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Some observations on immunologically mediated inhibited Teladorsagia circumcincta and their subsequent resumption of development in sheep
Veterinary Parasitology 147 (2007) 103–109 www.elsevier.com/locate/vetpar
Some observations on immunologically mediated inhibited Teladorsagia circumcincta and their subsequent resumption of development in sheep W.D. Smith * Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Scotland EH26 0PZ, UK Received 12 October 2006; received in revised form 15 March 2007; accepted 22 March 2007
Abstract Two similar experiments were conducted with groups of yearling sheep which had been trickle infected with Teladorsagia circumcincta for 6 weeks, treated with anthelmintic then challenged with a single dose of 50,000 larvae. Ten days after challenge these sheep contained significantly fewer worms and a significantly higher proportion of arrested early fourth stage larvae than controls which had not received the trickle infection. However, by 19 or 23 days after challenge most of the arrested worms had resumed development, a process which was accelerated in a group treated with corticosteroids. It was concluded that under these conditions arrested development of Teladorsagia was a short-lived, immunologically mediated phenomenon which could be reversed by immuno-suppressing the sheep. # 2007 Elsevier B.V. All rights reserved. Keywords: Teladorsagia circumcincta; Sheep; Arrested; Inhibited; Resumed; Development; Hypobiosis; Early fourth stage larvae; Corticosteroid
1. Introduction Teladorsagia circumcincta is one of several genera of gastro-intestinal nematodes of ruminants which can arrest its development at the early fourth larval stage. Excellent thought-provoking reviews have been written on the subject of arrested development in nematode parasites (Michel, 1978; Gibbs, 1986). Arrested development, also known as hypobiosis, has been defined as ‘‘ the temporary cessation of development of nematodes at a precise point in early parasitic development when such an interruption contains a facultative element occurring in certain hosts, certain circumstances or certain times of the year and often
* Tel.: +44 1314456131. E-mail address: [email protected]. 0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2007.03.026
affecting only a proportion of the worms’’ (Michel, 1978). Gibbs (1986) considered there to be two types of arrested development, an immune mediated type and one that was seasonally induced. He suggested that ‘‘inhibited’’ might be a better term to describe the immune mediated version. Compared to other abomasal species e.g. Ostertagia ostertagi in cattle and Haemonchus contortus in sheep, relatively little seems to have been published on hypobiosis or the subsequent resumption of development of Teladorsagia. The present paper further characterises a previously described Teladorsagia infection model which consistently induces a high proportion of challenge worms to become immunologically inhibited early fourth stage larvae (Smith et al., 1983; Smith et al., 1984). The experiments to be described indicate that this inhibition is short-lived and that resumption of development can be accelerated by immuno-suppressing the host.
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W.D. Smith / Veterinary Parasitology 147 (2007) 103–109
2. Materials and methods 2.1. Sheep Two experiments were conducted involving a total of 41 yearling Scottish Blackface cross sheep. All had been reared and housed since birth under conditions designed to exclude accidental infection with nematode parasites. 2.2. Infective larvae These were from an anthelmintic susceptible isolate of T. circumcincta which had been passaged through sheep at Moredun Research Institute for many years. The larvae had been stored at 4 8C for up to a month before use. All the challenge doses used within each experiment were from the same batch of larvae. 2.3. Post mortem procedures Shortly after each sheep was stunned with a captive bolt and exsanguinated, the abomasum was removed and opened along its lesser curvature. The contents were collected into a bucket and the abomasum was washed gently in two changes of warm saline. These washings were added to the contents. The abomasum was then placed in saline for 4 h at 37 8C as described before (Jackson et al., 1984). Abomasal contents and washings were made up to 5 l and fixed separately in 2% formalin. An approximate 5% aliquot of each was obtained by vigorously stirring and sampling with a ladle. The exact volume of the sub-sample was then measured. 2.4. Worm counting, measuring and staging Each sub-sample was stained by the addition of a concentrated iodine solution and the excess stain
removed by washing the sample over a 400 mesh sieve. A few ml at a time were poured onto a transparent tray etched with a 1 cm grid and searched under a stereomicroscope. The number of male, female and sexually undifferentiated early fourth stage larvae was recorded and a random sample of 40–50 were measured by a camera lucida under 10 magnification. A line was drawn down the centre of the magnified image of each worm. The line was measured by stepping a divider along it and along a line of known length which had also been drawn with the camera. Sexually undifferentiated worms measuring less than 1.5 mm were classified as early fourth stage larvae, longer parasites were considered to be undergoing development. 2.5. Statistical methods Arithmetic means with standard errors are shown throughout. Worm counts were compared by analysis of variance, followed by Dunnett’s multiple comparison test. Frequency distributions of male and female worm lengths were made for individual sheep and group mean distributions were calculated from these. 2.6. Design of experiments Both experiments compared the number and length of worms recovered from previously infected or control sheep killed 10, 19 or 23 days following a single challenge dose of 50,000 T. circumcincta larvae. The previously infected groups had received a trickle immunising infection for 6 weeks beforehand which had been cleared out with anthelmintic before challenge as shown by Smith et al. (1984). The anthelmintic type and hence the interval between this treatment and the timing of the challenge dose varied between experiment
Table 1 Design of Experiments 1 and 2 Experiment
Group
Treatments (relative to day of challenge) Trickle infection a
1
Previously infected Control
+
2
Previously infected Control
+
a b c
Ivermectin (200 mg/kg s.c.) ( 21)
Fenbendazole (5 mg/kg) ( 7)
Challenge infectionb (0)
(10)
(19)
+ +
+ +
5 5
5 5
+ +
5
+ +
2000 Teladorsagia circumcincta L3 5 per week for 6 weeks. 1 50,000 T. circumcincta L3. 5 of these sheep received 0.5 mg/kg intramuscular dexamethasone on Days 10, 13 and 16.
