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.     

The response of young Romney lambs to immunization with Trichostrongylus colubriformis larvae

1988. The response of young Romney lambs to immunization with Trichostrongylus colubriformis larvae. International Journal for Parasitology 18: 1035–1038. Groups of weaned Romney ewe lambs were immunized with two doses of 28,000, 35,000 or 42,000 (2000 kg⁻¹) infective larvae of Trichostrongylus colubriformis at 8, 12 or 16 weeks of age, respectively. Each group and helminthfree control lambs of similar age were challenged with T. colubriformis at the same dose rate as used for immunization. Faecal egg counts (FEC) and haematological observations were made during the experiment, and at slaughter, 42 days after challenge, worm burdens were determined and small intestinal histology was examined.

Lambs in each immunized group were identified as ‘responders’ or ‘non-responders’ on the basis of both FEC and worm burdens. A significant (P<0.001) decrease in the worm burdens recovered as a proportion of the challenge infections in both unimmunized and immunized lambs with increasing age was observed.

Globule leukocyte numbers increased with the age of lambs. In addition, within each age group globule leukocyte numbers reflected individual responsiveness to immunization, significantly (P<0.01) greater numbers being present in ‘responders’ than ‘non-responders’ or unimmunized lambs. No difference in haematological responses were found in relation to the lambs' responsiveness to immunization.     

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.     

Observations on the self-cure reaction and other forms of immunological responsiveness against Haemonchus contortus in sheep

Self-cure reactions and immunological responses preventing establishment of Haemonchus contortus in sheep may operate as separate entities. In one experiment, self-cure occurred when challenge infection with 5000 larvae was superimposed on an infection with 5000 larvae given to worm-free sheep 6 weeks previously. Resident worms were rejected and establishment of infection by incoming larvae was impeded. The latter effect was not observed in sheep treated similarly but with resident parasites removed by treatment with oxfendazole before challenge. In another experiment, younger worm-free sheep primed by three infections with 2000 larvae at intervals of 2 weeks or a single infection with 6000 larvae were challenged with 10,000 larvae 6 weeks after the first priming infection. Self-cure was not incited but establishment of infection was impeded in sheep primed with three divided doses of larvae whether or not priming infections had been removed by oxfendazole. Infection regimes used for priming did not influence numbers of arrested fourth-stage larvae derived from challenge infection. However, more arrested larvae were present when challenge was superimposed on extant infections, indicating that resident worms or a factor activated by their presence induced developmental arrest. In a third experiment, large burdens with H. contortus were established in sheep immunosuppressed with the corticosteroid, dexamethasone, at the time of infection. Self-cure was not triggered by a challenge infection given 32 days later either in these sheep, or in sheep with a smaller worm burden derived from infection given without immunosuppression. Faecal egg counts, however, indicated that development of the challenge infection was prevented in both groups of sheep.

Investigation of self-cure is restricted by lack of a predictable system for reproducing the phenomenon. Self-cure was induced by a single infection with 5000 larvae in mature sheep but not with 6000 larvae in immature sheep. Three infections with 3000 larvae given at intervals of 2 weeks to mature sheep did not prime for self-cure. Procedures aimed at heightening immediate hypersensitivity, i.e. treatment with pertussis vaccine or concurrent infections with Ostertagia circumcincta, did not promote self-cure reactivity in the latter situation.     

A study of the pathogenesis of Trichostrongylus colubriformis infection in lambs with observations on the contribution of gastrointestinal plasma loss

A study of the pathogenesis of Trichostrongylus colubriformis infection in lambs with observations on the contribution of gastrointestinal plasma loss. International Journal for Parasitology 3: 743–757. Increased effusion into the gut of⁵¹Cr-labelled plasma protein was observed in 20-week-old Merino lambs which showed signs of trichostrongylosis after administration of 100,000 T. colubriformis larvae. In another trial, similarly infected 12-week-old meat type crossbred lambs did not have elevated gastrointestinal plasma loss, or signs of trichostrongylosis, although they generally carried heavier worm burdens than the Merinos. Differences in breed susceptibility were considered a possible explanation. Nine-week-old Merino lambs inoculated with 85,000 T. colubriformis had increased plasma loss into the gut beginning 10–12 days after infection, coinciding with onset of inappetance, hypoproteinaemia and weight loss. Weight loss was considered largely due to reduced feed intake, but infected lambs during the period of protein effusion into the gut lost more weight, or gained less, than pair fed controls. Hypoproteinaemia was related mainly to effusion of plasma into the gut. One infected lamb, and its pair fed control, died, probably due to malnutrition. Slight anaemia, and depressed faecal dry matter per cent were observed in infected lambs.     

