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.     

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.     

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.     

Intestinal enzyme activity in lambs chronically infected with Trichostrongylus colubriformis: effect of anthelmintic treatment

After twenty weeks of continuous dosing with Trichostrongylus colubriformis larvae substantial, but declining, numbers of worms had persisted in most of the lambs examined, although there were wide inter-individual variations. Mucosal lesions were found in the proximal small intestines of all the infected animals, their severity being directly related to worm burden. Representative brush border enzyme activities analysed in intestinal mucosal extracts from the same lambs showed differing responses. Alkaline phosphatase and glycyl-L-leucine dipeptidase were significantly depleted, whereas maltase activity was only marginally reduced, and leucine aminopeptidase activity was normal. Mucosal acetylcholinesterase activity was significantly elevated in the parasitised animals and, interestingly in view of the postulated role of this enzyme in nematode pathogenicity, the level of activity was directly correlated with individual worm burdens. Intestinal trypsin and chymotrypsin activities were unaffected and the level of superoxide dismutase, an enzyme associated with the inflammatory response, was normal. There were also no consistent changes in the mucosal activities of several enzymes including lactic dehydrogenase, creatine phosphokinase, aldolase, and glutamic oxaloacetate transaminase, whose leakage from damaged or necrotic tissues has been well defined in terms of the concomitant increase in their activity in the circulation. Lambs treated orally with fenbendazole five and/or ten weeks before slaughter either in the presence or absence of continued larval intake, had negligible worm burdens, and showed little evidence of intestinal damage at post mortem. Brush border enzyme levels, with the exception of alkaline phosphatase and, in two cases dipeptidase, were normal in these animals. The activity of alkaline phosphatase was approximately double that in the continuously infected, untreated lambs, but remained markedly lower than in the uninfected controls. The activities of the other enzymes studied, including acetylcholinesterase, were within the control range. In summary, in chronic trichostrongylosis even relatively low nematode burdens were associated with marked pathological and biochemical damage in the intestine with both lesion severity and mucosal acetylcholinesterase activity being directly related to worm numbers. Although morphological integrity was completely restored after anthelmintic treatment, the persistent low activity of brush border alkaline phosphatase coupled with the enzymological findings in untreated, infected animals suggests that recovery of the full functional capability of the intestinal mucosa may take longer.     

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.     

The effect of daily challenge with Trichostrongylus colubriformis larvae on the nutrition and performance of immunologically-resistant sheep

Immunological resistance to Trichostrongylus colubriformis was developed in sheep by challenging them with 2500 larvae per day for 34 weeks. They were then rested for 24 weeks before being re-challenged with the same dose rate of the same larvae for 10 weeks. Nutritional, haematological and parasitological parameters were measured during the first 8 weeks of re-challenge infection. There were no faecal worm eggs excreted during the re-challenge infection. There was a small but significant increase in plasma-N leakage from Days 4 to 9 of dosing but this then declined to initial levels. There was no other detectable nutritional disturbance associated with re-infection. Re-challenge caused a rapid development of eosinophilia which peaked during Week 6 before declining to levels of the control animals by Week 8. Sheep resistant to T. colubriformis appear to be able to re-activate their immunological mechanisms when re-challenged with the parasite with little associated nutritional penalty.     

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.     

Immune response and performance of growing Santa Ines lambs to artificial Trichostrongylus colubriformis infections

This study was carried out to evaluate the immune response and the impact of Trichostrongylus colubriformis infections on the performance of growing Santa Ines lambs. Thirty male lambs, 3-4 months of age, were maintained in individual pens and restrictively randomised by weight into 3 treatment groups: (1) infected group, artificially infected with 2500 T. colubriformis larvae, three times a week, for 13 weeks, and fed ad libitum; (2) Pair-Fed Group, non-infected and fed with the same amount of food consumed by the infected animal of the same class on the previous day; and (3) control group, non-infected and fed ad libitum. Refused feed was weighed daily to assess the food intake of each lamb. Animals were weighed weekly and blood and fecal samples were collected. At the end of the trial, all lambs were euthanized to determine worm burden and collect intestinal tissues and mucus samples for histological and immunological analysis. The infected group presented eosinophilia, increased number of inflammatory cells in the mucosa, in addition to an increased production of specific immunoglobulins against T. colubriformis, which partially prevented the establishment of infective larvae. As a consequence of parasitism, the infected lambs presented reduced serum albumin concentrations and demonstrated severe small intestine lesions, such as villous atrophy and epithelial erosion, which impaired the digestion and absorption of nutrients, causing a significant loss in performance. The infected group presented a 37% reduction in daily weight gain (107.26 ± 10.8 g/day), when compared with the control group (171.07 ± 7.15 g/day). The infected lambs also demonstrated the worst food conversion, i.e., each animal needed to consume on average 10.05 ± 0.52 kg of food to gain 1 kg live weight. The voluntary hay intake depression in infected animals was small, and statistical difference (P<0.01) was seen only on two occasions (ninth and 12th weeks), in comparison with the control group. In conclusion, Santa Ines lambs infected with T. colubriformis demonstrated a reduction in performance, caused by the damages produced by the adult nematodes in intestinal mucosa, and also by the immunopathological changes associated with the infection.     

