Dietary impacts on the resistance of Merino lambs to Trichostrongylus colubriformis

AIM: To evaluate and compare the effects of a variety of diets on the resistance of young, lightweight Merino lambs to repeated low-dose infections of Trichostrongylus colubriformis.

METHODS: Ninety-six 12-week-old lambs were fed balanced or unbalanced diets, and given primary (at 19 weeks of age) or both primary (at 12 weeks) and secondary (at 19 weeks) infections of T. colubriformis. Suboptimal diets were low in total intake, rumen undegradable protein, total protein, readily available carbohydrate or minerals, or supplemented with cod liver oil. Liveweight was monitored fortnightly, protection was assessed from worm counts at the end of the study (25 weeks) and faecal nematode egg counts (FEC) conducted weekly, and mast-cell response assessed at the conclusion of the study from concentration of mast-cell protease in the jejunum. Concentrations of glucose in plasma were measured at 21 weeks.

RESULTS: Diets unbalanced for total protein, readily available carbohydrate, Mo or cod liver oil each increased the worm counts after 6 weeks of secondary infection (at 25 weeks of age) and FEC during secondary infection. Low intake of a highnutrient diet restricted liveweight gain but did not affect the development of resistance, as indicated by worm counts and FEC. Defi ciency of available carbohydrate resulted in reduced liveweight gain and reduced concentration of glucose in plasma. There was a reduction in jejunal mast-cell protease concentration in sheep fed unbalanced rations.

CONCLUSIONS: The parasitology results and reduction in the concentration of jejunal mast-cell protease observed in sheep fed unbalanced rations suggest that the nutrient defi ciencies and the fi sh-oil supplement may have acted via inhibition or retardation of the host's acquired immune response. Diets unbalanced for a range of components adversely affected the acquired protective response to T. colubriformis to a similar extent and appeared to involve a similar mechanism as each other. The quality of the diet was more important than quantity for the development of resistance, and soluble carbohydrate was an essential component.     

Trichostrongylus colubriformis and Haemonchus contortus infections in light bodyweight Merino lambs

OBJECTIVE; To show that low bodyweight is a predisposing cause of high Trichostrongylus colubriformis and Haemonchus contortus burdens and egg counts in Merino lambs. DESIGN: A comparison was made, among lambs of different bodyweights, on the effect on immunity of a primary or secondary viable infection with T colubriformis or H contortus larvae. PROCEDURE: Sixty-one Merino lambs, 1 or 6 months of age, were penned indoors and given primary, or both primary and secondary, infection of T colubriformis or H contortus. Faecal egg counts, worm counts and parasite-specific immunoglobulin concentrations were examined for their relationships with bodyweight. RESULTS: Bodyweight at the start of a primary infection was correlated with worm burden, worm fecundity and jejunal IgA antibody concentration. Merino lambs weighing less than 23 kg at the time of first exposure to T colubriformis or H contortus had impaired ability to develop protective mucosal immunity and to resist homologous challenge. CONCLUSION: If the goal is to ensure that lambs develop immunity before weaning, then every endeavour should be made to achieve the combination of critical bodyweight and exposure to moderate levels of nematode infection as soon as possible.     

Population dynamics of Trichostrongylus colubriformis in sheep following anthelmintic treatment

Three groups of five, three-and-a-half to four-month-old lambs were put to graze on three plots contaminated by Trichostrongylus colubriformis. One group (control) was free of parasites, while the other two were infected just before turning out with 3 X 700 third stage larvae. Depending on whether or not the lambs were treated with 50 mg kg-1 thiabendazole nine days after the first infection, the worm burden and the egg output increased or decreased during the grazing period when compared with the control group. In tracer animals put to graze on the same plots afterwards, it was found that the smaller the number of eggs excreted by the first lambs, the higher were the worm burdens of the tracer lambs.     

The influence of reproductive physiology and nutrient supply on the periparturient relaxation of immunity to the gastrointestinal nematode Trichostrongylus colubriformis in Merino ewes

