Trichostrongylus colubriformis infection in rabbits: Persistence of the distal adaptive response to parasitism after anthelmintic treatment

j . Comp. Path. 1995 Vol. 113, 145 153

Trichostrongylus colubriformis Infection

in Rabbits: P e r s i s t e n c e of the Distal Adaptive R e s p o n s e to P a r a s i t i s m after Anthelmintic Treatment H. H o s t e , S. M a l l e t a n d C. K o c h

Institut National de la Recherche Agronomique, Centre de Tours, Station de Pathologie Aviaire et de Parasitologie, F37380 Nouzil~, France

Summary Mucosal changes associated with anthelmintic treatment in Trichostrongylus colubriformis-infected rabbits were assessed along the entire length of the small intestine. The following groups, each of five rabbits, were compared: infected (group I); infected and treated on day 21 with fenbendazole (group IT); uninfected but given fenbendazole on day 21 (group C, controls). All animals were killed on day 28. In the proximal part of the small intestine of group I rabbits, the worms were associated with shortening of the villi and a significant depletion in alkaline phosphatase activity, which differed significantly from the findings in groups C and IT. In the same region, no difference was found between groups C and IT. In the distal small intestine, hypertrophy of villi and crypts (an adaptive response to the infection), coupled with an increase in enzymic activity, were present in both groups I and IT, in contrast to group C. These results suggest that a complete mucosal restoration occurred within 7 days of anthelmintic treatment in the parasitized part of the intestine. In contrast, the adaptive response observed beyond the main site of parasitism was not abolished by treatment. The functional significance of these findings is discussed in relation to the compensatory growth commonly observed after anthelmintic treatment in ruminants. 9 1995 Academic Press Limited

Introduction Studies on rabbits experimentally infected with the ruminant nematode Trichostrongylus colubriformis showed that, beyond the main site of infection in the small intestine, there was a region capable of adaptation to parasitism (Hoste et al., 1988). This adaptation, characterized by hypertrophy of the villi and crypts, may compensate for decreased nutrient absorption in the proximal region caused by worm damage to the mucosa (Sykes, 1978; Nielsen, 1982). The effect of antiparasitic treatment on the restoration of intestinal mucosa parasitized with T. colubriformis has been examined previously in sheep by Angus et al. (1979) and Angus and Coop (1984). These earlier workers described changes in the parasitized (i.e., proximal) region of the intestine, but did not report on possible changes in the adaptive, distal region. Information on such distal changes might be relevant to the compensatory growth that usually occurs after anthelmintic treatment (Horton, 1977). The present report describes a 0021-9975/95/060145+09 $12.00/0

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histological a n d b i o c h e m i c a l study o f the entire small intestine o f e x p e r i m e n t a l rabbits, designed to assess the effect o f anthelmintic t r e a t m e n t on the reg e n e r a t i o n o f m u c o s a d a m a g e d or c h a n g e d by T. colubriformis infection. Materials and Methods

Rabbits Fifteen male New Zealand White rabbits, aged 7 weeks, were reared indoors under parasite-free conditions, and given a feed concentrate. The rabbits were divided randomly into three groups of five, namely: group I (infected), group I T (infected, then treated with anthelmintic), and group C (control; uninfected but given anthelmintic).

Experimental Procedure On day 0, the rabbits in groups I and I T received 9000 T. colubriformis third-stage larvae (L3) in 5 ml of water by gastric cannula. The larvae had been cultured from eggs in faeces collected from an experimentally infected sheep, as described by Hubert and Kerboeuf (1984). On day 0, the rabbits in group C received 5 ml of water. On day 21, the rabbits in groups I T and C were given fenbendazole (FBZ) (Panacur ND; Distrivet Ltd) 5 mg/kg. On day 28, all rabbits in the three groups were killed by intracardiac injection of sodium pentobarbital (Dolethal ND; Vetoquinol Ltd). Immediately after death, two samples (A and B) of 2 cm length were taken from each of five segments of small intestine, at 50, 100, 150, 200 and 250 cm from the pylorus, for subsequent determination of histological (sample A) and biochemical (sample B) parameters. The small intestines of the rabbits in groups I and IT were divided into five 50-cm segments and frozen at --20~ for worm counts. After thawing, the segments were flushed with saline and the luminal contents collected. A 10% volume of these contents was used to estimate the total worm burden of each rabbit. The sex and stages of development of T. colubriformis were identified according to Douvres (1957).

Histological Examination Samples collected from five segments of small intestine (see above) for histological examination were opened and fixed in aqueous Bouin's fluid for 2 days. After dehydration, the tissues were trimmed longitudinally and embedded in paraffin wax; sections (5 gin) were then cut and stained with haematoxylin and eosin. Four separate slides were prepared from each sample, for histological measurements. The two measurements made were (1) the mean villous length (indicative of the mucosal absorptive surface), calculated on 40 villi/segmental sample, and (2) the mean crypt surface area (indicative of the intensity of the proliferative process in glands of Lieberkt~hn), measured on 40 crypts. The measurements were made with an Analytical Measuring System VIDS III (Shirehill Industrial Estate, Saffron Walden, UK).

