R e s p ir a t o r y t r a c t c r y p t o s p o r id io s is in im m u n o s u p p r e s s e d RAT IS ASSOCIATED WITH AN EPITHELIAL METAPLASIA

ROUSSEL F.*, LEMETEIL D.**, FAVENNEC L.**,****, TAYOT J .***, BALLET J.J.**** & BRASSEUR P.**

Summary :

Cryptosporidium parvum is an opportunistic protozoa that chroni­

cally infects the digestive tract of immunocompromised hosts.

Respiratory cryptosporidiosis, which was reported in AIDS

patients, is an uncommon feature of mammalian cryptosporidiosis

models. In this study, we document the respiratory lesions obser­

ved in an immunosuppressed rat model of cryptosporidiosis.

Twenty rats were immunosuppressed with corticosteroids and low

protein diet. They were challenged intratracheally with 106

C. parvum sporozoites. Lungs and ileums were examinated on

D3, D6, D10, D14. On D 10 and D14, C. parvum were present

in the respiratory tract of all animals in association with the pro­

gressive appearance of an immature malpighian metaplasia. On

D14, an intestinal infection was also detected in 2 /4 animals.

The respiratory tract appears to be a fully permissive area for the

protozoa in immunosuppressed rats. Introduction of parasites on

the respiratory mucosa seems a requisite to induce respiratory

cryptosporidiosis. This experimental protocol yields a low mortality

rate, and so modelizes late and/or chronic stages of respiratory

cryptosporidiosis.

KEY WORDS : immunosuppression, corticosteroids. Cryptosporidium parvum. rat. respiratory tract.

Abbreviations : DMEM : Dulbecco Modified Eagle Medium. D : Day.

Résumé : La C r y p t o s p o r id io s e p u lm o n a ir e d u r a t im m u n o d é pr im é

EST ASSOCIÉE À UNE MÉTAPLASIE ÉPITHÉLIALE

Cryptosporidium parvum est un protozoaire opportuniste respon­

sable d'infections chroniques de l'appareil digestif chez les hôtes

immunodéprimés. Une cryptosporidiose respiratoire a été décrite

chez des patients atteints de SIDA mais est une manifestation rare

de la cryptosporidiose expérimentale des mammifères. Nous décri­

vons ici les lésions respiratoires d'un modèle de cryptosporidiose

chez le rat immunodéprimé. Vingt rats immunodéprimés par traite­

ment aux corticoïdes et régime carencé en protéines ont été infestés

par voie intratrachéale à l'aide de 106 sporozoïtes de C. parvum.

tes poumons et les iléons sont étudiés à J4, J6, J 10, et J14 après

l'infestation. AJ10 et à J14, C. parvum est retrouvé dans l'appareil

respiratoire de tous les animaux, et sa présence est associée à

l'apparition progressive d'une métaplasie malpighienne immature. A

J14, deux rats sur quatre présentent également une cryptosporidiose

intestinale. L'arbre bronchique apparaît donc réceptif à C. parvum

chez le rat immunodéprimé, et la voie aérienne d'introduction des

parasites au niveau de la muqueuse respiratoire semble indispen­

sable au développement d'une cryptosporidiose respiratoire. Le pro­

tocole décrit ici, qui s'accompagne d'un faible taux de mortalité,

constitue un modèle qui reproduit les stades tardifs et/ou chro­

niques de la cryptosporidiose respiratoire.

MOTS CLES : immunosuppression, corticostéroïdes, Cryptosporidium parvum.

rat. appareil respiratoire.