A pen experiment was conducted to investigate the interaction of early-weaning and nutrient supply on the periparturient relaxation of immunity to the gastrointestinal nematode (GIN) Trichostrongylus colubriformis in Merino ewes. Mixed-age pregnant and non-pregnant (dry) ewes were infected with 8,000 T. colubriformis L(3)/week, and fed either a high or low quality diet. Following parturition, lambs were either removed from their mothers at 2 days of age or allowed to continue suckling. Systemic immunity began to wane during late pregnancy with circulating eosinophils and plasma total antibody (Ab) levels declining from day -37 (relative to the midpoint of lambing) and day -24, respectively. Pregnant ewes fed the low quality diet exhibited an increasing faecal worm egg count (WEC) from day -24 and had higher intestinal worm burdens on day 13, whereas ewes fed the high quality diet had a delayed transient rise in WEC of lower magnitude. Dry and early-weaned ewes remained highly resistant to T. colubriformis at all times. In the post-lambing/lactation period, ewes fed the high quality diet had higher levels of local total Ab and numbers of goblet cells (GC) in the small intestine on days 13 and 41. Lactating/suckled ewes had a lower anti-parasite local immune response as indicated by reduced titres of total Ab, IgG(1), IgM and IgA and lower numbers of mucosal mast cells (MMC), globule leukocytes (GL) and GC in small intestinal tissue compared to their dry and early-weaned counterparts. Early-weaning resulted in rapid recovery of blood eosinophils and total Ab. On day 13 post-lambing, titres of total Ab, IgG(1), IgM, IgA and IgE, and numbers of MMC and GL were greater than those measured in dry and suckled ewes. When fed the high quality diet, ewes had a higher dry matter (DM) intake, maternal weight, fat score, greater fat depth and eye muscle depth, birthed heavier lambs that had higher growth rates, and produced more milk. The physiological status of pregnancy resulted in a higher DM intake but lower measures of fat depth and eye muscle depth, and suckling led to an increase in DM intake but a reduction in body weight and fat score through mobilisation of fat and muscle reserves. Despite the marked effect of diet quality on production traits, some inconsistencies were observed between body composition and apparent parasite resistance, measured by WEC and worm counts, suggesting that the nutritional influence was not necessarily always mediated through changes in body composition. Although reproductive status affected blood leptin levels, diet had no effect within suckled ewes and therefore it was concluded that leptin has no causative role in maintaining the periparturient relaxation of immunity to T. colubriformis.     

Efficacy of abamectin against ivermectin-resistant strain of Trichostrongylus colubriformis in sheep

The efficacy of two formulations of abamectin, i.e. oral and injectable was determined against ivermectin-resistant strain of T. colubriformis in sheep. Twenty-four lambs were infected with 10,000 third stage larvae of ivermectin-resistant strain of T. colubriformis. Twenty-four days post-infection, the lambs were divided randomly into four groups of six animals each according to egg counts. The first group was left untreated and kept as a control. The second group was treated with ivermectin (oral) at 0.2mg kg(-1) body weight. The third group was treated with oral formulation of abamectin at 0.2mg kg(-1) body weight. The fourth group was treated with injectable formulation of abamectin at 0.2mg kg(-1) body weight. Fecal egg count and controlled slaughter tests were employed to determine the efficacy of abamectin (oral and injection) against ivermectin-resistant strain of T. colubriformis in sheep. Reduction in arithmetic mean fecal egg counts achieved by ivermectin (oral), abamectin (oral) and abamectin (injection) was 66, 98 and 76%, respectively 10 days after treatment. Ivermectin (oral), abamectin (oral) and abamectin (injection) reduced arithmetic mean worm burden by 63, 97 and 74%, respectively. The findings demonstrated that abamectin oral formulation was more effective than abamectin injection against ivermectin-resistant strain of T. colubriformis in sheep.     

Development of resistance to Trichostrongylus colubriformis in goats

A high level of host resistance to Trichostrongylus colubriformis was established in a group of Saanen goats infected weekly with 10,000 infective larvae for 10 weeks, treated with an anthelmintic and then challenged twice with 50,000 larvae, with a further anthelmintic treatment 28 days after the first challenge. A second group of goats exposed only to the two challenge infections developed worm burdens intermediate between, and significantly different from, the first group and a third control group given only the second challenge infection. Nematode fecundity (eggs per female) and male:female ratios were also significantly decreased in the first two groups. Faecal egg counts and worm counts were highly correlated and this relationship was dominated by the influence of the number of eggs/female nematode. The goats were approximately 9 months old at the start of the experiment.     

