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.     

Population dynamics of the parasitic stages of Ostertagia spp. in sheep

Callinan A. P. L. and Arundel J. H. 1982. Population dynamics of the parasitic stages of Ostertagia spp. in sheep. International Journal for Parasitology12: 531–535. The development and survival of continuing infections of Ostertagia spp. in weaner sheep were studied in order to develop a general model of the parasitic stages of the life cycle of these sheep nematodes. After 10 days, 13.8% of infective larvae (L₃) given at the rate of 1 dose of 1000 L₃ twice per week (group 1) and 20.8% of L₃ given at the rate of 10,000 L₃ twice per week (group 2) were recovered in the first of the serial nematode counts. In the final counts at 137 days, 7.7 and 0.7% were recovered in these groups. The build up and maintenance of nematode populations was regulated and related to the level of infection. A model in which the death rate of the parasitic stages was a function of the time of exposure to infection and rate of infection was used to describe the serial total nematode counts. During the experiment there was no noticeable trend in numbers of fourth stage larvae (L₄) in nematode counts, the size of adult nematodes, nematode egg counts (EPG) and egg output per female nematode (EPF). After 112 days, liveweight gains were significantly reduced in group 2 only, but increases in wool lengths were significantly reduced in both groups.     

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.     

Cholecystokinin and anorexia in sheep infected by the intestinal nematode Trichostrongylus colubriformis

Symons L. E. A. and Hennessy D. R. 1981. Cholecystokinin and anorexia in sheep infected by the intestinal nematode Trichostrongylus colubriformis. Internationaljournal for Parasitology11: 55–58. It was postulated that there is a correlation between anorexia in intestinal nematode infection and the plasma concentration of cholecystokinin (CCK). In the first experiment plasma concentration of CCK rose as food consumption fell until, when sheep infected with Trichostrongylus colubriformis were almost completely anorexic, it had increased by 65%. Plasma CCK and food consumption returned to pre-infection levels in from four to six days after administration of an anthelmintic. In the second experiment food consumption by uninfected sheep was reduced in the first ten minutes after intravenous infusion of 150–300 μg of the octapeptide of CCK. It was concluded that anorexia in these infections may be due to or be mediated by higher concentrations of CCK.     

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.     

Haemonchus contortus and Trichostrongylus colubriformis did not adapt to long-term exposure to sheep that were genetically resistant or susceptible to nematode infections

We tested the hypothesis that Haemonchus contortus and Trichostrongylus colubriformis would adapt to long-term exposure to sheep that were either genetically resistant or susceptible to H. contortus. Sheep genotypes were from lines with 10 years prior selection for low (resistant, R) or high (susceptible, S) faecal worm egg count (WEC) following H. contortus infection. Long-term exposure of H. contortus and T.colubriformis to R or S genotypes was achieved using serial passage for up to 30 nematode generations. Thus, we generated four nematode strains; one strain of each species solely exposed to R sheep and one strain of each species solely exposed to S sheep. Considerable host genotype differences in mean WEC during serial passage confirmed adequate nematode selection pressure for both H. contortus (R 4900 eggs per gram (epg), S 19,900 epg) and T. colubriformis (R 5300 epg, S 13,500 epg). Adaptation of nematode strain to host genotype was tested using seven cross-classified tests for H. contortus, and two cross-classified and one outbred genotype test for T. colubriformis. In the cross-classified design, where each strain infects groups of R, S or randomly bred control sheep, parasite adaptation would be indicated by a significant host genotype by nematode strain interaction for traits indicating parasite reproductive success; specifically WEC and, for H. contortus strains, packed cell volume. We found no significant evidence of parasite adaptation to host genotype (P > 0.05) for either the H. contortus or T. colubriformis strains. Therefore, we argue that nematodes will not adapt quickly to sheep bred for nematode resistance, where selection is based on low WEC, although selecting sheep using a subset of immune functions may increase adaptation risk. Our results support the hypothesis that nematode resistance is determined by many genes each with relatively small effect. In conclusion, selection of sheep for nematode resistance using WEC should be sustainable in the medium to long-term.     

