HISTOPATHOLOGICAL APPEARANCES IN PRIMARY AND SECONDARY INFECTIONS WITH STRONG YLOIDES RATTI IN MICE

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1 International Journnl for Parasitolog~v. Vol. Il. pp Prrpamon Press Ltd Printed Great Britain. 0 Australian Society for Parasitology. OOZO-7519/81/ $02.00/O HISTOPATHOLOGICAL APPEARANCES IN PRIMARY AND SECONDARY INFECTIONS WITH STRONG YLOIDES RATTI IN MICE H. J. S. DAWKINS, G. M. MUIR and D. I. GROVE Department of Medicine, University of Western Australia and the Repatriation General Hospital, Nedlands, W.A (Received 29 May 1980) Abstract-HAWKINS H. J. S., MUIR G. M. & GROVE D. I. 198 I. Histopathological appearances in primary and secondary infections with Strongyloides rarti in mice. International Journal for Parasitology 11: The histological appearances of the skin, lungs and small intestines of mice with primary and secondary infections with S. ratfi are described. When the skins of mice with a primary infection were examined, larvae were seen scattered throughout the dermis. An inflammatory reaction of neutrophils and eosinophils was first noted around larvae I2 h after infection. By 48 h, mononuclear cells were prominent. The intensity of the inflammatory reaction gradually increased to a maximum on the fifth day and the larvae were destroyed. Very few larvae were seen in the lungs; those observed were located in the alveolar spaces and were not surrounded by an inflammatory infiltrate. Worms in the small intestines were found mostly in the crypts of Leiberkuhn, and were probably located within the epithelial layer; there was no significant villous atrophy or cellular infiltration. Marked differences were found in the tissues of mice with secondary infections. In the skin, oedema and neutrophils and eosinophils were seen around worms as early as 2 h after infection. By 24 h after infection, there was a mixed inflammatory infiltrate and worms were undergoing disintegration. Larvae in the lungs were surrounded by polymorphonuclear and mononuclear cells 48 h and 72 h after infection and the engulfed larvae were undergoing lysis. Only a few worms were seen in the intestines of mice with a secondary infection; the histological appearances were similar to that found in animals with primary infections. It is suggested that the rapid development of an oedematous reaction in the skins of immune mice may facilitate the entry of larvae into the bloodstream and that inflammatory cells destroy many larvae in the lungs of immune mice. INDEX KEY WORDS: Strongyloides rutfi; histology; inflammatory infiltrate; primary infection; secondary infection, skin; lungs; small intestines. INTRODUCTION IN THE preceding paper, we have quantitated the course of primary and secondary infections with Strongyloides vatti in C57B1/6 mice. It was shown that prior exposure to S. ratti induced considerable resistance to reinfection but the mechanism by which this occurred is uncertain. In order to provide a morphological background for these events, we now describe the histological appearances in the skin, lungs and small intestine at various times after primary and secondary infections of these animals with S. mtti. MATERIALS AND METHODS The animals and the parasite have been described previously (Dawkins &Grove, 19810). Thirty-three mice were infected percutaneously (p.c.) with 500 filariform larvae of S. rntti and patent infections were confirmed Address for correspondence: Dr. D. I. Grove, Department of Medicine, University of Western Australia, Nedlands, W.A seven days later by the demonstration of larvae in the faeces. Four weeks after the initial infection, 33 previously exposed mice and 33 control littermates were infected p.c. at the same site with 3000 unsterilised filariform larvae. At various intervals after infection, 3 mice from each group were killed by cervical dislocation and the organs removed. An area approx. 10x IO mm of the anterior abdominal wall was removed and placed in a 10% buffered formalin solution (BFS) at room temperature. Lungs, inflated with BFS, were removed with the heart intact and placed in BFS. The small intestine was removed and I cm sections of the gut were taken 10 cm from the pylorus and IO cm from thecaecum. Both piecesofintestinewereslit longitudinally, laid flat on a piece of filter paper, and fixed in BFS. After fixation, the tissue samples were mounted in paraffin blocks, and thin 5 pm sections were taken from 3 levels in each block. The sections were mounted on glass slides and stained with haematoxylin and eosin. RESULTS Skin Skins were examined 2, 12 and 24 h and 2,3,5,6,7 and 8 days after infection. Incontrol mice, larvae were seen throughout the dermis two hours after infection;

