H.J.S. Dawkins, T.A. Robertson, J.M. Papadimitriou and D.I. Grove

Transcription

1 Z Parasitenkd (1983)69: Zeitschrift fur Parasitenkunde Parasitorogy Research 9 Springer-Verlag 1983 Light and Electron Microscopical Studies of the Location of Strongyloides ratti in the Mouse Intestine H.J.S. Dawkins, T.A. Robertson, J.M. Papadimitriou and D.I. Grove Departments of Medicine and Pathology, University of Western Australia, Queen Elizabeth II Medical Centre, Nedlands 6009, Western Australia Abstract. This study was designed to define the precise anatomical location of Strongyloides ratti in the intestinal mucosa of the mouse. Light microscopy showed adult worms in vacuoles in close relationship with the columnar epithelium. Serial sections indicated that the adults wound their way circuitously through the mucosa, usually close to the crypts. Portions of worms were sometimes seen in the intestinal lumen. Electron microscopy demonstrated that adult worms were situated between the epithelial cells. They were never observed to penetrate the basement lamina and enter the lamina propria. Enterocytes were in close proximity to the cephalic end of worms, suggesting that the head of the moving worm forced the cells apart. More posteriorly along the worm, a fluidfilled vacuole surrounded the nematode. The surrounding epithelial cells were compressed and distorted but there was never any evidence of syncytial cell formation. The external cortical layer of worms was seen in some vacuoles, suggesting that ecdysis may occur in tunnels in the epithelium. It appears that S. ratti may create epithelial tunnels through which it moves, moults and deposits eggs. Since S. ratti is a mucosaldwelling parasite, it is susceptible to attack by cellular elements of the host's defences. Introduction During the course of an autopsy, Askanazy (1900) discovered that Strongyloides stercoralis invaded the intestinal wall. He noted that worms were found mostly in the crypts of Lieberkfihn; they were sometimes seen in the lumen of the crypt but were usually located in the glandular epithelium. Furthermore, he noted that parasites, especially embryos, were sometimes encountered in the lamina propria but rarely penetrated the muscularis Offprint requests to. D.I. Grove

2 358 H.J.S. Dawkins et al.

3 Intestinal location of Strongyloides ratti 359 Figs. 1-8, Sections of Strongyloides ratti in the intestine 5 days after infection. Arrows indicate portions of the same worm. Each scale bar represents 100 btm

4 360 H.J.S, Dawkins et al. mucosae. Confusion as to the precise location has led to textbooks merely stating that S. stercoralis invades the intestinal mucosa (Belding 1942; Faust et al. 1970; Wilcocks and Manson-Bahr 1972; Hunter et al, 1976). The location of S. ratti in the intestinal mucosa of rats is similarly uncertain. Abadie (1963) considered that these worms invaded and developed in the gut mucosa. Wertheim and Lengy (1965) thought that larvae burrowed into the mucosa rapidly after reaching the intestine, In contrast, Genta and Ward (1980) were of the view that adult worms were located deep in the crypts but did not penetrate the mucosa. We have recently shown that S. ratti infects certain inbred strains of mice (Dawkins et al. 1980; Dawkins and Grove 1981 a; Dawkins et al. 1981). We have used this model to investigate, by light and electron microscopy, the exact location of adult Strongyloides in the intestinal wall. Materials and Methods Animals. Female C57B 1/6J mice, g in weight, and female Sprague-Dawley rats, g in weight, were supplied by the Animal Resources Centre, Murdoch, Western Australia. Parasite. An homogonic strain of Strongyloides ratti was maintained by serial passage in rats as described previously (Dawkins et al. 1980). Mice were infected percutaneously with 3000 filariform larvae of S. ratti. The method of percutaneous infection has been described elsewhere (Dawkins and Grove 1981 a). The development of patent infections was confirmed by the presence of newborn larvae in the feces of mice 5 days after infection. Histology. Mice were killed by cervical dislocation 5 days after infection. Pieces of small intestine were removed from 5 cm and 10 cm below the pylorus and were then cut transversely into approximately 2 mm lengths. The tissue was fixed in 4% buffered formaldehyde solution (ph 7.2) at room temperature. The fixed tissues were embedded in wax and serial 5-6 gm sections were cut and stained with hematoxylin and eosin. Electron Microscopy. Mice were killed by cervical dislocation, the abdominal cavity opened and the small intestine separated from the mesentery by blunt dissection. The lumen of the small intestine was filled immediately with 2.5% glutaraldehyde in 0.05 M cacodylate buffer (ph 7.4) by injecting 2 ml into the duodenum using a syringe and a 23 gauge needle. The glutaraldehyde solution was also poured into the peritoneal cavity to assist in the rapid fixation of the intestinal tissues. The small intestine was then removed and placed in a petri dish containing glutaraldehyde solution. Approximately 1 mm transverse pieces were cut from the intestine 5cm and 10 cm below the pylorus. The samples were post-fixed in 1% osmium tetroxide in 0.1 M phosphate buffer (ph 7.4) for 1 h and then stained in 2% uranyl acetate for 20 min. Specimens were then dehydrated in graded alcohol solutions of ethanol and embedded in araldite. Sections were cut on a LKB III ultramicrotome, stained with lead hydroxide and then examined on a Philips 201 electron microscope at an accelerating voltage of 80 KV. Results Histology. Female adult Strongyloides ratti were found in the small intestine of mice. It is known that worms isolated from similarly infected animals measure mm in length (Dawkins and Grove 1981 a). Serial sections of intestine indicated that these worms were circuitously embedded in the