Biochemical Examination The intestinal segments for biochemical determinations (see above) were treated as previously described (Hoste and Mallet, 1990) to determine alkaline phosphatase (AP) activity. Briefly, they were first washed with 0" 15 M NaCI and then stored at - 20~ in 5 ml of saline. After thawing, the samples were homogenized with an Ultra Turrax Homogenizer (Janke and Kunkel KG, Staufen in Breisgau, Germany). After centrifugation (15min at 4000g), the supernates were stored in 2ml aliquots at -20~ until assayed. Protein concentrations were measured in a BCA (bicinchoninic acid) protein assay by a micromethod, with 0"01 ml samples of test material and bovine serum albumin

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A d a p t i v e R e s p o n s e t o T. c o l u b r i f o r m i s

Table 1 D i s t r i b u t i o n o f T. eolubriformis along the e n t i r e l e n g t h of the s m a l l i n t e s t i n e in g r o u p I and group IT rabbits

Segment of smaU intestine

Mean number of worms+S.D. in ~ I

I 2 3 4 5

832 + 268"4 740-1-250 668___271-8 528_+750 108_+ 194

~ 15 ___12-9 12• 18-9 25___30 10 -

as a reference (Pierce Technical Bulletin, 1984). Absorbance was read at 550 nm with a Titertek Multiscan MC Spectrophotometer (Flow Laboratories, McLean, USA). AP activity was assayed by incubating 0-1 ml of test material (1 in 10 dilution of the supernate) with 0"2 ml of substrate (8.8 ~mol of p-nitrophenyl phosphate per ml of glycine buffer, pH 8"8) at 37~ The yellow coloured product of the reaction (pnitrophenol) was measured directly at time intervals with the Titertek Multiscan MC Spectrophotometer at 405 nm. Results were expressed as Ix mol of p-nitrophenol released/h/cm of intestine.

Statistics The Student's t-test was used for statistical analysis of the data. In addition, correlation coefficients were calculated between the length of villi, surface of crypts, AP activity and the distance from the pylorus.

Results

Worm Populations The mean worm burden of the parasitized group I was 2876 adult worms, which represented approximately 32% of the original dose. T. colubriJbrmis worms were distributed evenly in the first four segments, and a residual population (approximately 4% of the total number) was found in the last intestinal segment, 250 cm from the pylorus (Table 1). In group IT, a mean burden of 62 worms was calculated at necropsy, which represented 0"69% of the infective dose administered and a drug efficacy of 97-8%.

Protein Contents and Enzymatic Activity No significant difference was found between the three groups of rabbits in the mucosal protein content except between group C and the other two groups

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at 150 cm from the pylorus (P<0"02) (Fig. la). By comparison with the control group C, the AP activity was lower in group I at 50 (P<0.07) and 100 (P<0"02) cm from the pylorus. In contrast, in the distal intestine (200 and 250 cm from the pylorus), the AP activity was increased in group I, though only in the last segment was this difference significant (P<0"05) (Fig. lb).

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In group IT, the AP activity did not differ from that of the controls in the proximal intestine (50 and 100cm from the pylorus), but was significantly higher than in the group I animals. In contrast, distally, at 200 and 250 cm from the pylorus, no statistical difference was found between groups IT and I; however, the AP activity in group IT was significantly higher than in group C at 200 cm (P<0"01) (Fig. lb).

Histological Measurements In the group I rabbits, a significant shortening of villi was observed in the proximal small intestine, 50 and 100 cm from the pylorus (P<0"01). In contrast, villous hypertrophy occurred in the distal region, representing a 16"9 to 24"5 % increase above the control values; the difference was significant at 200 cm from the pylorus (P<0"05) (Fig. 2a). The modifications in villous morphology observed in the group I rabbits were associated with a significant hypertrophy of the glands of Lieberktihn along the entire length of the small intestine (Fig. 2b). Minor histological differences were found between groups C and IT in the proximal small intestine. In contrast, distal to the second segment, increases in villous size, ranging from 18"5 to 22"2%, were observed. These increases were close to significance at 150cm from the pylorus (P<0"06); they were associated with non-significant increases in the crypt surface (Fig. 2b). In groups C and IT, there was a significant negative relationship between the mean length of villi and the distance from the pylorus (group C: r--- -- 0.896, d.f.=22, P<0.01; group IT: r----0"675, d.f.=23, P<0.01). In contrast, a significant positive relationship was found in group I (r= + 0"468, d.f. = 22, P<0-05). There was no correlation between the crypt surface and the distance from the pylorus in any of the three groups of rabbits. Discussion