2 98 H. J. S. HAWKINS, G. M. MUIR and D. I. GROVE I.J.P. VOL they were not closely associated with the hair follicles suggesting that they may have penetrated the skin directly but no worms were actually seen in the process of penetration. At this stage there was no sign of oedema or inflammation around the larvae (Fig. I). By 12 h, after infection a clear space was noted around some worms (Fig. 2) while others were surrounded by a mild inflammatory infiltrate, composed mostly of neutrophils and eosinophils (Fig. 3). Twenty-four hours after infection many of the worms were engulfed by a leucocytic infiltrate, predominately neutrophils and eosinophils. A few worms showed signs of lysis. By 48 h, a marked in~anlmatory reaction containing more mononuclear cells, was noted around nearly all the worms; many larvae were undergoing lysis as evidenced by a hazy outline and a poor uptake of stain (Fig. 4). With increasing duration of infection the size of the inflammatory infiltrate and degrceofwormdisintegrationprogressivelyincreased. Five days after infection large granulomas composed of dense mononuclear cellular infiltrates were seen throughout the dermis and subcutaneous tissues but very few worms could be identified (Fig. 5). Thereafter the inflammation slowly subsided. In theskins of mice previously infected with S. rafti, there was a marked dif-terence in the speed and intensity of the in~am~atory response in comparison with mice receiving a primary infection. Two hours after infection, there were signs of oedema and many worms were surrounded by inflammatory cells consisting mainly of neutrophils and eosinophils (Figs. 6 and 7). By 12 h, the intensity of the cellular infiltrate had increased and in addition to the polymorphonuclear cells, there were now many mononuclear ceils. Most larvae were engulfed by the cellular infiltrate and signs of worm lysis were becoming apparent. By 24 h after infection there was a very dense mixed inflammatory infiltrate of neutrophils, eosinophils and mononuclear cells with the worms showing increased signs of disintegration (Fig. 8). By 48 h, the predominantly mononuclear cellular infiltrate was very dense throughout the dermis with large numbers of cells surrounding disintegrating worms. Similar observations were noted 3 to 5 days after infection; thereafter the inflammatory reaction gradually subsided. Lungs The lungs were examined 1 to 8 days after infection. Larvae were very sparse in the lungs of control mice; an occasional worm was noted in the lungs at 24 h, but more were seen 48 h after infection. The worms lay within the alveolar spaces and no inflammatory cells were present (Fig. 9). No larvae were seen at 72 h or beyond. In contrast, more larvae were found in the lungs of previously exposed animals. A few worms were seen at 24 h but there was no obvious inflammatory reaction. By 48 h, however, most of the larvae were surrounded by polymorphonuclear leucocytes (Fig. IO). Similar findings were noted after 3 days except that the cellular infiltrate was predominantly mononuclear. At both 48 and 72 h the worms were showing signs of disintegration. No larvae could be identified after 3 days. Smut1 intestines The small intestines were examined 3 to 8 days after infection. In control mice, most of the worms were seen in the anterior sections of the small intestines. Worms were in the intestinal mucosa when first examined 3 days after infection. They were seen in the bases of the crypts of Lieberkuhn (Fig. 1 I) and were probably located in the epithelial layer (Fig. 12). Worms were surrounded by a clear space; it is uncertain whether this is real or an artefact. Five days after infection a mild inflanin~dtory infiltrate, with prominent eosinophils, was observed in the lamina propria. There was no obvious flattening of the