No killed (23)
11 c 4
W.D. Smith / Veterinary Parasitology 147 (2007) 103–109
105
which 39% of the worms measured were less than 1.5 mm. The situation was very different in the previously infected sheep. At Day 10 virtually all the worms measured less than 1.5 mm (Fig. 1C). However, by Day 19 the distribution was much more spread out and suggestive of two broad peaks of 2–3 and 5.5–6.5 mm, respectively (Fig. 1D).
(Table 1), but within each experiment control and previously infected groups were otherwise treated identically. 3. Results 3.1. Experiment 1 The main purpose of this experiment was to compare the size and the state of development of the worm populations of previously infected and control sheep 10 or 19 days after challenge with 50,000 larvae (Table 1). The mean worm count of the control sheep on Day 10 was 25,552, only about 2% of which were early fourth stage larvae (EL4). By Day 19 the mean total count had declined significantly ( p < 0.001) to 8107, although there was marked individual variation (Table 2). Ten days after challenge strikingly fewer worms were found in the previously infected sheep compared to the controls ( p < 0.001), yet the mean number of EL4s was higher (Table 2). There was no evidence of further worm loss between Days 10 and 19 in the previously infected group, though the proportion at the early fourth stage declined from 95 to 54%. Mean length frequency distributions for female worms recovered from each group are plotted in Fig. 1. Very similar profiles were observed with the male parasites (not shown). Ten days after infection the control worm lengths were normally distributed, peaking at 4.5–5 mm (Fig. 1A). By Day 19 the peak length had increased to 6.5–7 mm, although a smaller EL4 peak was also observed at 1–1.5 mm (Fig. 1B), caused entirely by the population from a single sheep in
3.2. Experiment 2 This was done partly to confirm some of the observations made in Experiment 1, but also to investigate whether resumption of Teladorsagia development was influenced by the host response. For this purpose five previously infected sheep were immunosuppressed with corticosteroid from Day 10 after challenge to determine whether this would accelerate re-development (Table 1). The original plan was to replicate the trickle infection regime used in Experiment 1 exactly, but in the event, ivermectin, not fenbendazole, was erroneously used to terminate it. In order to cater for the longer half life of ivermectin (Prichard et al., 1985), the interval between anthelmintic and challenge was extended to 3 weeks (Table 1). As in Experiment 1, there was no evidence of significant worm loss between Days 10 and 23 in the previously infected sheep. The proportion of EL4s found on Day 23 was low in most controls (one exception had 25.5%, see Table 3). In contrast the populations recovered from the previously infected on Day 10 were almost entirely EL4 (an exception had 52.5%). By Day 23 the proportion of EL4s in the previously infected group had dropped significantly to a
Table 2 Experiment 1: individual and group mean total and early fourth stage worm counts Group Control
Previously infected
a
a
(10 )
Mean S.E. a
(10a)
(19 )
(19a)
Total
EL4
Total
EL4
Total
EL4
Total
EL4
29,880 27,640 24,720 22,760 22,760
687 332 1236 91 273
19,080 15,360 4,680 4,480 4,480 560
248 200 – 399 – –
2358 1895 1511 884 311
2358 1769 1511 884 267
9334 4126 1642 771 122
6058 3494 1516 64 24
169 76.5
1392 362
1358 361.1
3199 1678
2231 1147.1
25,552 1,403
Value in parenthesis is kill day.
524 202.6
8,107 2,989
106
W.D. Smith / Veterinary Parasitology 147 (2007) 103–109
Fig. 1. Experiment 1: frequency distribution of female worm lengths (includes 50% of unsexable EL4s).
development (Fig. 2B) with about 65% more than 5 mm long and therefore similar in size to the bulk of the control group (Fig. 2C). Interestingly, no early or retarded L4s were measured in the samples from the immuno-suppressed group (Fig. 2D). It was concluded that this experiment had confirmed one of the main findings of Experiment 1, namely that, although the trickle infection induced most of the challenge worms to inhibit their development at the early fourth stage, re-development rapidly occurred. Experiment 2 also showed that corticosteroid treatment
mean of 14%, but this was significantly higher than in the immuno-suppressed group, where hardly any EL4s were recovered (Table 3). Population frequency distribution graphs of female worms recovered from each group are plotted in Fig. 2. Ten days after challenge of the previously infected sheep the great majority of the worms were less than 1.5 mm (Fig. 2A), in agreement with the visual assessment just described. Thirteen days later more than 85% of these worms were more than 1.5 mm indicating that the great majority had resumed their
Table 3 Experiment 2: individual and group mean total and early fourth stage worm counts Group (kill day) Previously infected (10)
Mean S.E.