The study showed that the distal adaptive response to nematode parasitism persisted after anthelmintic treatment, whereas in the proximal region of the intestine the mucosa returned to normal. The rate of establishment and intestinal distribution of T. colubriformis were as previously observed in rabbits (Bezubik et al., 1988; Hoste et al., 1988). Moreover, the 97"8% reduction in worm population after anthelmintic treatment confirmed the efficacy of FBZ against adult T. colubriformis in rabbits (Herlich, 1976). The histological and enzymatic changes in the proximal part of the T. colubriformis-infected small intestine corresponded to those previously observed in sheep and rabbits (Barker, 1975a and b; Jones, 1983; Hoste et al., 1988). After FBZ treatment, no morphological or enzymological differences were detected between the control and infected groups in the proximal intestine. This finding indicated a complete restoration of the mucosa within 7 days ofanthelmintic administration. The rate of recovery was rapid compared

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Comparison of the histological measurements of the intestinal mucosa in group C (V'l), group I ([]), and group IT (m) rabbits. (a) Villous length. (b) Crypt surface area. Mean values-l-standard deviation (five animals/group). T h e presence of different superscripts indicates a significant difference between groups (P<0"05) (Student's t-test). Shared superscript indicates absence of difference.

with that observed in previous studies in chronically infected sheep, especially in regard to the epithelial alkaline phosphatase activity (Angus et al., 1979; Jones 1983). This discrepancy may have been the result of differences in the level and mode of infection. Indeed, a relationship between the duration of the pathogenic stimuli and the rate of recovery of the mucosa has been

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reported in coccidiosis of chickens (Michael, 1974; Enigk and Dey Hazra, 1976). On the other hand, it is also possible that differences in the rate of mucosal restitution in nematode infection are associated with differences in response in host species. The hypertrophy of villi and crypts in the distal intestine was similar to the adaptive response previously described in nematode-infected rabbits (Hoste et al., 1988, 1993). However, an increase in the epithelial AP activity was not observed in T. colubriformis-infected rabbits (Hoste and Mallet, 1988). This difference may have been related to a difference in the stage of infection. However, in nematode infections in rodents and ruminants, a similar increase in brush border enzyme activity, beyond the main site of parasitism, has also been described (Dharsana et al., 1976; Weesner et al., 1988; Smith and Lloyd, 1989). The functional significance of this process remains unclear but may be related to an increase in the digestive capacity of the distal intestine. In the distal small intestine the histological and enzymatic changes in group IT did not differ from those in group I but were significantly different from those in group C. This indicated that the mucosal changes due to infection persisted after anthelmintic administration, in contrast to the response to treatment in the proximal parasitized region. In avian coccidiosis, a villous hypertrophy has also been observed distally during infection and was maintained during the recovery phase (Fernando and McCraw, 1973). However, this differed from the present study with nematodes in that the proximal infected intestine had not yet returned to normal morphology. The present results were acquired only at one time point after drug administration and it is therefore difficult to give them any functional interpretation. However, it is important to emphasize that distal compensatory absorption in parasitized sheep has often been suggested (Symons, 1976; Sykes, 1978; Nielsen, 1982). Moreover, structural evidence for an intestinal adaptive response, with characteristics comparable with those observed in rabbits, has also been shown in sheep (Roy et al., 1994). Examination of the mucosal recovery along the entire length of the small intestine in sheep, after anthelmintic treatment, might help to explain the compensatory growth known to occur after anthelmintic treatment in ruminants. Sprinz (1962) suggested that the intestinal mucosa tends to respond nonspecifically to various stimuli, and Riecken (1988) classified these mucosal structural responses into the following three types: "mucosal atrophy", which corresponds to the damage caused by the action of antimitotic agents or irradiation on stem cells in the glands of Lieberktihn; "mucosal hypertrophy and hyperplasia", as found in the distal gut remnant after a proximal resection; and "mucosal transformation of the hyper-regenerative type", which occurs in situations where hyper-proliferation of the mucosa is associated with lesions of the surface epithelium, as in coeliac disease. According to this classification, the changes found in the proximal intestine in T. colubriformis infection corresponded to a mucosal transformation of the "hyper-regenerative type". On the other hand, beyond the main site of parasitism, the modifications were similar to the "mucosal hyper-proliferation type". With this model, it is possible to control the number and distribution

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of T. colubriformis in rabbits, and, by use of anthelmintics, to eliminate the worms. Consequently, this experimental model may prove valuable in studying these two types of responses in the intestinal mucosa, their inter-relationship, and the rate of recovery after treatment. Acknowledgments

Special thanks are due to Dr R. Gasser, Department of Veterinary Science, Melbourne University, for helpful criticism of the manuscript. References

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I Received, January 5th, 1995] Accepted, March 24th, 1995J