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.     

Concomitant and protective immunity in mice exposed to repeated infections with Echinostoma malayanum

Concomitant immunity and its consequence against infection play roles in regulating worm burdens in helminthiasis. Under natural conditions, this immunity is generated by exposure to repeated low dose or trickle infection. In this study, concomitant immunity was induced in mice exposed repeatedly to infection with Echinostoma malayanum and its protective effect on a challenge infection evaluated. A profile of worm burden from exposure to 10 metacercariae/mouse/week rose rapidly during the first 2 weeks reaching a plateau from week 3 to 8 post infection. Based on a cumulative dose of infection, worm recoveries were around 75% in the first 2 weeks, dropped to 50% at week 3 and 19% at week 8. After week 2, adult worm burden was constant and no juvenile worms were found after week 3 of the experiment. To examine the effect of resistance against reinfection, mice in the experimental group were primarily infected with 10 metacercariae/week for 5 weeks, treated with praziquantel and were challenged with 75 metacercariae/animal. The number of worms recovered from the experimental groups was significantly lower than that from naïve control groups beginning from 24 h to 28 days post challenge. The worms in the experimental group showed growth retardation and the proportion of adult worms was lower than that in the control animals especially during the first 3 weeks of the experiment. Parasite fecundity was also suppressed compared with that in the control group. The selective effects of protective immunity on establishment, growth, and fecundity of challenged worms affected the population dynamics of E. malayanum which is a similar phenomenon to concomitant immunity in schistosomiasis.     

Induction of immunity in sheep to Fasciola hepatica with mimotopes of cathepsin L selected from a phage display library

An M13 phage random 12-mers peptide library was used to screen cathepsin L mimotopes of Fasciola hepatica and to evaluate their immunogenicity in sheep. Seven clones showed positive reactivity to a rabbit anti-cathepsin L1/L2 antiserum in ELISA, and their amino acid sequences deduced by DNA sequencing were tentatively mapped on the protein. Twenty sheep were randomly allocated into 4 groups of 5 animals each, for immunization with 1x10(14) phage particles of clones 1, 20, a mixture of 7 clones and PBS, without adjuvant at the beginning, and 4 weeks later. All groups were challenged with 300 metacercariae at week 6 and slaughtered 16 weeks later. The mean worm burdens after challenge were reduced by 47.61% and 33.91% in sheep vaccinated with clones 1 and 20, respectively; no effect was observed in animals inoculated with the clone mixture. Also, a significant reduction in worm size and burden was observed for those sheep immunized with clone 1. Animals receiving clone 20, showed a significant reduction in egg output. Immunization induced a reduction of egg viability ranging from 58.92 to 82.11%. Furthermore, vaccinated animals produced clone-specific antibodies which were boosted after challenge with metacercariae of F. hepatica.     

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.     

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.     

Experimental American leishmaniasis and Chagas'' disease in the Brazilian squirrel monkey: cross immunity and electrocardiographic studies of monkeys infected with Leishmania braziliensis and Trypanosoma cruzi