Gastric dysfunction in sheep infected with Trichostrongylus colubriformis, a nematode inhabiting the small intestine

Barker I. K. and Titchen D. A. 1982. Gastric dysfunction in sheep infected with Trichostrongylus colubriformis, a nematode inhabiting the small intestine. International Journal for Parasitology12: 345–356. Six of 12 lambs infected with Trichostrongylus colubriformis had reduced abomasal acidification (pH 4.0–8.1) in comparison with uninfected pair-fed and replete controls (pH <3.5), though less than 0.8% of the worm burden was in the abomasum. Loss of prominence of parietal cells and encroachment of mucous cells deep into fundic glands was seen by light microscopy. Under the electron microscope, parietal cells had little canalicular or tubulovesicular development, had large vacuoles, many polyribosomes and few mitochondria in comparison with those in controls. In a further 8 sheep prepared with abomasal fistulae and separated fundic pouches and inoculated orally with T. colubriformis, the volume of fundic pouch secretion declined as feed intake dropped and in 7 out of 8 animals H⁺ concentration in fundic pouch secretion also fell. Infection generally reduced volume and acidity of pouch secretion more than did a pre-inoculation fast. In 5 sheep, abomasal content exceeded pH 4. Inoculation of T. colubriformis by enterotomy and Ostertagia circumcincta per os, in a lamb with a separated fundic pouch, caused depression of volume and acidity of pouch secretion characteristic of T. colubriformis infection, rather than the hypersecretion typical of abomasal infection with Ostertagia. Factors inhibitory to parietal cell differentiation and gastric acid secretion may be released from the small intestine of some sheep in response to changes in the gut induced by the presence of T. colubriformis. Abomasal dysfunction is a manifestation of severe intestinal trichostrongylosis.     

In vitro bioassay of sheep gastrointestinal mucus for nematode paralysing activity mediated by substances with some properties characteristic of SRS-A

A bioassay is described for determining the inhibition of nematode larval migration from agar by substances exerting a paralysing action.In the assay, larval migration was completely inhibited by the anthelmintic levamisole (25 μg/ml) whereas biogenic amines, and prostaglandins E₁ and E₂ at 50 μg/ml, were without effect. Mucus from the gastrointestinal tract of sheep resistant to nematode infection inhibited larval migration by up to 93% whereas mucus from heavily infected sheep or sheep reared helminth free did not significantly inhibit larval migration. Mucus from sheep resistant to Trichostrongylus colubriformis inhibited the migration of larvae of other nematode species.The larval migration inhibitory (LMI) activity of mucus from resistant sheep was associated with components having some properties of slow reacting substance of anaphylaxis (SRS-A).Faecal samples from resistant sheep possessed significantly more LMI activity than faecal samples from heavily infected sheep or sheep reared helminth free. The level of LMI activity in the faeces of sheep undergoing challenge infection may be a useful indicator of the sheep's resistance status.The presence of the larval migration inhibitory activity in sheep mucus is discussed in relation to resistance to infection.     

Acetylcholinesterase secretion by parasitic nematodes. II. Trichostrongylus spp

Unusually high levels of acetylcholinesterase (AChE) were found in the nematode parasites Trichostrongylus axei, T. colubriformis and T, retortaeformis. In T. colubriformis the enzyme was located in the oesophageal and excretory glands of the parasitic stages. The highest level/unit wt was found in the fourth-stage larvae, which per worm had a comparable level to that in adult worms because the excretory gland was fully developed in the fourth-stage larvae. In acrylamide gel electrophoresis, T. axei and T. colubriformis AChE and esterases were similar but differed from those present in T. retortaeformis. Globulins prepared from the sera of sheep and guinea-pigs infected with T. colubriformis complexed with T. colubriformis and T. axei AChE, but not with esterases nor with AChE from T. retortaeformis, Nippostrongylus brasiliensis, Oesophagostomum radiatum or O. venulosum. Complexing of AChE to globulins did not inhibit the enzymic function of this enzyme.     

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.     