The influence of Ostertagia circumcincta and Trichostrongylus colubriformis infections on the pharmacokinetics of febantel in lambs

Plasma concentrations of febantel and its major metabolites fenbendazole, oxfendazole and oxfendazole sulphone were determined after oral administration of 7.5 mg/kg febantel in lambs before and 28 days after infection with 50 000 L₃ larvae of Ostertagia circumcincta or Trichostrongylus colubriformis. The febantel concentrations were always very low and only in a few samples higher than the detection limit. The mean decrease in AUC for the three metabolites for the infected sheep in comparison to the parasite naive sheep was 13.9%± 4.1% (mean± SEM) and 23.7%± 5.3% in the 0. circumcincta infected and the T. colubriformis infected lambs respectively. This reduction was only significant for the T. colubriformis infected group.
In order to determine a more complete pharmacokinetic profile, febantel was injected intravenously at a dose of 2.5 mg/kg in a further study.     

Effect of condensed tannins on egg hatching and larval development of Trichostrongylus colubriformis in vitro

The effects of condensed tannins extracted from seven forages on the viability of the eggs and first stage (L1) larvae of the sheep nematode Trichostrongylus colubriformis were evaluated in in vitro assays. The extracts of condensed tannins were obtained from Lotus pedunculatus (LP), Lotus corniculatus (LC), sulla (Hedysarum coronarium), sainfoin (Onobrychus viciifolia), Dorycnium pentaphylum (DP), Dorycnium rectum (DR) and dock (Rumex obtusifolius). Extracts containing 200 to 500 microg/ml reduced the proportion of eggs that hatched. The larval development assay was used to evaluate the effect of the extracts on the development of either eggs or L1 larvae to L3 infective larvae. Development was allowed to proceed for seven days by which time the larvae in control incubations had reached the infective L3 stage. Extracts containing 200 microg/ml from LP, DP, DR or dock prevented egg development, and only 11, 8 and 2 per cent of the eggs developed to L3 larvae with extracts from LC, sulla and sainfoin, respectively. When the concentration was 400 microg/ml no eggs developed to L3 larvae. The addition of the extracts after hatching also inhibited the development of L1 to L3 larvae; 200 microg/ml extracted from LP, LC, sulla, sainfoin, DP, DR and dock resulted in only 14, 18, 17, 15, 14, 16 and 4 per cent of L1 larvae developing to the L3 stage compared with 85 per cent for controls, and 400 microg/ml further reduced the development of L1 larvae. Statistical analyses showed that when the extracts were added before hatching they were significantly (P<0.001) more effective at inhibiting the larval development than when they were added after hatching. The condensed tannins from dock had the greatest inhibitory effect on egg development followed by the tannins from DR, sainfoin, DP, LP, sulla and LC.     

The development of Trichostrongylus colubriformis larvae on a range of herbage species or on plots of differing topographical aspect