, and 1988. Experimental American leishmaniasis and Chagas' disease in the Brazilian squirrel monkey: cross immunity and electrocardiographic studies of monkeys infected with Leishmania braziliensis and Trypanosoma cruzi. International Journal for Parasitology 18: 1053–1059. Adult, laboratory-bred squirrel monkeys (Saimiri sciureus) previously infected with either Leishmania braziliensis braziliensis or L. b. panamensis were challenge infected with blood-form trypomastigotes of Trypanosoma cruzi (Brazil strain). Monkeys previously infected with T. cruzi were challenged with stationary phase promastigote forms of L. b. braziliensis. Monkeys were examined during the course of challenge for evidence of infection, electrocardiographic alterations and parasite-specific antibody responses. T. cruzi epimastigotes were cultured from the blood of monkeys up to 3 months after challenge with this parasite. Unulcerated cutaneous lesions appeared and persisted in monkeys challenged with L. b. braziliensis. The formation of satellite lesions was observed in one monkey. Increased QRS intervals were not observed in T. cruzi challenged monkeys without prior cardiac irregularities and QRS left axis shifts were observed in only two of these monkeys. Elevated titers of parasite binding IgM and IgG specific for both T. cruzi and L. braziliensis were observed in all monkeys following challenge. These results indicate that prior infection with T. cruzi or L. braziliensis does not protect against heterologous challenge infection with these organisms. However, prior infection with Leishmania parasites may provide some protection against chagasic cardiopathies.     

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.     

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.     

Population dynamics of Trichostrongylus colubriformis in sheep: the effect of infection rate on the establishment of infective larvae and parasite fecundity

The establishment of Trichostrongylus colubriformis was estimated in helminthologically naive 20-week-old Merino sheep given third stage infective larvae (L3) at rates of 2000, 632 or 200 L3 per day, 5 days per week. After varying periods of continuous L3 intake, a levamisole-susceptible strain of T. colubriformis was replaced with a highly resistant strain for 1 week. The animals were then treated with levamisole to remove the susceptible population, and establishment of the cohort of resistant worms was estimated. In previously uninfected sheep, approximately 65% of the L3 given in the first week became established as adults. This fell to low levels (less than 5%) after 7, 10 and 14 weeks of continuous L3 intake for the high, medium and low infection rates, respectively. At the low infection rate, establishment remained at maximum levels for the first 4 weeks, but then fell at a rate similar to that observed for the higher infection rates. This implied that a threshold of worm exposure was required before resistance to establishment developed. Parasite egg production, expressed as eggs per gram of faeces, was proportional to infection rate and is explained by higher worm burdens occurring at high infection rates. However, estimates of fecundity in eggs per female per day showed the opposite relationship with rate of infection. Fecundity stayed high (approximately 600) for 5 weeks at the low infection rate but only maintained this level for 3 weeks and 1 week at the medium and high rates, respectively. This suggests that fecundity, like establishment, was similarly affected at threshold levels of immunological recognition.     

Elucidation of Schistosoma japonicum population dynamics in pigs using PCR-based identification of individuals representing distinct cohorts

The study reported here investigated the interactions of successive infections and acquired resistance of pigs to challenge infections of Schistosoma japonicum. Two morphologically indistinguishable geographical isolates from China (from Anhui and Zhejiang provinces) were used for the infections. The worms of the two isolates were distinguishable by PCR-linked restriction fragment length polymorphism analysis of the nicotinamide adenine dinucleotide phosphate dehydrogenase I gene of the mitochondrial genome. Thirty-two pigs divided into seven groups were used in the experiment. Two groups received a single infection by either the Anhui or the Zhejiang isolate. In Challenge Groups 1, 4, 6, 8 and 12, a primary infection of the Zhejiang isolate was followed by a challenge infection with the Anhui isolate at week 1, 4, 6, 8 or 12 after the primary infection. In this way it was possible to determine whether worms recovered by perfusion originated from the primary or the challenge infection. Only the challenge infection at week 1 resulted in a higher worm burden when compared with a single primary infection with the Zhejiang isolate. The results showed that challenge worms were able to establish, and that the proportion of worms originating from challenge infection increased at the later challenge infections, however without an increase in the total number of worms. In addition, mixed pairs of the two isolates were found in all challenge-infected groups. The results indicate that pigs are able to mount a partial resistance against re-infection with S. japonicum by 4 weeks after a primary infection, but that worms of the challenge infections eventually replace the primary infection. The finding of mixed pairs of the two isolates indicates that worms of S. japonicum are either polygamous or able to wait in solitude for up to 12 weeks for a partner.     