5 q Intestinal location of Strongyloides ratti jum ~.~ Fig. 9. Diagrammatic representation of S. ratti and its relative size to intestinal villi. The approximate positions are indicated from which sections a-h in Fig. I were taken O SCALE INjure i 9, " * mucosal tissues (Figs. 1-8). The sections pictured in this figure were taken at various intervals along the intestine (Fig. 9). In Fig. 1, the egg-bearing region of the worm was seen within the mucosa and near the crypts of Lieberkuhn. At least four pieces of the same parasite can be seen in this section. The helminth contained developing and developed eggs in utero and was surrounded by epithelium. A vacuole was evident around the worm while the surrounding epithelial cells were compressed. The same worm may be found both within the mucosa and in the lumen. Figures 24 show the worm completely surrounded by host tissue, whereas it can be seen free in the intestinal lumen and in contact with adjacent villi (Fig. 5-8). The tail of the worm was ultimately found in the lumen of the intestine approximately 216 lam from the section in Fig. 1. It was not common, however, to find parts of the helminth in the intestinal lumen. Adult worms were never seen in the muscularis. Electron Microscopy. The head of the worm was seen in close contact with the surrounding epithelial cells (Figs. 10 and 11). Figure 10 shows a complete cross-section of the anterior end of the worm and its location between several enterocytes. An electron-dense fibrillo-granular deposit was noted

6 362 H.J.S. Dawkins et al. Fig. 10. Cephalic region of adult S. ratti separating and distorting epithelial cells of the intestinal mucosa, c cuticle; ecru epithelial cell membrane; edd electron dense deposit; n nucleus. Arrows indicate epithelial cell membrane junctions. Scale bar represents 1 gm

7 Intestinal location of Strongyloides ratti 363 Fig. 11. Cephalic region of S. ratti showing the depth of the surrounding enterocytes. The lumen (lu) of the small intestine is evident, c cuticle; ecru epithelial cell membrane; n nucleus. Arrows indicate cellular interdigitations. Scale bar represents 1 gm

8 364 H.J,S. Dawkins et al. Fig. 12. Oesophageal region of the worm surrounded by a vacuole (v) in the epithelium. Several epithelial cells border the vacuole. Extensive cell compression and distortion is evident (arrows). c cuticle; edd electron dense deposit; lu lumen; n nucleus. Scale bar represents 1 gm

9 Intestinal location of Strongyloides ratti 365 Fig. 13. High magnification of the vacuolar membrane. The electron dense deposit (eda) on the epithelial cell membrane (ecru) of the vacuole (v) is evident. An autophagosome (a) is evident near the distorted nucleus (n) of the cell. Scale bar represents 1 pm

10 366 H.J.S. Dawkins et al. Fig. 14. Anterior portion of S. ratti still within a vacuole within the epithelium, ap anal pore; edd electron dense deposit and cell debris; lu lumen; v vacuole. Scale bar represents 1 gm

11 Intestinal location of Strongyloides ratti 367 Fig. 15. Vacuole containing a cuticular body from a moulted S. ratti worm. ecl external cortical layer of cuticle; edd electron dense deposit and cell debris; lu lumen; v vacuole. Arrows indicate epithelial cell interdigitations. Scale bar represents 1 gm