villi. By day 8 the inflammatory infiltrate had increased marginally and the worms themselves still appeared intact. Again there was no significant partial villous atrophy. Eggs were not seen in the mucosa. When immune mice were examined, the appearances of the small intestines were similar to that seen in primary infections except that very few worms were found. FIG. I. Skin 2 h after primary infection (Scale bar 50 pm). FIG. 2. Skin I2 h after primary infection (Scale bar 50 pm). FIG. 3. Skin 12 h after primary infection (Scale bar 50 pm). FIG. 4. Skin 48 h after primary infection (Scale bar 25 h&m). FIG. 5. Skin 5 days after primary infection (Scale bar 100 pm). FIG. 6. Skin 2 h after secondary infection (Scale bar 2.5 pm). FIG. 7. Skin 2 h after secondary infection (Scale bar 25 pm). Frc. 8. Skin 24 h after secondary infection (Scale bar 100 pm). FIG. 9. Lung 48 h after primary infection (Scale bar 25 pm). FIG. 10. Lung 48 h after secondary infection (Scale bar 25 pm). FIG. 11. Small intestine 6 days after primary infection (Scale bar 100 pm). FIG. 12. Small intestine 6 days after primary infection (Scale bar 25 pm).

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6 102 H. J. S. DAWKINS, G. M. MUIR and D. I. GROVE I.J.P. VOL. 1 I DISCUSSION The present histopathological study has provided an insight into the host reactions which occur during the first and second exposures to S. ratti. The only extensive study in which all stages of the life cycle of this organism have been examined is that of Abadie (1963). Furthermore, that report was restricted to the events which occurred during a primary infection with S. ratti in rats. We shall attempt to interpret the changes we observed in the light of the kinetics of infection described quantitatively in the previous paper. The mechanism by which Strongy/oides larvae penetrates skin is controversial. In perhaps, the first histological study, Van Durme (1902) considered that penetration of S. stercoralis via hair follicles appeared the most reasonable mechanism, but stated that he had never actually seen a single larva beside a hair. According to Goodey (1925), Kosuge (1924) found that, in the case of thin skinned animals, S. stercoralis gained entry via the epidermal scales, whereas in thick skinned animals, it was through the hair follicles. Abadie (1963), studying the penetration of thickskinned rat tails by S. ratti, considered that the hair follicle was the usual portal of entry. We observed larvae scattered throughout the dermis and only infrequently were they seen in close proximity to the hair follicles. This suggests that in the case of the relatively thin-skinned anterior abdominal wall of the mouse, infective larvae may be able to penetrate the epidermal scales directly. Indeed, direct penetration of the mouse skin by Nippostrongylus hrasiliensis has been shown by Lee (1972). Furthermore, we have shown in a scanning electron microscopical study that S. ratti infective larvae are able to penetrate the stratum corneum of new born mouse skin directly (Zaman, Dawkins & Grove, 1980). Nevertheless, these observations do not exclude the additional possibility of penetration via the hair follicles. There was no inflammatory reaction around worms in the first few hours after their penetration. By I2 h, a clear space was frequently observed around the larvae and this may indicate the secretion of proteolytic enzymes. Most worms appeared to have left the skin by 24 h, but the small proportion remaining were trapped and were ultimately destroyed by a surrounding inflammatory infiltrate. Very few larvae were found in the lungs, but those observed were located in the alveolar spaces and were not surrounded by an inflammatory reaction. Similar observations were made in rats infected with S. rotti (Abadie, 1963; Moqbel, 1980). Worms in the small intestine reside predominantly in the crypts of Lieberkuhn; they appeared to be situated within the epithelial layer, but ultrastructural studies are needed to define their location precisely. There was no significant partial villous atrophy, as has been described in other intestinal parasitic infections (Roberts-Thomson, Grove, Stevens &Warren, 1976). As in strongyloidiasis in rats (Moqbel, 1980), the inflammatory