The ecology of the free-living stages of Trichostrongylus axei

Callinan A.P.L. 1978. The ecology of the free-living stages of Trichostrongylus axei. International Journal for Parasitology8: 453–456. The development and survival of the free-living stages of Trichostrongylus axei was studied in western Victoria in 1974–1976. For all plots in which development occurred, preinfective larvae (L₁-L₂) were recovered within 0–5 days, infective larvae (L₃) in faeces within 4–28 days and L₃ on herbage and soil within 4–21 days. The mean minimum development time to L₃ on herbage and soil was 12.3 ± 0.7 days and the mean development time to maximum yield of these was 33.8 ± 7.4 days. A mean of 66.7 ± 6.6% of L₃ on herbage and soil were actually found on herbage. Yields of L₃ on herbage and soil varied from 0 to 8.9 % of the number of eggs put out on each plot. Yields varied approximately inversely as the mean daily temperatures for the period until maximum yield. No L₃ were observed to survive over summer.     

The ecology of the free-living stages of Trichostrongylus vitrinus

Callinan A. P. L. 1979. The ecology of the free-living stages of Trichostrongylus vitrinus. International Journal for Parasitology9: 133–136. The ecology of the free-living stages of Trichostrongylus vitrinus was studied in western Victoria in 1975–1976. Where development of eggs (E₁–E₂) did occur, preinfective larvae (L₁–L₂) were recovered within 0–8 days, infective larvae (L₃) in faeces within 10–39 days and on L₃ herbage and soil within 4–26 days. The mean development time to L₃ on herbage and soil was 12.5 ± 4.8 days and the mean development time to maximum yield of these was 37.9 ± 9.7 days. The mean percentage of L₃ on herbage and soil actually on herbage was 70.8 ± 6.5 %. L₃ persisted in faeces for up to 106 days and on herbage and soil for up to 208 days. Yields of L₃ on herbage and soil varied from 0 to 5.05 % of the number of eggs deposited. Yields were highest from those eggs deposited in autumn-winter. No L₃ were observed to survive over summer.     

Control of nematode parasites by grazing management—II. Decontamination of sheep and cattle pastures by varying periods of grazing with the alternate host

Commencing in December 1970, paddocks of a uniform series of sheep pastures were grazed for 6, 12 or 24 weeks by either yearling steers is or yearling ewes. Cattle pastures were treated similarly. All ewes and steers were pre-dosed with anthelmintic.At the conclusion of alternate grazing the effectiveness of the grazing treatments was evaluated by grazing each paddock for 1 month with either ten worm-free lambs (sheep pastures) or three wormfree calves (cattle pastures). The test animals were then slaughtered for total worm counts. The grazing of sheep pastures with cattle for 6, 12 or 24 weeks from December onwards resulted in reductions in numbers of Haemonchus contortus and Trichostrongylus colubriformis in test lambs. In comparison with continuous grazing by sheep, Nematodirus spp was only reduced after 24 weeks grazing by cattle. Cattle pastures grazed by sheep for 6 weeks showed no reduction in numbers of Ostertagia ostertagi or Cooperia oncophora in test calves. However after 12 weeks with sheep, numbers of O. ostertagi though not C. oncophora were reduced and after 24 weeks of alternate grazing both these species were reduced.Calves following the 6 week sheep treatments acquired both Haemonchus contortus and Trichostrongylus colubriformis and the calves from the 12 week sheep treatment paddock also carried H. contortus. For sheep the only evidence of cross-transmission was the occurrence of small numbers of Cooperia oncophora in test lambs from the 24 week cattle grazing treatment.The results provide evidence that sequential stocking with cattle and sheep in conjunction with anthelmintic treatment is an effective management strategy for preparing parasitologically safer pastures, but further information is required to determine the optimum timing of sequential stocking in farming situations.     

