostrongylus colubriformis infective larvae (L₃) from immune sheep. Specific antagonists of leukotrienes (plroxicam), prostaglandin (indomethacin) and the immunosuppressant cyclosporin A were given orally, while injectable preparations of dexamethasone, chlorpheniramine, BN 52051, WEB 2086 (anti-platelet-activating factor) and theophylline were administered as directed. The drugs were given for 5 days prior to and 4 days after challenge with 20,000 L₃, when worm counts were done. Corticosteroids inhibited rejection by around 70% in two experiments, and none of the remaining compounds were effective. In the third study, six of the drugs were given to susceptible sheep and did not affect the establishment of a primary infection with T. colubriformis.