High genetic correlation between resistance to Haemonchus contortus and to Trichostrongylus colubriformis in INRA 401 sheep

To compare the protection acquired by infection with Haemonchus contortus or Trichostrongylus colubriformis, successive cross infections with the two species were conducted in two genetically similar groups of 150 ewe lambs from 30 sires of INRA 401 breed. At 6 and 7.5 months of age, each group received two doses of 10000 L3 separated by a treatment of one of the species, followed by a similar infection with the second species at 9 and 11.5 months. Faecal egg counts (FECs) were done 28 and 35 days after each dose. There were very high genetic correlation within and between species suggesting that the environmental conditions were very similar. The heritability of FEC of H. contortus ranged from 0.39 to 0.48 and the genetic correlations between FEC after the first and second infections with the same or different species were all near 1. Selection against this species could be done on the response to only one dose. For T. colubriformis, only the response to challenge infection had a similar heritability (0.47); the genetic correlations within and among species were also near one. These results will assist the selection of sheep resistant to nematode parasites.     

Evidence for the presence of nematode-derived acetylcholinesterase in sheep infected with Trichostrongylus colubriformis

The distribution of acetylcholinesterase isoenzymes in the ovine intestinal nematode Trichostrongylus colubriformis was compared with that in chronically infected and worm-free lambs. Total acetylcholinesterase activity in homogenates of adult T colubriformis was resolved into five isoenzyme peaks following gel electrophoresis and specific esterase staining. Two isoenzymes with electrophoretic mobilities similar to those present in adult worm homogenates were detected in mucosal homogenates and plasma extracts from all of six sheep chronically infected with T colubriformis, but not in similar preparations from two uninfected animals.     

Selection of ivermectin-resistant Trichostrongylus colubriformis in lambs

A mixed population of Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus colubriformis was passed for four generations through lambs. In successive passages and on Day 6 (D6), lambs were dosed subcutaneously with ivermectin at 100, 200, 250 and 225 micrograms kg-1 body weight, respectively. No larvae of H. contortus were recovered after the first passage and following the third drug exposure only T. colubriformis was present. Larvae of the selected strain of T. colubriformis developed to third stage, sheathed larvae in vitro in concentrations of ivermectin that inhibited development of larvae from the original strain of T. colubriformis. A definitive control test was then run with 45 lambs, to compare this selected strain to the original strain. The treatments were 150 micrograms kg-1 subcutaneous (SC) D6, 200 micrograms kg-1 SC D6, 200 micrograms kg-1 intraruminal (IR) D6 and 200 micrograms kg-1 SC D21; all treatments were compared to non-treated controls, selected or original strain as applicable. The D6 IR and D6 SC 150 micrograms kg-1 treatments were greater than 99% effective in both strains, although the D6 SC 200 micrograms kg-1 treatment was 85 and 48% effective in the original and selected strain, respectively. The D21 200 micrograms kg-1 was only 58% effective against the selected strain. There was significant (P less than 0.001) differences between the original and selected strains of T. colubriformis for both fecal egg output and worm counts. The exposure of four generations of immature T. colubriformis to ivermectin selected for partial resistance.     

Testing for direct anthelmintic effects of bioactive forages against Trichostrongylus colubriformis in grazing sheep

The aim of the present study was to investigate potential direct anthelmintic effects of forages that contain plant secondary metabolites (PSM) towards the intestinal nematode Trichostrongylus colubriformis. For this purpose, we introduced an experimental design, which enabled us to investigate the direct anthelmintic effects of PSM-rich forages, without significant interference of possible indirect immunological effects of PSM. Sixty parasite naive sheep were infected with 8000 T. colubriformis L(3) on day 1 of the experiment. On day 28, sheep entered one of the experimental plots, which consisted of four PSM-rich forages and one control: Lotus pedunculatus (lotus), Hedysarium coronarium (sulla), Onobrychis viciifolia (sainfoin), Cichorium intybus (chicory) and Lolium perenne/Trifolium repens (grass/clover). On day 35 of the experiment, all sheep were re-infected with 8000 T. colubriformis L(3) and were killed on day 42. By day 42, all parasites of the primary infection would have been recovered as adults, whereas those of the secondary challenge would have only developed to the fourth stage larvae within a week (i.e. days 35-42). The first batch of larvae would enable us to investigate possible direct anthelmintic effects of PSM-rich forages against adult T. colubriformis, whereas the second one whether grazing on the PSM-rich forages could affect the establishment of the incoming infective larvae. Sheep grazing on lotus tended to have a lower FEC compared to sheep grazing on grass/clover (P = 0.06), whereas daily faecal output was higher in sheep grazing lotus compared to those grazing on the other forages (P < 0.05). As a consequence, daily egg output was similar in all sheep. Grazing on the PSM-rich forages for a period of 2 weeks did not affect the immature and adult parasite populations. Although the present experimental design enabled us to test the experimental hypothesis, the lack of evidence on a direct anthelmintic effect of PSM-rich forages can not be considered conclusive, as the composition of PSM-rich forages is variable within and across grazing seasons.     