12 368 H.J.S. Dawkins et al. between the external cortical layer of the worm's cuticle and the cellular membrane of the surrounding epithelial cells. There was no evidence of syncytial formation and the worms were invariably surrounded by distinct mononuclear cells, most of which were epithelial. The distance between the enterocytes' contact with the worm and the microvilli at the luminal surface was of the order of several micrometres (Fig. 11). Cellular interdigitations between the opposed portions of epithelial cells were also evident. Figure 12 shows a cross-section of the worm through its posterior oesophageal region. There was a large, clearly defined vacuole surrounding the parasite. On the left of the electromicrograph, part of the vacuole surrounding another portion of this same worm can be seen. Compression, distortion and atrophy of the enterocytes adjoining the worm was seen in most profiles of the worm's habitat. The cellular compression and distortion, however, was particularly evident between the successive segments of the same worm as depicted in this figure. Several epithelial cells contributed to the border of the vacuole, which was invariably lined by an electron-dense fibrillogranular deposit. Again, cellular interdigitations were evident. Syncytia were not detected. Figure 13, taken from approximately the mid-length of the worm, shows a higher magnification of the border region of the vacuole. The latter was delineated by the plasma membranes of the compressed and distorted epithelial cells together with a layer of closely adherent electron-dense material. A section through the anal pore of the worm (Fig. 14) showed that the vacuole was still easily indentifiable at that level. A large deposit of electron-dense material and cellular debris was evident within the vacuole, albeit compressed to the vacuolar wall. The worm was, in all instances, positioned between the border of adjacent epithelial cells and was never seen within the cytoplasm of intact enterocytes or free in the lamina propria. Figure 15 shows a cuticular shell in a vacuolar tunnel. As with intact worms, electron-dense deposits were found in close relation with the epithelial cells. There was also some cell debris present in the vacuole. Discussion This study has clearly shown that Strongyloides does penetrate the intestinal mucosa and that adult female worms are located within the glandular epithelium. Larvae of S. ratti first reach the small intestine of mice h after percutaneous infection (Dawkins and Grove 1981 a; Dawkins et al. 1982). By 4 days after infection they moult twice to become adult female worms and soon produce rhabditiform larvae. Light microscopical examination of serial histological sections revealed that S. ratti wound its way through the mucosal tissues. Adult worms appeared to be in vacuoles situated in close relationship to the columnar epithelium. Occasionally, portions of adult worms were seen in the lumen of the intestine. In the instance illustrated in this paper, in which the posterior end of the worm was seen in the lumen, the vulval region of the nematode was embedded in the mucosa. Eggs seemed to lie in a similar location

13 Intestinal location of Strongyloides ratti 369 to the adult worms. This suggested that they might be deposited by adult worms in tunnels near the epithelial layer. On a light microscopical level, S. ratti appears to occupy a very similar niche to that of Trichinella spiralis. For example, Gardiner (1976) considered that adult T. spiralis, which were mostly found at the bases of the villi and in the crypts, were embedded in the epithelial layer of the mucosa. Despommier et al. (1978) thought that adult T. spiralis were located beneath the columnar epithelium and above the lamina propria. Furthermore, both demonstrated that larvae were released directly into the mucosa by adult worms. Recently, Wright (1979) examined the intestinal location of T. spiralis by electron microscopy. He considered that the adult worms were intracetlular rather than extracellular in location, and occupied both absorptive and goblet epithelial cells. Although our electron micrographs are very similar to those by Wright (1979) of T. spiralis, we have concluded that S. ratti adult worms lie between distorted, compressed and displaced columnar epithelial cells, with several enterocytes surrounding each worm. Interdigitations were noted between the epithelial cells surrounding worms, thus confirming their separate identity. Moreover, it would seem unlikely that an enterocyte could survive the continuous plasmalemmal disruption and direct continuity with the extracellular space that intracytoplasmic invasion would require. Electronmicrographs of the head end of the adult worm showed epithelial cells in close proximity to the worm cuticle and suggested that the cells were being forced apart. The worm thus appears able to fracture junctional complexes between adjoining enterocytes and move actively between them. As the nematode moved through the epithelium, however, a vacuole formed around the worm. The vacuole had several epithelial cells contributing to its wall. The actual vacuole was delineated by a single cell membrane from the surrounding enterocytes which often interdigitated with each other. It seems unlikely that the vacuole is an artifact, as it was not present around the cephalic end of the worm, yet in the same araldite section, vacuoles were evident around the posterior oesophageal region of another worm. It is reasonable to assume that the vacuole represents a fluid-filled space bathing the worm and results from its movement through the epithelium. There was a fibrillar and granular electron-dense deposit on the vacuolar surface of the enterocytes surrounding the worms. The nature of this deposit is uncertain, but may be derived from helminth, host or both. Vacant tunnels, similar to those occupied by the worms, were occasionally found in the epithelium of the small intestine. In some instances, unusual bodies were also found in these tunnels, which under high magnification had an identical structure to that of the external cortical layer of the cuticle of whole S. ratti worms. It would seem that these bodies are cuticles shed by worms moulting in epithelial tunnels. Thus, it appears that Strongyloides larvae moult within and move through the epithelium creating tunnels lined by enterocytes. It seems probable that they exit into the lumen and re-enter the epithelial layer. They fail, however, to traverse the basement lamina and do not enter the lamina