C ryptosporid ium p arv u m is a protozoa respon­sible for opportunistic chronic infection in immunocompromised hosts with an intestinal tropism (Current and Garcia, 1991). Extra digestive

and extra biliary m anifestations w ere uncom m only observed in man, and most o f them involve the respi­ratory tract (Brady et a l ., 1984; Forsacs et al., 1983; Goodstein et a l., 1989; Hojyling and Jen sen , 1988; Kibbler et al., 1987; Kocoshis et al., 1984; Ma et al., 1984; Travis et al., 1990; Weitz et al., 1986). In birds, C. b a i le y i is responsible for respiratory infections (Blagburn et al., 1987). Respiratory cryptosporidiosis

* L a b o r a to ire s d ’H is to lo g ie , ** L a b o r a to ire d e P a ra s ito lo g ieExpérim entale, *** Laboratoire d'Anatom ie Pathologique B, G roupede recherches ERPUR, CHU de Rouen F-76000.**** Laboratoire d 'im m un ologie et d 'Im m unopathologie, CHU de Caen, F- 14033.Correspondance : Philippe Brasseur, Laboratoire de Parasitologie, Hôpital Charles N icolle, Centre H ospitalier Universitaire, F-76031 Rouen cédex. Tél. : 35 08 80 15 - Fax : 35 08 80 17.

w as n o t o fte n re p o rte d in m am m al m o d els (Meulbroek et al., 1991).

In this study, we document the lesions observed in a immunosuppressed rat model o f respiratory crypto­sporidiosis.

Sprague D aw ley rats w eighting betw een 300 and 350 g and free o f C. p arv u m infection were immuno­su p p ressed as previou sly d escrib ed (B rasseu r et al., 1988). Briefly, rats were fed a low protein (7%) diet (exclusively white bread) and given a regimen of 25 mg o f hydrocortisone acetate injected subcuta- neously tw ice w eekly, five w eeks before and two w eeks after C. p a rv u m challenge (i.e. from D35 to D 14). Sulfam ethoxazole (30 mg/kg/day) and trime­th o p rim (6m g/kg/day) (E u sap rim ® , W e llco m e , France) w ere given in drinking water from D35 to D3 to avoid P neum ocystis c a r in ii infection. Animals were challenged at D0 with human C. p arv u m sporo­zoites. Oocysts w ere obtained from the stools o f a

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Article available at http://www.parasite-journal.org or http://dx.doi.org/10.1051/parasite/1995021085

ROUSSEL F., LEMETEIL D„ FAVENNEC L , TAYOT J„ BALLET J.J. & BRASSEUR P.

Fig. 1. - Large bronchi at D 10. Cilia are absent from m any cells. Som e o f them are bearing apical parasites, closely resem bling the intestinal parasitism (arrow h ead s). Som e foam y m aterial (arrow ) contain ing m acrop h ages is filling som e bron chial lum ens. (Stain: H em atein-Eosin-Saffron. Bar: 20 m m .)

single AIDS patient attending the medical clinic o f the hospital, purified in a sucrose gradient, and excysta- ted as previously described (Buraud et a l., 1991). Parasites in suspension were washed in DMEM and sporozoites were separated from oocysts by filtration through 5 (Am filters. Seventeen animals were challen­ged intratracheally with 106 sporozoites. The trachea of anaesthetized animals was punctured with a 18G needle. A 22G catheter was inserted through the needle and blocked in a medium size bronchus of the right lung, and sporozoites were instillated in 2 0 0

|Al warm saline. In three control animals, a sham ins­tillation (saline) was performed. At D2, two control and 16 challenged animals survived. They were ran­domized for programmed sacrifices at D3, D6 , D 10, and D14. Oocysts shedded in stools were counted as p rev iou sly (H ein e , 1982; B rasseu r e t a l . , 1988). Control rats w ere killed at D14. All anim als w ere sacrificed by ether overdose. The trachea, the lungs, the terminal portion of the ileum were fixed in 1 0 % buffered paraform aldehyde, em bedded in paraffin and sectionned (4 μm). Sections w ere stained with Hematein-Eosin-Saffron.

On D3, no oocyst was detected in stools. A few para­sites were observed in the respiratory tract o f two out of four rats. They appeared tightened to the cilia of the respiratory epithelial cells. No C. p a r v u m was seen in the ileum.

On D 6 , some parasites were seen in the trachea of 2/4 rats. Oocysts were present in the feces o f 1/4 ani­mal, and small numbers o f C. p a rv u m oocysts were also observed in the ileal mucosa.