Regulation of Trichostrongylus vitrinus and T colubriformis populations in naturally infected sheep in the Netherlands

During a study on the epidemiology of gastrointestinal helminth infections in sheep at the University of Utrecht (1974 to 1977) information was obtained on the population regulation of small intestinal Trichostrongylus species. Comparison of the cumulative burdens in tracer lambs with those in flock lambs showed an accumulation of Trichostrongylus species in the latter until autumn followed by an expulsion of worms. The dominating species, T vitrinus, was expelled earlier than T colubriformis. The proportion of inhibited early third stage Trichostrongylus species larvae in tracer lambs was low, but it tended to increase in autumn. In most flock lambs the levels of inhibition were also low. In non-lactating ewes, populations consisted predominantly of inhibited early third stage larvae but when adults were present they tended to be mainly T colubriformis. Similar populations were seen in some lactating ewes. In other lactating ewes Trichostrongylus species populations with low proportions of inhibited early third stage larvae which were dominated by T vitrinus, indicated a relaxation of resistance around parturition.     

Biological parameters of Trichostrongylus colubriformis in Meriones unguiculatus

In a series of three experiments, 64 jirds, Meriones unguiculatus, were infected with 700 infective larvae of Trichostrongylus colubriformis for the study of several biological parameters of this laboratory host-parasite model. In jirds the third stage larvae of T. colubriformis were shown to reach the fourth larval stage by the Day 6 post-infection (PI). By Day 10 PI, all the worms harvested had reached the immature adult stage. All immature adult stages of T. colubriformis developed to sexually mature adult stage by Day 12 PI when the female worms showed developing eggs in their uteri. Developed eggs were observed in the uteri of females on Days 13 and 14 PI. The first eggs of T. colubriformis appeared in the faeces of jirds on Day 13 PI. The peaks of egg production were recorded between Day 21 and Day 31 PI. Immunosuppression of jirds infected with 700 L3, by administration of dexamethasone from Day 57 to Day 94 PI led to increased faecal egg count when compared to untreated controls. All the worms were located in the small intestine. The jirds of dexamethasone treated group harboured higher number of adult worms than those of the untreated group. The number of adult worms was significantly higher in the first part of the small intestine than in the three other similar parts of the small intestine. The coefficient of correlation between the faecal egg count and worm number on the day of necropsy of jirds ranged between r = 0.58 and r = 0.89. Patent infections in jirds were maintained till the end of experiment on Day 100 PI, indicating that in this host and unlike other laboratory hosts, T. colubriformis is responsible for long lasting infections similar to what happens in domestic ruminants. The results of the present study suggest that the jird is a suitable laboratory model to study various aspects of this host-parasite relationship.     

Influence of adjuvants on immunity in rabbits vaccinated with infective larval somatic proteins of Trichostrongylus colubriformis

Adult New Zealand rabbits were vaccinated at 2-week intervals with three doses of 100 micrograms of infective larval somatic proteins (L3SP) administered subcutaneously with Freund's complete adjuvant (FCA) or beryllium hydroxide and then challenged orally with 10,000 L3. Groups of rabbits immunized orally with three doses of 5000 or 2000 L3 served as vaccination controls. Intestinal worm burdens on Day 21 after challenge revealed that beryllium hydroxide effectively potentiates the protective immunogenicity of L3SP. The level of protection obtained using the beryllium adjuvant (94.8%) was nearly as high as that in rabbits immunized with three doses of 5000 L3 (99.8%). Rabbits vaccinated using FCA showed very poor immunity (29.5%). Local and systemic antibody levels detected by radioimmunoprecipitation tests using 125I-L3SP showed very little correlation with the degree of protection. The beryllium hydroxide-treated group demonstrated significantly higher bile IgA antibody levels than other experimental rabbits. FCA-treated rabbits developed a much higher serum precipitating antibody response, detectable using gel double diffusion tests, than the beryllium group. Also, mucosal IgA antibody levels detected on Day 21 after challenge were significantly higher in the FCA group than in other groups.     

The establishment and development of Trichostrongylus colubriformis in goats

Twelve goats were inoculated with 20,000 infective larvae of Trichostrongylus colubriformis and two were killed on each of Days 4, 7, 11, 14, 18 and 21 after inoculation (DAI). The number of worms that established, and the site of development were recorded. Most of the worms established within the first 3 m of the small intestine. There was little relocation or loss of nematodes after establishment. The worms started to migrate from the mucosa to the lumen 11 days after infection. At 4 DAI, 63% of the worms were still at the late L3 stage; the remainder of the worm population had completed the third moult to the L4 stage. The population at 11 DAI comprised largely young adults. When 21 DAI was reached, about 57% of the female worms had eggs in their uteri.     