Previously infected (23)
Previously infected and steroid (23)
Control (23)
Total
EL4
Total
EL4
Total
EL4
Total
EL4
5580 4720 3200 1000 160
5460 4660 1680 980 160
7080 4000 3740 320 280 160
180 1660 640 40 0 20
13420 3840 3820 4880 1540
0 0 0 20 0
5000 3460 3300 2420
0 40 840 40
2932 1164.5
2588 1168.5
2597 1262.8
3545 619.1
230 235.0
423 291.5
5500 2296.3
4 4.5
W.D. Smith / Veterinary Parasitology 147 (2007) 103–109
107
Fig. 2. Experiment 2: frequency distribution of female worm lengths (includes 50% of unsexable EL4s).
of the sheep accelerated the re-development process, suggesting that it was controlled by the host response. 4. Discussion Factors which influence the induction of arrested development of gastro-intestinal nematode parasites of ruminants could be politely described as incomplete. Even less is known about the factors which trigger the resumption of development of such arrested parasites. The present study is of significance because it adds to our general knowledge on these topics, especially on immunologically induced arrestment and subsequent re-development of Teladorsagia in the sheep, a host parasite relationship in which this subject has received little attention. The first experiment in this paper showed that after worm naı¨ve sheep were given a dose of 50,000 larvae, significant numbers of Teladorsagia were lost from some animals between Days 10 and 19, but only about 2% of the worms present at these times were arrested at the early fourth stage. In contrast, Dunsmore (1960) found 20–75% arrested Teladorsagia after a primary infection of 100,000 larvae but less than 2% when the dose was reduced to 1000 larvae. Immuno-suppressing the sheep substantially reduced arrestment, and so he suggested that it was caused by a rapid host response
triggered by the earliest arriving worms affecting later incomers (Dunsmore, 1961). Hong et al. (1986) confirmed the dose effect on arrested development observed by Dunsmore with single doses of 3000, 10,000 or 33,000 larvae given to worm free sheep. Their Weybridge isolate of Teladorsagia showed a greater propensity to arrest after a primary infection than our Moredun culture, indicating genetic variation for this trait between isolates. Some of the arrested larvae observed by Hong et al. (1986) resumed development rapidly i.e. by 15 days after infection and almost all had done so by 8 weeks. Relatively few studies on arrested Teladorsagia seem to have been conducted in previously infected, immune sheep. The present experiment design was used before with similar results, namely, that compared to controls, many fewer worms were recovered 10 days after challenge from previously infected sheep and nearly all these were inhibited early fourth stage larvae (Smith et al., 1983, 1984). The mechanism must have been immune mediated because the proportion of arrested worms in the control sheep was very much lower (Smith et al., 1983, 1984). Gibbs (1986) questioned whether this was genuine arrestment because the interval between challenge and kill was short. Indeed, the results from both experiments in the present paper indicate that most of the worms had resumed
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W.D. Smith / Veterinary Parasitology 147 (2007) 103–109
development some 3 weeks after challenge, confirming Gibbs’ suspicions that it might be a short-lived phenomenon. Yet Michel’s widely accepted definition of arrested development quoted in the introduction does not define a minimum period of time for delayed development at the early fourth stage to be classified as ‘‘arrested’’. Indeed, results from his laboratory showed that populations of ‘‘arrested’’ Teladorsagia or O. ostertagi resumed development more or less continuously, with the earliest worms emerging from arrestment after only a few weeks (Michel et al., 1976; Hong et al., 1986). It was unfortunate that the unplanned switch in anthelmintic meant that there was a longer interval between termination of the trickle infection and challenge in Experiment 2 compared to Experiment 1. However, by Day 10 post challenge the proportion of EL4s was very high in both experiments suggesting that the mechanism which stimulated arrest had not waned significantly between 1 and 3 weeks post treatment. On the other hand, the proportion of EL4s in the previously infected sheep on Day 23 appeared to be substantially lower in Experiment 2 than on Day 19 in Experiment 1 (Table 2 versus Table 1). Perhaps the longer treatment to challenge interval in Experiment 2 allowed a greater proportion of the challenge worms to resume development, but the possibility that this difference was simply because the sampling time was 4 days later in Experiment 2 cannot be ruled out either. In the previously infected sheep described in the present trials, it is possible to speculate that any of numerous factors turned on by the sensitising infections could have been responsible for inducing arrest. However, earlier work showed that if the challenge dose was reduced from 50,000 to 1000 larvae, hardly any worms arrested and the smaller dose failed to induce a detectable local immune response (Smith et al., 1984). If the arrestment mechanism was due to a passive state induced by the trickle infection, it would have been expected to affect large and small challenges alike, supporting the view that an active, response was required and, since Teladorsagia only remain as sexually undifferentiated larvae until Day 4, it must have happened before then. Elements of the immediate type hypersensitivity reaction triggered by the arrival of the challenge larvae and mediated by the numerous mast cells present in the mucosa are obvious candidates for such a mechanism (Smith et al., 1984). In natural infections, continuously ingested incoming larvae (as well as the presence of adult or developing worms) would be expected to maintain a near continuous hypersensitive reaction which might maintain arrestment for longer than