On D 10 (Fig. 1), C. p a rv u m were present in the respi­ratory tract o f 4/4 rats. Their presence was restricted

Fig. 2. - Trachea at D14. The respiratory epithelium is metaplastic. T h e a p ica l ce lls are b ea r in g n u m ero u s p aras ites (a rro w h ea d s). (Stain : Hem atein-Eosin-Saffron. Bar: 10 mm.)

to the trachea and the main bronchus. In one animal, a foam y material filled the bronchial lum ens. The parasites w ere scarce in three anim als, in one rat however, high densities o f C. p a r v u m w ere locally found in both the respiratory tract and the ileum, and associated with low grade oocyst shedding.

On D14 (Fig. 2) very high densities o f C. p a r v u m were found in the respiratory tracts o f 3/4 animals, of which two exhibited parasites in the ileum and shed oocysts in the stools. Parasites were mainly located in the epithelium of the trachea, and of the hilar and lobar bronchi. In the proximal territories, the density o f parasites was high. The epithelium bearing the parasites was transformed with immature, non kerati­nizing, malpighian metaplasia (Fig. 2). Densities o f parasites was much lower in zones with residual cilia­ted cells. In more distal bronchi, epithelial cells were characterized by a patchy loss o f apical ciliary expen­sions. Some parasites were attached on residual cilia. The alveolar related areas were filled with a retentio­nal foamy material containing macrophages. In one animal very low numbers o f parasites were seen in the respiratory tract, and the aspect was close from what was observed on D10.

Non specific findings consisted mainly o f aspergillo- mas : 1/4 at D3, 1/4 at D10 and 2/4 at D14. A pleural involvement was observed in 2/4

In our previous experiments, respiratory infestation of digestive origin was never observed in more than one thousand rats challenged per os. For the study o f res­piratory cryp tosp orid iosis, w e have d esign ed an experimental protocol which required intra-bronchic sporozoite instillation, and resulted in a respiratory infection in 4/4 animals. Thus introduction of para-

Parasite, 1995, 2, 85-8786 Note de recherche

R e s p ir a t o r y c r y p t o s p o r id io s is in r a t

sites on the respiratory mucosa seems a requisite to induce respiratory cryptosporidiosis.

In 2/4 animals, an intestinal infection was also detec­ted. The occurence o f a digestive infestation from the respiratory tract can be considered as an evidence of com plete intrabronchic parasite cycles since a puri­fied sporozoite preparation was administered intratra- cheally, and it has been shown that sporozoites per os do not in fect the ileum (Riggs and Perrym an, 1987). The presence o f some contaminating oocysts in sporozoite inocula cannot be excluded however. The time interval before the appearance of parasites in the feces was longer than after per os challenge, which is also consistent with the ingestion of oocysts from the trachea and further developm ent o f the parasite in the gut.

Altogether data confirm that the respiratory tract is a fully permissive area for C. p arv u m . In our model, the permissive territory consists more specifically of ciliated respiratory epithelium. Persisting Cryptospori­d iu m infestation was associated with the progressive appearance of an immature malpighian metaplasia (D 10, D14). Such lesions were not present in normal and immunosuppressed rats (Brasseur et al., 1988). Malpighian metaplasia is generally considered as a response to local irritations and it is remarkable that in in fected rats, m etap lastic areas exh ib ited the highest parasite density. Thus m etaplastic process does not seem to contribute to protection.

In our model, the development o f the bronchic infec­tio n se e m e d s lo w e r th an in th e rat m o d el o f M eu lbroek e t a l . (1 9 9 1 ). This w as presum ably a co n seq u en ce o f the use o f sporozoites instead of oocysts. Our protocol yields a low mortality rate, and m odelizes late and/or chronic stages o f respiratory cryptosporidiosis. In both models, data indicate that it is possible to mimick features of the human respira­tory cryptosporidiosis in experimental animals.

ACKNOWLEDGEMENTS

We thank Mrs J . Elouard for her skilful technical assistance. The work was sup­ported by grants from ANRS (1993) and a

subvention from Lions’ Club, Deauville-Trouville and Fondation Luc, Deauville, France.

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Accepté le 13 octobre 1994

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