The ecology of the free-living stages of Ostertagia circumcincta

Callinan A. P. L. 1978. The ecology of the free-living stages of Ostertagia circumcincta. Internationaljournal for Parasitology8: 233–237. The development and survival of the free-living stages O. circumcincta was studied in western Victoria in 1974–1976. For all plots in which development occurred, pre-infective larvae (L₁-L₂) were recovered within 0–8 days, infective larvae (L₃) within 7–30 days and L₃ on herbage and soil within 4–27 days. The mean minimum development time to L₃ on herbage and soil was 14·3 ± 3·5 days and the mean development time to maximum yield of these was 33·3 ± 6·3 days. There were no distinct differences in development rates between plots. Plot yields of L₃ on herbage and soil varied inversely as the mean of daily temperatures for the period until maximum yield of these. Yields varied from 0 to 16% of the number of eggs put out on each plot and highest yields were obtained from eggs put out during late autumn-winter. No L₃ were observed to survive over summer. The migratory habits of L₃ were such that a mean of 75·1 ± 5·6% of L₃ on herbage and soil were actually on herbage; the soil was always a significant source of L₃.     

Histamine and leukotriene concentrations in duodenal tissue and mucus of lambs selected for high and low responsiveness to vaccination and challenge with Trichostrongylus colubriformis

Lambs selectively bred for high responsiveness or low responsiveness to vaccination with irradiated Trichostrongylus colubriformis were vaccinated and challenged. Duodenal tissue histamine concentrations in both high and low responder lambs were lower at 3 days than at 28 days after challenge. At 3 days after challenge, histamine concentrations were higher in both male and female high responder lambs than in low responder lambs whereas at 28 days concentrations were increased only in high responder females. At 3 days after challenge, histamine concentrations were generally lower in mucus than in tissues, but levels were again higher in mucus from high responder groups. In duodenal tissue at 3 days after challenge, leukotriene C₄ and B₄ concentrations were similar in high and low responder animals. At the same time, concentrations of both leukotrienes were higher in mucus than in tissues, with high responder female lambs having the highest concentration. It is suggested that increased levels of histamine and leukotrienes in mucus and tissue are associated with larval rejection or exclusion.     

Acetylcholinesterase secretion by parasitic nematodes. IV. Antibodies against the enzyme in Trichostrongylus colubriformis infected sheep

Sheep infected with the nematode parasite Trichostrongylus colubriformis showed anti-T. colubriformis acetylcholinesterase. (AChE) antibodies in the IgG₁ but not the IgG₂ or IgM fractions prepared from their serum. Using the fluorescent antibody technique with representative sera, antibodies in the IgG₁ fraction exhibited specificity for antigens in the subventral glands of the worm excretory system. IgA antibody specificity for antigens in the excretory glands and intestine of the worm was also demonstrated.     

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.     

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 role of immunologically specific and non-specific components of resistance in cross-protection to intestinal nematodes

Vaccination of 6–8-month-old Merino sheep with irradiated Trichostrongylus colubriformis larvae gave a high level of protection (81 %) against single-species challenge with normal infective larvae of the same species. The level of protection (34%) was substantially reduced against challenge with a closely related species (T. vitrinus) and no significant protection occurred against single-species challenge with a generically unrelated nematode (Nematodirus spathiger). These results suggest that antigen(s) which stimulate protective immunity are shared by the related Trichostrongylus species but not by N. spathiger. By contrast with the results obtained for single-species challenge, vaccination with irradiated T. colubriformis produced 98–100% protection against all 3 species in animals challenged simultaneously with infective larvae of the 3 species. Comparison of the levels of protection recorded following the 2 types of challenge indicate that although a specific antigenic trigger is required to provoke an appropriate response, the results obtained, particularly in the case of N. spathiger, suggest that the terminal effector mechanism is not immunologically specific. The implications of these conclusions are discussed in relation to theories of the mechanism of resistance to gastrointestinal nematodes and the potential efficacy of vaccination in the field.     

The response of the regional lymph node of guinea-pigs to primary and challenge infection with the nematode, Trichostrongylus colubriformis

Responses characteristic of both cell-mediated immunity and the production of humoral antibodies occurred in the regional lymph node (mesenteric) of guinea-pigs following primary and challenge infection with the nematode Trichostrongylus colubriformis. In addition, depletion of lymphocytes in the paracortical areas and pronounced infiltration with basophil and eosinophil leucocytes were observed. All these changes were chronologically related to parasite rejection.