was observed in the current trials. Data from continuous or interrupted trickle challenges have provided experimental evidence, in the form of increased local pepsinogen concentrations, to support this prolonged hypersensitivity theory (Smith et al., 1987). Obviously the cause of arrested development after a primary infection of Teladorsagia must be different, but by definition it also has to occur by Day 4 after infection. It is also dose dependent (Dunsmore, 1960; Hong et al., 1986) and sensitive to immunosuppression of the sheep (Dunsmore, 1961). At least two cell signalling pathways have been discovered in free living C. elegans through which development is arrested as ‘‘dauer’’ larvae (reviewed by Nisbet et al., 2004). Could cell signalling molecules or certain chemokines be upregulated rapidly by the ovine innate immune response after infection and consequently arrest the development of Teladorsagia? The results of the second experiment in the present paper indicated that resumption of development of arrested T. circumcincta could be accelerated by immuno-suppressing the sheep. In contrast, earlier attempts to reactivate arrested H. contortus (Gibbs, 1968) or O. ostertagi (Prichard et al., 1974) by immunosuppressing the host were unsuccessful. One explanation for this difference is that, whilst the mechanism of induction of arrest was clearly immune mediated in the current experiments, in those reported by Gibbs and Pritchard et al., arrestment was seasonally induced and less likely to respond to immunosuppression of the host. In support of this explanation Behnke and Parish (1979) reported re-development of immunologically arrested Nematospiroides dubious following cortisone treatment of mice. Acknowledgements The late Robert Graham is thanked for able technical assistance. The work was funded by the Scottish Executive Environment and Rural Affairs Department. References Behnke, J.M., Parish, H.A., 1979. Nematospiroides dubious arrested development of larvae in immune mice. Exp. Parasitol. 47, 116–127. Dunsmore, J.D., 1960. Retarded development of Ostertagia species in sheep. Nature 186, 986. Dunsmore, J.D., 1961. Effect of whole body irradiation and cortisone on the development of Ostertagia in sheep. Nature 192, 139–140. Gibbs, H.C., 1968. Some factors involved in the ‘‘spring rise’’ phenomenon in sheep. In: Soulsby, E.J.L. (Ed.), The reaction of the Host to Parasitism. N.G. Elwert, Univeritats and Verlagsbuchand, Marburg, Lahn, pp. 160–173.
W.D. Smith / Veterinary Parasitology 147 (2007) 103–109 Gibbs, H.C., 1986. Hypobiosis in parasitic nematodes—an update. Adv. Parasitol. 25, 129–174. Hong, C., Lancaster, M.B., Michel, J.F., 1986. Populations of Ostertagia circumcincta in lambs following a single infection. Int. J. Parasitol. 16, 63–67. Jackson, E., Jackson, F.J., Smith, W.D., 1984. Comparison of saline incubation and pepsin digestion as methods for recovering Ostertagia circumcincta larvae from the abomasum of sheep. Res. Vet. Sci. 36, 380–381. Michel, J.F., 1978. Topical themes in the study of arrested development. In: Borgsteede, F.H. (Ed.), Facts and Reflections. III. Workshop on Arrested Development of Nematodes of Sheep and Cattle. Central Veterinary Institute, Lelystad, Netherlands, pp. 7–17. Michel, J.F., Lancaster, M.B., Hong, C., 1976. The resumed development of arrested Ostertagia ostertagi in experimentally infected calves. J. Comp. Path. 86, 615–619. Nisbet, A.J., Cottee, P., Gasser, R.B., 2004. Molecular biology of reproduction and development in parasitic nematodes: progress and opportunities. Int. J. Parasitol. 34, 125–138.
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Prichard, R.K., Donald, A.D., Hennessy, D.R., 1974. The effect of corticosteroid treatment on the development of inhibited Ostertagia ostertagi larvae. Res. Vet. Sci. 13, 225–229. Prichard, R.K., Steel, J.W., Lacey, E., Hennessy, D.R., 1985. Pharmacokinetics of ivermectin in sheep following intravenous, intraabomasal or intraruminal administration. J. Vet. Pharmacol. Ther. 8, 88–94. Smith, W.D., Jackson, F.J., Jackson, E., Williams, J., 1983. Local Immunity and Ostertagia circumcincta: changes in the gastric lymph of immune sheep after a challenge infection. J. Comp. Path. 93, 479–488. Smith, W.D., Jackson, F.J., Jackson, E., Williams, J., Miller, H.R.P., 1984. Manifestations of resistance to ovine ostertagiasis associated with immunological responses in the gastric lymph. J. Comp. Path. 94, 591–601. Smith, W.D., Jackson, F.J., Graham, R., Jackson, E., Williams, J., 1987. Mucosal IgA production and lymph cell traffic following prolonged low level infections of Ostertagia circumcincta in sheep. Res. Vet. Sci. 43, 320–326.