Five "contaminations", where faeces containing Trichostrongylus colubriformis eggs were deposited on pasture and serially recovered, were used to compare the rate of decline of faecal mass and larval development. In the first three contaminations, faeces from a common source were deposited on swards of browntop (Agrostis capillaris cv Grasslands Muster), ryegrass (Lolium perenne cv Grassland Nui), white clover (Trifolium pratense cv Grassland Tahora), or onto bare ground in the late spring, summer or autumn. The last two contaminations were done on the north facing aspect or south facing aspect of hill country pastures in summer and autumn. Number of free-living nematodes (first- and second-stage larvae (L(1) and L(2)) and soil dwelling nematodes) and third stage larvae (L(3)) recovered from faeces were counted. In spring there was a significant (P<0.01) effect of sward type on the mass of faeces remaining, with greatest mass remaining on browntop and ryegrass 28 days later, and less on bare ground and white clover. In summer there were more (P<0.05) faeces remaining on browntop than on other herbages which had little faeces remaining and which did not differ one from another. In autumn there was a rapid decline in faecal mass. All faeces were gone from white clover and ryegrass swards by day 10 and from browntop and bare ground by day 14. The number of free-living nematodes did not differ markedly between seasons, ranging from 5 to 8.5% of eggs deposited. The number of L(3) recovered was low in spring ( approximately 0.4% of eggs deposited) and did not differ between swards. In summer, more (P<0.05) L(3) were recovered from faeces deposited on swards of ryegrass and white clover than from bare ground or browntop. Most L(3) were recovered from days 7 to 14 ( approximately 1.3% of eggs deposited). In the autumn, low numbers of L(3) were recovered from browntop on day 3 and ryegrass on day 7 (0.2% of eggs deposited) with virtually no L(3) recovered from faeces placed on white clover or bare ground. There were significant (P<0.001) effects of aspect on the amount of faecal mass remaining in both summer and autumn with less faeces remaining on the south facing aspect than on the north. This was particularly evident during the summer when virtually all of the faeces were intact on the north facing aspect but only 40% was remaining on the south on day 28. In the autumn, while faeces were completely gone from both aspects by day 28 but there were less (P<0.05) faeces remaining on the south facing aspect from days 3 to 18 than from the north. There was no aspect effect in either season on the number of free-living nematodes recovered which averaged 8-11% of eggs deposited. In both seasons a greater number of L(3) were recovered from faeces on the south facing aspect than on the north, particularly 3-10 days after faecal deposition. In summer the rise in L(3) recovered in faeces was more rapid on south facing aspect than on the north but both attained a maximum level of approximately 4% of eggs deposited. In autumn on day 3 there was a rapid rise on south facing aspect to approximately 21% of eggs deposited followed by a gradual decline on day 10 while on the north facing aspect numbers of L(3) recovered only attained 10% of eggs deposited.     

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.     

Effect of prolonged subclinical infection with Trichostrongylus colubriformis on the performance and nitrogen metabolism of growing lambs

Ten 5-month-old lambs, reared free of parasites and prepared with abomasal and ileal cannulas, were paired and given rations of pelleted dried grass by means of continuous feeders. From 7 months of age one of each pair was dosed daily with 2500 Trichostrongylus colubriformis larvae for 34 weeks. An untreated animal served as a pair-fed control to each infected lamb. During 5 experimental periods (pre-infection, and Weeks 6-8, 12-14, 18-20 and 29-34 of dosing) measurements were made of the amounts of N leaving the small intestine, the digestibility of 35[S]-labelled microbial protein in the small intestine, and the amounts of plasma protein leaking into the digestive tract. The infection caused a growth check (P less than 0.05) from Week 6 to approximately Week 13 of dosing. This resulted from inappetence, but also from a disruption in the N digestion and metabolism of the lambs. There was a marked eosinophilia at this time. Measurements of ileal flow rates during Weeks 6 and 12 indicated that significantly more N flowed to the terminal ileum of infected lambs during this period (Week 6, 3.1 g N day-1 P less than 0.05; Week 12, 5.0 g N day-1 P less than 0.001). Plasma N leakage into the intestine during Weeks 8 and 14 was higher in infected lambs (1.2 g N day-1 P less than 0.01 and 0.9 g N day-1 P less than 0.05, respectively) but the digestibility of 35[S]-labelled microbial protein was depressed below that of the controls during the later part of this period, i.e. during Week 13. Continued larval challenge beyond this point caused a less severe nutritional penalty. Voluntary feed intake increased and the animals started to gain weight. The amount of extra N flowing to the terminal ileum of infected lambs was less at Week 18 of dosing (3.5 g N day-1 P less than 0.01) than it had been at Week 12. Similarly, extra plasma N leakage into the tract of infected lambs was less during Week 20 (0.26 g N day-1) than during Week 14. There was however, still a detectable reduction in the coefficient of digestibility of 35S microbial protein in infected lambs during Week 19. By Weeks 29-34 the only detectable difference between the infected and control lambs was in their eosinophil counts.