14 370 H.J.S. Dawkins et al. propria. Eggs could be deposited either directly into the lumen or in the epithelial tunnels and find their way through them into the intestinal lumen. These findings have implications for our understanding of the host defence mechanisms in strongyloidiasis. Adult Strongyloides are expelled spontaneously from the intestine in primary infection and more rapidly in secondary infections (Dawkins and Grove 1981 a). There is evidence to suggest that this expulsion is immunologically mediated (Dawkins and Grove 1981b). Since these worms are located in the intestinal mucosa between epithelial cells rather than within them, they are open to attack by inflammatory cells. Acknowledgements. Dr. Dawkins holds a Wyn Spence Fellowship for Medical Research. This study was supported by a grant from the National Health and MedicaI Research Council of Australia. References Abadie SH (1963) The life cycle of Strongyloides ratti. J Parasitol 49: Askanazy M (1900) Ueber Art und Zweck der Invasion der Anguillula intestinalis in die Darmwand. Centralblatt fiir Bakteriologie nnd Parasitenkunde und Infektionskrankheiten, Abteilung I. Originale 27: (translated In Tropical Medicine and Parasitology. Classic investigations (1978). Kean BH, Mott KE, Russell AJ, (eds). Cornell University Press, London p 332 Belding DL (1942) Text of Clinical Parasitology. Second edition. Appleton Century, New York Dawkins HJS, Grove DI (1981 a) K~netics of primary and secondary infections with Strongyloides ratti in mice. Int J Parasitol 11 : Dawkins HJS, Grove DI (1981b) Transfer by serum and cells of resistance to infection with Strongyloides ratti in mice. Immunology 43: Dawkins HJS, Grove DI, Dunsmore JD, Mitchell GF (1980) Strongyloides ratti: susceptibility to infection and resistance to reinfection in inbred strains of mice as assessed by excretion of larvae. Int J Parasitol 10: Dawkins HJS, Muir GM, Grove DI (1981) Histopathological appearances in primary and secondary infections with Strongyloides ratti in mice. Int J Parasitol 11: Dawkins HJS, Thomason HJ, Grove DI (1982) The occurrence of Strongyloides ratti in the tissues of mice after percutaneous infection. J Helminthol 56: Despommier DD, Sukhdeo M, Meerovitch E (1978) Trichinella spiralis: Site selection by the larvae during the enteral phase of infection in mice. Exp Parasitol 44: Faust EC, Russell PF, Jung RC (1970) Craig and Faust's Clinical Parasitology. Eighth edition. Lea and Febiger, Philadelphia Gardiner CH (1976) Habitat and reproductive behavior of Trichinella spiralis. J Parasitol 62: Genta RM, Ward PA (1980) The histopathology of experimental strongyloidiasis. Am J Pathol 99 : Hunter GW, Swartzwelder JC, Clyde DF (eds) (1976) Tropical Medicine Fifth edition, WB Saunders Company, Philadelphia Wertheim G, Lengy J (1965) Growth and development of Strongyloides ratti Sandground, 1925, in the albino rat. J Parasitol 51: Wilcocks C, Manson-Bahr PEC (1972) Tropical Diseases. Seventeenth edition. Bailliere Tindall, London Wright KA (1979) Triehinella spiralis: An intracellular parasite in the intestinal phase. J Parasitol 65: Accepted February t, 1983