Some observations on immunologically mediated inhibited Teladorsagia circumcincta and their subsequent resumption of development in sheep W.D. Smith * Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Scotland EH26 0PZ, UK Received 12 October 2006; received in revised form 15 March 2007; accepted 22 March 2007
Abstract Two similar experiments were conducted with groups of yearling sheep which had been trickle infected with Teladorsagia circumcincta for 6 weeks, treated with anthelmintic then challenged with a single dose of 50,000 larvae. Ten days after challenge these sheep contained significantly fewer worms and a significantly higher proportion of arrested early fourth stage larvae than controls which had not received the trickle infection. However, by 19 or 23 days after challenge most of the arrested worms had resumed development, a process which was accelerated in a group treated with corticosteroids. It was concluded that under these conditions arrested development of Teladorsagia was a short-lived, immunologically mediated phenomenon which could be reversed by immuno-suppressing the sheep. # 2007 Elsevier B.V. All rights reserved. Keywords: Teladorsagia circumcincta; Sheep; Arrested; Inhibited; Resumed; Development; Hypobiosis; Early fourth stage larvae; Corticosteroid
1. Introduction Teladorsagia circumcincta is one of several genera of gastro-intestinal nematodes of ruminants which can arrest its development at the early fourth larval stage. Excellent thought-provoking reviews have been written on the subject of arrested development in nematode parasites (Michel, 1978; Gibbs, 1986). Arrested development, also known as hypobiosis, has been defined as ‘‘ the temporary cessation of development of nematodes at a precise point in early parasitic development when such an interruption contains a facultative element occurring in certain hosts, certain circumstances or certain times of the year and often
* Tel.: +44 1314456131. E-mail address: [email protected]. 0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2007.03.026
affecting only a proportion of the worms’’ (Michel, 1978). Gibbs (1986) considered there to be two types of arrested development, an immune mediated type and one that was seasonally induced. He suggested that ‘‘inhibited’’ might be a better term to describe the immune mediated version. Compared to other abomasal species e.g. Ostertagia ostertagi in cattle and Haemonchus contortus in sheep, relatively little seems to have been published on hypobiosis or the subsequent resumption of development of Teladorsagia. The present paper further characterises a previously described Teladorsagia infection model which consistently induces a high proportion of challenge worms to become immunologically inhibited early fourth stage larvae (Smith et al., 1983; Smith et al., 1984). The experiments to be described indicate that this inhibition is short-lived and that resumption of development can be accelerated by immuno-suppressing the host.
104
W.D. Smith / Veterinary Parasitology 147 (2007) 103–109
2. Materials and methods 2.1. Sheep Two experiments were conducted involving a total of 41 yearling Scottish Blackface cross sheep. All had been reared and housed since birth under conditions designed to exclude accidental infection with nematode parasites. 2.2. Infective larvae These were from an anthelmintic susceptible isolate of T. circumcincta which had been passaged through sheep at Moredun Research Institute for many years. The larvae had been stored at 4 8C for up to a month before use. All the challenge doses used within each experiment were from the same batch of larvae. 2.3. Post mortem procedures Shortly after each sheep was stunned with a captive bolt and exsanguinated, the abomasum was removed and opened along its lesser curvature. The contents were collected into a bucket and the abomasum was washed gently in two changes of warm saline. These washings were added to the contents. The abomasum was then placed in saline for 4 h at 37 8C as described before (Jackson et al., 1984). Abomasal contents and washings were made up to 5 l and fixed separately in 2% formalin. An approximate 5% aliquot of each was obtained by vigorously stirring and sampling with a ladle. The exact volume of the sub-sample was then measured. 2.4. Worm counting, measuring and staging Each sub-sample was stained by the addition of a concentrated iodine solution and the excess stain
removed by washing the sample over a 400 mesh sieve. A few ml at a time were poured onto a transparent tray etched with a 1 cm grid and searched under a stereomicroscope. The number of male, female and sexually undifferentiated early fourth stage larvae was recorded and a random sample of 40–50 were measured by a camera lucida under 10 magnification. A line was drawn down the centre of the magnified image of each worm. The line was measured by stepping a divider along it and along a line of known length which had also been drawn with the camera. Sexually undifferentiated worms measuring less than 1.5 mm were classified as early fourth stage larvae, longer parasites were considered to be undergoing development. 2.5. Statistical methods Arithmetic means with standard errors are shown throughout. Worm counts were compared by analysis of variance, followed by Dunnett’s multiple comparison test. Frequency distributions of male and female worm lengths were made for individual sheep and group mean distributions were calculated from these. 2.6. Design of experiments Both experiments compared the number and length of worms recovered from previously infected or control sheep killed 10, 19 or 23 days following a single challenge dose of 50,000 T. circumcincta larvae. The previously infected groups had received a trickle immunising infection for 6 weeks beforehand which had been cleared out with anthelmintic before challenge as shown by Smith et al. (1984). The anthelmintic type and hence the interval between this treatment and the timing of the challenge dose varied between experiment
Table 1 Design of Experiments 1 and 2 Experiment
Group
Treatments (relative to day of challenge) Trickle infection a
1
Previously infected Control
+
2
Previously infected Control
+
a b c
Ivermectin (200 mg/kg s.c.) ( 21)
Fenbendazole (5 mg/kg) ( 7)
Challenge infectionb (0)
(10)
(19)
+ +
+ +
5 5
5 5
+ +
5
+ +
2000 Teladorsagia circumcincta L3 5 per week for 6 weeks. 1 50,000 T. circumcincta L3. 5 of these sheep received 0.5 mg/kg intramuscular dexamethasone on Days 10, 13 and 16.