infiltrate was only moderate in degree. Marked differences in histological appearances were found when the skins of previously exposed animals were studied. Oedema and a mild inflammatory infiltrate were noted early after infection. This reaction may well have facilitated a more rapid departure of larvae from the skin. As with mice which were given a primary infection, a proportion of the larvae were trapped in the cutaneous tissues; the inflammatory reaction around these worms was accelerated and more intense. This contrast between primary and secondary infection has also been observed in rats (Moqbel, 1980). No significant inflammatory reaction occurred in primary infections, but a rapid infiltration of the dermis by eosinophils, polymorphonuclear leucocytes and mononuclear cells was seen in challenge infections. Similarly, marked changes were found in the lungs of previously exposed animals. Whereas no inflammatory reaction was ever seen around worms in the lungs of animals with a primary infection, an intense infiltrate was seen around worms in the lungs of previously exposed mice. Initially, the infiltrate was composed mainly of polymorphonuclear cells, then subsequently, mononuclear cells were in predominance. The worms within these granulomas were disintegrating and no worms could be indentified beyond the third day. Moqbel (1980) noted intense lymphoid infiltrates in the lungs in secondary infections with S. ratti in rats, but in contrast to our studies, did not see cells in contact with larvae. These observations confirm the hypothesis expressed in the previous paper, on the basis of quantitative changes, that many larvae were destroyed in the lungs of immune animals. Asmallproportionofwormsreached theintestines; they were also located in the epithelial layer of the crypts of Lieberkuhn. We were not able to discern any marked differences in the host reaction to them, either in terms of inflammatory infiltration or partial villous atrophy. In conclusion, we havedescribed the morphological changes which are seen around S. ratti in the skin, lungs and small intestines of mice with a primary infection. Marked differences were seen in animals with asecondary infection, and it is believed that these responses are consistent with rapid migration of larvae from the skin and destruction of many of them in the lungs. Acknowlrdgenlents-This study was supported by grants from the Rockefeller Foundation and the Australian Department of Veterans Affairs. We thank Mr. H. Upenieks for the photographic illustrations. REFERENCES ABADIE S. H The life cycle of Strongyloides ratfi. Journal qf Parasitology 49 : 24 I-248. DAWKINS H. J. S. & GROVE D Kinetics of primary and secondary infections with Strongyloides

7 I.J.P. VOL Histopathology of murine strongyloidiasis 103 ratti in mice. International Journal for Parasitology 11: primary, secondary and repeated infections of rats with Strongyloides ratti, with special reference to tissue eosinophils. Parmire fmmunology 2: I l-27. GOODEY T Observations on certain conditions requisite for skin penetration by the infective larvae of ROBERTS-THOMSON I. C., GROVE D. I., STEVENS D. P. & Strongyloides and ankylostomes. Journal of Helrnintho- WARREN K. S Suppression of giardiasis by the Ioav 3: intestinal phase of trichinosis in the mouse. Gut 17: KOSUGE I Histologische untersuchungen iiber das eindrigen von Strongyloides stercoralis in die Haut von VAN DURME P Quelques notes sur les embryons de versuchstieren. Archic fiir Schiffs und Tropenhygiene Sfrongyloides infesfinalis et leur p&n&ration par la peau. 28: Thompson Yates Laboratories Report 4: ZAMAN V., DAWKINS H. J. S. & GROVE D. I LEE D. L Penetration of mammalian skin by the Scanning electron microscopy of the penetration of infective larva of Nippostrongylus brasiliensis. Parasitonewborn mouse skin by Strongyloides rarti and logy 65 : Ancylostoma caninum larvae. Southeast Asian Journal OJ M~QBEL R Histopathological changes following Tropical Medicine andpublic Health. 11:

Effects of prednisolone on murine strongyloidiasis

Parasitology (1981), 83, 401-409 With 2 figures in the text Effects of prednisolone on murine strongyloidiasis D. I. GROVE and H. J. S. DAWKINS Department of Medicine, University of Western Australia and