Effects of molybdenum intake on primary infection and subsequent challenge by the nematode parasite Trichostrongylus colubriformis in weaned Merino lambs.

In a study designed to test the effect of molybdenum (Mo) on resistance to trichostrongylosis, the Mo content of the diet during primary infection of 8-month-old Merino lambs with Trichostrongylus colubriformis was found to affect the number of worm eggs found in the faeces during challenge and the total number of worms in the jejunum after six weeks of challenge infection. The optimal intake of Mo in this experiment was in the range of 4-8 mg sheep(-1)day(-1), approximately equivalent to feeding a diet containing 6-10 mg Mo kg(-1)dry matter (DM) and to 0.15-0.30 mg Mo kg(-1)liveweight. Lambs fed Mo at this rate showed a 90 per cent reduction in faecal egg count and total worm count six weeks after challenge compared with lambs fed quantities of Mo outside this range. The mechanism by which Mo exerted these effects was not defined, although the interactions of molybdenum, worm establishment and faecal egg count suggested that this trace element may be acting via an effect on the host's acquired immune response. This hypothesis is supported by the observed enhancement of immune responses (intestinal antibody and granulocyte numbers and in vitro worm-specific proliferation of lymphocytes) associated with Mo intake of 0.15-0.3 mg kg(-1)Lwt.     

Regulation of Teladorsagia circumcincta and Trichostrongylus colubriformis worm populations by grazing sheep with differing resistance status

In an experiment lasting 4 years, changes in the Teladorsagia circumcincta and Trichostrongylus colubriformis populations were compared in lambs and adult sheep with differing resistance statuses. Two flocks of 30 rams (resistant R and susceptible S) grazed separate pastures and 8 rams were slaughtered in the middle and at the end of each grazing season. Five groups of tracer lambs were added each year to estimate the pasture infectivity and were killed for worm counts. The availability of animals with differing resistance statuses (rams and tracer lambs) and differing levels of infection made it possible to investigate the number, size and fecundity of worms of these two species. The inflammatory response was measured in the rams by counting the globule leukocytes, mast cells and eosinophils in the fundic, pyloric and intestinal mucosa. In the tracer lambs, the daily egg production by the female worms of both species was negatively correlated with the worm burden. Worm length accounted for 60 and 70% of the variation in the number of eggs in utero for T. circumcincta and T. colubriformis respectively. Worm length was closely associated with the resistance status of the host; there were greater differences between lambs, and S and R rams for T. colubriformis. T. circumcincta worm lengths were not affected by the worm number. Globule leukocyte counts were related to the worm burdens, and mast cell counts to worm length in the R and S rams. The number, size and fecundity of the worms may well be regulated by similar mechanisms in both species, but T. colubriformis seemed to be more intensively regulated than T. circumcincta. This finding could be useful in devising more effective methods of parasite control.     

Measurement in vitro of a larval migration inhibitory factor in gastrointestinal mucus of sheep made resistant to the roundworm Trichostrongylus colubriformis

Lambs were challenged by dosing with 2500 T. colubriformis larvae per day for 34 weeks, rested for 4 weeks and then re-challenged with the infective larvae for a further 10 weeks. A technique for the measurement of inhibition of larval migration from agar gels was used to investigate the antiparasitic activity of mucus, obtained from the small intestine and abomasum of the lambs, at the end of the re-challenge period. Measurements were also made on ileal digesta samples collected at different times during the development of the initial resistance, the rest period and the re-challenge period, and on faeces collected during the re-challenge infection. The mucus from the small intestine and abomasum paralysed and inhibited larval migration from agar gels significantly more (P less than 0.01 and P less than 0.05, respectively) than corresponding mucus from parasite-free control animals. This inhibitory activity was also detected (P less than 0.01) in the ileal digesta of infected animals from Week 6 of primary dosing. The magnitude of the inhibition in the ileal digesta increased with time during the infection period and was again detected during re-infection (P less than 0.01). It was not detectable in resistant sheep towards the end of the rest period. Slight inhibitory activity was detected in faeces after 2 weeks of re-infection. These observations suggest that substances secreted into the lumen of the small intestine of infected animals are responsible for resistance against ingested larvae.