No killed (23)
11 c 4
W.D. Smith / Veterinary Parasitology 147 (2007) 103–109
105
which 39% of the worms measured were less than 1.5 mm. The situation was very different in the previously infected sheep. At Day 10 virtually all the worms measured less than 1.5 mm (Fig. 1C). However, by Day 19 the distribution was much more spread out and suggestive of two broad peaks of 2–3 and 5.5–6.5 mm, respectively (Fig. 1D).
(Table 1), but within each experiment control and previously infected groups were otherwise treated identically. 3. Results 3.1. Experiment 1 The main purpose of this experiment was to compare the size and the state of development of the worm populations of previously infected and control sheep 10 or 19 days after challenge with 50,000 larvae (Table 1). The mean worm count of the control sheep on Day 10 was 25,552, only about 2% of which were early fourth stage larvae (EL4). By Day 19 the mean total count had declined significantly ( p < 0.001) to 8107, although there was marked individual variation (Table 2). Ten days after challenge strikingly fewer worms were found in the previously infected sheep compared to the controls ( p < 0.001), yet the mean number of EL4s was higher (Table 2). There was no evidence of further worm loss between Days 10 and 19 in the previously infected group, though the proportion at the early fourth stage declined from 95 to 54%. Mean length frequency distributions for female worms recovered from each group are plotted in Fig. 1. Very similar profiles were observed with the male parasites (not shown). Ten days after infection the control worm lengths were normally distributed, peaking at 4.5–5 mm (Fig. 1A). By Day 19 the peak length had increased to 6.5–7 mm, although a smaller EL4 peak was also observed at 1–1.5 mm (Fig. 1B), caused entirely by the population from a single sheep in
3.2. Experiment 2 This was done partly to confirm some of the observations made in Experiment 1, but also to investigate whether resumption of Teladorsagia development was influenced by the host response. For this purpose five previously infected sheep were immunosuppressed with corticosteroid from Day 10 after challenge to determine whether this would accelerate re-development (Table 1). The original plan was to replicate the trickle infection regime used in Experiment 1 exactly, but in the event, ivermectin, not fenbendazole, was erroneously used to terminate it. In order to cater for the longer half life of ivermectin (Prichard et al., 1985), the interval between anthelmintic and challenge was extended to 3 weeks (Table 1). As in Experiment 1, there was no evidence of significant worm loss between Days 10 and 23 in the previously infected sheep. The proportion of EL4s found on Day 23 was low in most controls (one exception had 25.5%, see Table 3). In contrast the populations recovered from the previously infected on Day 10 were almost entirely EL4 (an exception had 52.5%). By Day 23 the proportion of EL4s in the previously infected group had dropped significantly to a
Table 2 Experiment 1: individual and group mean total and early fourth stage worm counts Group Control
Previously infected
a
a
(10 )
Mean S.E. a
(10a)
(19 )
(19a)
Total
EL4
Total
EL4
Total
EL4
Total
EL4
29,880 27,640 24,720 22,760 22,760
687 332 1236 91 273
19,080 15,360 4,680 4,480 4,480 560
248 200 – 399 – –
2358 1895 1511 884 311
2358 1769 1511 884 267
9334 4126 1642 771 122
6058 3494 1516 64 24
169 76.5
1392 362
1358 361.1
3199 1678
2231 1147.1
25,552 1,403
Value in parenthesis is kill day.
524 202.6
8,107 2,989
106
W.D. Smith / Veterinary Parasitology 147 (2007) 103–109
Fig. 1. Experiment 1: frequency distribution of female worm lengths (includes 50% of unsexable EL4s).
development (Fig. 2B) with about 65% more than 5 mm long and therefore similar in size to the bulk of the control group (Fig. 2C). Interestingly, no early or retarded L4s were measured in the samples from the immuno-suppressed group (Fig. 2D). It was concluded that this experiment had confirmed one of the main findings of Experiment 1, namely that, although the trickle infection induced most of the challenge worms to inhibit their development at the early fourth stage, re-development rapidly occurred. Experiment 2 also showed that corticosteroid treatment
mean of 14%, but this was significantly higher than in the immuno-suppressed group, where hardly any EL4s were recovered (Table 3). Population frequency distribution graphs of female worms recovered from each group are plotted in Fig. 2. Ten days after challenge of the previously infected sheep the great majority of the worms were less than 1.5 mm (Fig. 2A), in agreement with the visual assessment just described. Thirteen days later more than 85% of these worms were more than 1.5 mm indicating that the great majority had resumed their
Table 3 Experiment 2: individual and group mean total and early fourth stage worm counts Group (kill day) Previously infected (10)
Mean S.E.