Failure of low level gamma-irradiation of infective larvae to influence establishment or expulsion of Trichostrongylus colubriformis in guinea pigs

Trichostrongylus colubriformis infective larvae were subjected to various levels of γ-irradiation and administered to guinea pigs. The worms surviving were subsequently counted. Irradiation with 12.9 C kg⁻ inhibited worm establishment but lower doses neither influenced worm establishment nor survival.     

The molecular size of nematode acetylcholinesterases and their separation from nematode allergens

The molecular size of nematode acetylcholinesterases, and their separation from nematode allergens. International Journal for Parasitology 3: 735–741. Acetylcholinesterases and allergens were derived from two parasitic nematodes, Nippostrongylus brasiliensis which parasitises rats and Trichostrongylus colubriformis which infects sheep and guinea pigs.

Chromatographic studies indicated that the mol. wt of nematode acetylcholinesterases lies between 65,000 and 75,000. The acetylcholinesterases of both species were separated from nematode allergens whose mol. wt is in the range of 10,000–50,000.

γG binding antigens of T. colubriformis were located in fractions with a mol. wt range of 30,000–150,000. γE binding activity was confined to allergenic material with a mol. wt of less than 70,000.     

The effect of specific immunization or infection with Trichostrongylus colubriformis on production of eosinophil differentiation factors in guinea pigs.

The cultivation of bone marrow was used to quantitate the levels of eosinophil differentiation factors (EDF) produced in conditioned medium (CM) by incubation of mesenteric lymph node cells (MLNC) with mitogens or specific antigens from the intestinal nematode parasite, Trichostrongylus colubriformis. In liquid cultures with 20 units ml⁻¹ recombinant murine interleukin-5 (IL-5), bone marrow cells (BMC) from either normal or infected donors contained <5% eosinophils and differentiated to> 50% eosinophils over 2–3 weeks. Conditioned medium from 3–4 week infected donors produced between 20 and 50% eosinophils when donor MLNC were stimulated with the specific antigen preparation SP3, but macrophages predominated when using CM from MLNC incubated with Concanavalin A (ConA). CM from MLNC of challenged donors incubated with SP3 produced 30–70% eosinophils in BMC assays, with highest levels induced by CM from high responder (HR) donors. Marrow from parasitized or normal donors gave rise to comparable proportions of eosinophils. CM was also produced from LNC of donors given protein or parasite antigens in adjuvant where between 28 and 35% eosinophils were produced in culture. There were no differences between activities attributable to the antigen, but Freund's complete adjuvant induced earlier differentiation of BMC than alum-induced CM. The results confirm that high levels of EDF activity are specifically induced by parasitic infection, and can also be produced by intraperitoneal and subcutaneous inoculation of adjuvanted antigens. Consistent with the greater eosinophilia exhibited by HR guinea pigs to infection with T.colubriformis L3, their MLNC also produced the highest levels of EDF activity.     

Bile and bile salts and exsheathment of the intestinal nematodes Trichostrongylus colubriformis and Nematodirus battus

1972. Bile and bile salts and exsheathment of the intestinal nematodes Trichostrongylus colubriformis and Nematodirus battus. International Journal for Parasitology, 2: 433–438. Exsheathment of T. colubriformis was potentiated at physiological levels of CO₂ by bile and bile salts. Lamb bile and crude sodium taurocholate preparations produced the greatest increase in exsheathment while rabbit bile, sodium glycocholate and deoxycholate had less pronounced effects. Exsheathment in bile and taurocholate occurred at a pH above 4 and it is therefore suggested that infective larvae which fail to exsheath in the abomasum could well do so in the proximal part of the small intestine. Pure sodium taurocholate was found to greatly potentiate exsheathment of N. battus in vitro but this occurred at a pH below 3 and thus the action of this bile salt could not affect exsheathment in the host.     

Lack of intraspecific variation in the second Internal Transcribed Spacer (ITS-2) of Trichostrongylus colubriformis ribosomal DNA

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