Previously infected (23)
Previously infected and steroid (23)
Control (23)
Total
EL4
Total
EL4
Total
EL4
Total
EL4
5580 4720 3200 1000 160
5460 4660 1680 980 160
7080 4000 3740 320 280 160
180 1660 640 40 0 20
13420 3840 3820 4880 1540
0 0 0 20 0
5000 3460 3300 2420
0 40 840 40
2932 1164.5
2588 1168.5
2597 1262.8
3545 619.1
230 235.0
423 291.5
5500 2296.3
4 4.5
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Fig. 2. Experiment 2: frequency distribution of female worm lengths (includes 50% of unsexable EL4s).
of the sheep accelerated the re-development process, suggesting that it was controlled by the host response. 4. Discussion Factors which influence the induction of arrested development of gastro-intestinal nematode parasites of ruminants could be politely described as incomplete. Even less is known about the factors which trigger the resumption of development of such arrested parasites. The present study is of significance because it adds to our general knowledge on these topics, especially on immunologically induced arrestment and subsequent re-development of Teladorsagia in the sheep, a host parasite relationship in which this subject has received little attention. The first experiment in this paper showed that after worm naı¨ve sheep were given a dose of 50,000 larvae, significant numbers of Teladorsagia were lost from some animals between Days 10 and 19, but only about 2% of the worms present at these times were arrested at the early fourth stage. In contrast, Dunsmore (1960) found 20–75% arrested Teladorsagia after a primary infection of 100,000 larvae but less than 2% when the dose was reduced to 1000 larvae. Immuno-suppressing the sheep substantially reduced arrestment, and so he suggested that it was caused by a rapid host response
triggered by the earliest arriving worms affecting later incomers (Dunsmore, 1961). Hong et al. (1986) confirmed the dose effect on arrested development observed by Dunsmore with single doses of 3000, 10,000 or 33,000 larvae given to worm free sheep. Their Weybridge isolate of Teladorsagia showed a greater propensity to arrest after a primary infection than our Moredun culture, indicating genetic variation for this trait between isolates. Some of the arrested larvae observed by Hong et al. (1986) resumed development rapidly i.e. by 15 days after infection and almost all had done so by 8 weeks. Relatively few studies on arrested Teladorsagia seem to have been conducted in previously infected, immune sheep. The present experiment design was used before with similar results, namely, that compared to controls, many fewer worms were recovered 10 days after challenge from previously infected sheep and nearly all these were inhibited early fourth stage larvae (Smith et al., 1983, 1984). The mechanism must have been immune mediated because the proportion of arrested worms in the control sheep was very much lower (Smith et al., 1983, 1984). Gibbs (1986) questioned whether this was genuine arrestment because the interval between challenge and kill was short. Indeed, the results from both experiments in the present paper indicate that most of the worms had resumed
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development some 3 weeks after challenge, confirming Gibbs’ suspicions that it might be a short-lived phenomenon. Yet Michel’s widely accepted definition of arrested development quoted in the introduction does not define a minimum period of time for delayed development at the early fourth stage to be classified as ‘‘arrested’’. Indeed, results from his laboratory showed that populations of ‘‘arrested’’ Teladorsagia or O. ostertagi resumed development more or less continuously, with the earliest worms emerging from arrestment after only a few weeks (Michel et al., 1976; Hong et al., 1986). It was unfortunate that the unplanned switch in anthelmintic meant that there was a longer interval between termination of the trickle infection and challenge in Experiment 2 compared to Experiment 1. However, by Day 10 post challenge the proportion of EL4s was very high in both experiments suggesting that the mechanism which stimulated arrest had not waned significantly between 1 and 3 weeks post treatment. On the other hand, the proportion of EL4s in the previously infected sheep on Day 23 appeared to be substantially lower in Experiment 2 than on Day 19 in Experiment 1 (Table 2 versus Table 1). Perhaps the longer treatment to challenge interval in Experiment 2 allowed a greater proportion of the challenge worms to resume development, but the possibility that this difference was simply because the sampling time was 4 days later in Experiment 2 cannot be ruled out either. In the previously infected sheep described in the present trials, it is possible to speculate that any of numerous factors turned on by the sensitising infections could have been responsible for inducing arrest. However, earlier work showed that if the challenge dose was reduced from 50,000 to 1000 larvae, hardly any worms arrested and the smaller dose failed to induce a detectable local immune response (Smith et al., 1984). If the arrestment mechanism was due to a passive state induced by the trickle infection, it would have been expected to affect large and small challenges alike, supporting the view that an active, response was required and, since Teladorsagia only remain as sexually undifferentiated larvae until Day 4, it must have happened before then. Elements of the immediate type hypersensitivity reaction triggered by the arrival of the challenge larvae and mediated by the numerous mast cells present in the mucosa are obvious candidates for such a mechanism (Smith et al., 1984). In natural infections, continuously ingested incoming larvae (as well as the presence of adult or developing worms) would be expected to maintain a near continuous hypersensitive reaction which might maintain arrestment for longer than
was observed in the current trials. Data from continuous or interrupted trickle challenges have provided experimental evidence, in the form of increased local pepsinogen concentrations, to support this prolonged hypersensitivity theory (Smith et al., 1987). Obviously the cause of arrested development after a primary infection of Teladorsagia must be different, but by definition it also has to occur by Day 4 after infection. It is also dose dependent (Dunsmore, 1960; Hong et al., 1986) and sensitive to immunosuppression of the sheep (Dunsmore, 1961). At least two cell signalling pathways have been discovered in free living C. elegans through which development is arrested as ‘‘dauer’’ larvae (reviewed by Nisbet et al., 2004). Could cell signalling molecules or certain chemokines be upregulated rapidly by the ovine innate immune response after infection and consequently arrest the development of Teladorsagia? The results of the second experiment in the present paper indicated that resumption of development of arrested T. circumcincta could be accelerated by immuno-suppressing the sheep. In contrast, earlier attempts to reactivate arrested H. contortus (Gibbs, 1968) or O. ostertagi (Prichard et al., 1974) by immunosuppressing the host were unsuccessful. One explanation for this difference is that, whilst the mechanism of induction of arrest was clearly immune mediated in the current experiments, in those reported by Gibbs and Pritchard et al., arrestment was seasonally induced and less likely to respond to immunosuppression of the host. In support of this explanation Behnke and Parish (1979) reported re-development of immunologically arrested Nematospiroides dubious following cortisone treatment of mice. Acknowledgements The late Robert Graham is thanked for able technical assistance. The work was funded by the Scottish Executive Environment and Rural Affairs Department. References Behnke, J.M., Parish, H.A., 1979. Nematospiroides dubious arrested development of larvae in immune mice. Exp. Parasitol. 47, 116–127. Dunsmore, J.D., 1960. Retarded development of Ostertagia species in sheep. Nature 186, 986. Dunsmore, J.D., 1961. Effect of whole body irradiation and cortisone on the development of Ostertagia in sheep. Nature 192, 139–140. Gibbs, H.C., 1968. Some factors involved in the ‘‘spring rise’’ phenomenon in sheep. In: Soulsby, E.J.L. (Ed.), The reaction of the Host to Parasitism. N.G. Elwert, Univeritats and Verlagsbuchand, Marburg, Lahn, pp. 160–173.
W.D. Smith / Veterinary Parasitology 147 (2007) 103–109 Gibbs, H.C., 1986. Hypobiosis in parasitic nematodes—an update. Adv. Parasitol. 25, 129–174. Hong, C., Lancaster, M.B., Michel, J.F., 1986. Populations of Ostertagia circumcincta in lambs following a single infection. Int. J. Parasitol. 16, 63–67. Jackson, E., Jackson, F.J., Smith, W.D., 1984. Comparison of saline incubation and pepsin digestion as methods for recovering Ostertagia circumcincta larvae from the abomasum of sheep. Res. Vet. Sci. 36, 380–381. Michel, J.F., 1978. Topical themes in the study of arrested development. In: Borgsteede, F.H. (Ed.), Facts and Reflections. III. Workshop on Arrested Development of Nematodes of Sheep and Cattle. Central Veterinary Institute, Lelystad, Netherlands, pp. 7–17. Michel, J.F., Lancaster, M.B., Hong, C., 1976. The resumed development of arrested Ostertagia ostertagi in experimentally infected calves. J. Comp. Path. 86, 615–619. Nisbet, A.J., Cottee, P., Gasser, R.B., 2004. Molecular biology of reproduction and development in parasitic nematodes: progress and opportunities. Int. J. Parasitol. 34, 125–138.
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Prichard, R.K., Donald, A.D., Hennessy, D.R., 1974. The effect of corticosteroid treatment on the development of inhibited Ostertagia ostertagi larvae. Res. Vet. Sci. 13, 225–229. Prichard, R.K., Steel, J.W., Lacey, E., Hennessy, D.R., 1985. Pharmacokinetics of ivermectin in sheep following intravenous, intraabomasal or intraruminal administration. J. Vet. Pharmacol. Ther. 8, 88–94. Smith, W.D., Jackson, F.J., Jackson, E., Williams, J., 1983. Local Immunity and Ostertagia circumcincta: changes in the gastric lymph of immune sheep after a challenge infection. J. Comp. Path. 93, 479–488. Smith, W.D., Jackson, F.J., Jackson, E., Williams, J., Miller, H.R.P., 1984. Manifestations of resistance to ovine ostertagiasis associated with immunological responses in the gastric lymph. J. Comp. Path. 94, 591–601. Smith, W.D., Jackson, F.J., Graham, R., Jackson, E., Williams, J., 1987. Mucosal IgA production and lymph cell traffic following prolonged low level infections of Ostertagia circumcincta in sheep. Res. Vet. Sci. 43, 320–326.