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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 10 6 C. parvum sporozoites. Lungs and ileums were examinated on D3, D6, D 10, 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 : D M EM : Dulbecco Modified Eagle Medium. D : Day.

Résumé : La Cr y p t o s p o r id io s ep u l m o n a ir ed u r a tim m u n o d é p r 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. A J 1 0 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,

rat. appareil respiratoire.

C

sible for o pportun istic ch ron ic in fectio n in im m unocom prom ised hosts with an intestinal

tropism (Current and G arcia, 1991). Extra digestive and extra biliary m anifestations w ere uncom m only

observed in man, and m ost o f them involve the respi­

ratory tract (Brady et a l ., 1984; Forsacs et al., 1983;

G ood stein et a l., 1989; H ojyling and Je n se n , 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 il e y i is re sp o n sib le fo r respiratory in fectio n s (Blagburn et al., 1987). Respiratory cryptosporidiosis

* L a b o r a to ir e s d ’H is to lo g ie , ** L a b o r a to ir e d e P a r a s ito lo g ie Expérim en tale, *** Laboratoire d'A natom ie P ath olog iq u e B, G roupe d e re ch erch es ERPUR, CHU de R ouen F -76000.

**** Lab oratoire d 'im m u n o lo g ie et d 'Im m u n o p ath o lo g ie, CHU de Caen, F- 14033.

C orresp on d an ce : P hilippe B rasseu r, Laboratoire d e Parasitologie, H ôpital C harles N icolle, C en tre H osp italier U niversitaire, F -76031 R ouen céd e x . T él. : 35 0 8 8 0 15 - Fax : 35 0 8 8 0 17.

w as n o t o fte n re p o r te d in m am m al m o d e ls (M eulbroek et al., 1991).

In this study, w e docum ent the lesions observed in a im m unosuppressed rat m odel o f respiratory crypto­

sporidiosis.

Sprague D aw ley rats w eigh tin g b etw ee n 3 00 and 350 g and free o f C. p a r v u m infection w ere immuno­

su p p re ssed as p re v io u sly d e sc rib e d (B ra sse u r et al., 1988). Briefly, rats w ere fed a low protein (7% ) diet (exclusively white bread) and given a regimen o f 25 mg o f hyd rocortisone acetate injected subcuta- neou sly tw ice w eekly, five w eeks b efo re and tw o w eeks after C. p a r v u m challenge (i.e. from D35 to D 14). Sulfam ethoxazole (30 mg/kg/day) and trim e­

th o p rim (6m g / k g / d ay ) (E u s a p rim ® , W e llc o m e , France) w ere given in drinking w ater from D35 to D3 to avoid P n eu m o cy stis c a r in ii infection. Animals w ere challenged at D0 with human C. p a r v u m sporo­

zoites. O ocysts w ere obtained from the stools o f a

Parasite, 1995, 2, 85-87

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Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1995021085

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

Fig. 1. - Large b ro n ch i at D 1 0 . Cilia are ab sen t fro m m any cells.

Som e o f th em are bearin g apical parasites, closely resem b lin g the in testin al p arasitism (a rro w h e a d s). S o m e fo am y m aterial (a rro w ) co n ta in in g m a cro p h a g e s is filling so m e b ro n c h ia l lu m en s. (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 d escribed (Bu rau d et a l., 1991).

Parasites in suspension w ere w ashed in DMEM and sporozoites w ere separated from oocysts by filtration through 5 (Am filters. Seventeen animals w ere challen­

ged intratracheally with 106 sporozoites. The trachea o f anaesthetized animals was punctured with a 18G n eed le. A 22G cath eter w as in serted throu gh the needle and blocked in a medium size bronchus o f the right lung, and sporozoites w ere instillated in ^{2 0 0}

|Al warm saline. In three control animals, a sham ins­

tillation (saline) w as performed. At D2, two control and 16 challenged animals survived. They w ere ran­

domized for programmed sacrifices at D3, D6, D 10, and D14. O ocysts shedded in stools w ere counted as p re v io u sly (H e in e , 1 9 8 2 ; B ra s s e u r e t a l ., 1 9 8 8 ).

C ontrol rats w ere k illed at D 14. All anim als w ere sacrificed by ether overdose. The trachea, the lungs, the terminal portion o f the ileum w ere fixed in 1 0% bu ffered p araform ald ehyd e, em bed d ed in paraffin and section ned (4 μm). Sections w ere stained with Hematein-Eosin-Saffron.

On D3, no oocyst was detected in stools. A few para­

sites w ere observed in the respiratory tract o f two out of four rats. They appeared tightened to the cilia of the respiratory ep ithelial cells. No C. p a r v u m was seen in the ileum.

On D6, som e parasites w ere seen in the trachea o f 2/4 rats. Oocysts w ere present in the feces o f 1/4 ani­

mal, and small num bers o f C. p a r v u m oocysts were also observed in the ileal mucosa.

O n D 10 (Fig. 1), C. p a r v u m w ere present in the respi­

ratory tract o f 4/4 rats. Their presence was restricted

Fig. 2. - T rach ea at D 14. T h e respiratory epithelium is m etaplastic.

T h e a p ic a l c e lls a re b e a r in g n u m e ro u s p a ra s ite s (a r ro w h e a d s ).

(Stain : H em atein-Eosin-Saffron. Bar: 10 m m .)

to the trachea and the main bronchus. In one animal, a foam y m aterial filled the bro n ch ial lum ens. T h e parasites w ere scarce in three anim als, in o n e rat how ever, 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.

O n D 14 (Fig. 2) very high densities o f C. p a r v u m w ere found in the respiratory tracts o f 3/4 animals, o f which two exhibited parasites in the ileum and shed oocysts in the stools. Parasites w ere mainly located in the epithelium o f the trachea, and o f the hilar and lobar bronchi. In the proximal territories, the density o f parasites w as high. T he epithelium bearin g the parasites was transformed with immature, non kerati­

nizing, m alpighian m etaplasia (Fig. 2). D ensities o f parasites was much low er in zones with residual cilia­

ted cells. In more distal bronchi, epithelial cells w ere characterized by a patchy loss o f apical ciliary exp en ­ sions. Some parasites w ere attached on residual cilia.

The alveolar related areas w ere filled with a retentio­

nal foamy material containing m acrophages. In one animal very low num bers o f parasites w ere seen in the respiratory tract, and the aspect was close from w hat was observed on D10.

Non specific findings consisted mainly o f aspergillo- mas : 1/4 at D3, 1/4 at D 10 and 2/4 at D14. A pleural involvement was observed in 2/4

In our previous experim ents, respiratory infestation o f digestive origin was never observed in more than one thousand rats challenged per os. For the study o f res­

p irato ry cry p to sp o rid io sis, w e h av e d esig n e d an experim ental protocol w hich required intra-bronchic sporozoite instillation, and resulted in a respiratory infection in 4/4 animals. Thus introduction o f para-

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Re s p ir a t o r yc r y p t o s p o r i d io s i sin r a t

sites on the respiratory mucosa seem s a requisite to induce respiratory cryptosporidiosis.

In 2/4 animals, an intestinal infection was also detec­

ted. The occurence o f a digestive infestation from the respiratory tract can be considered as an evidence o f com plete intrabronchic parasite cycles since a puri­

fied sporozoite preparation was administered intratra- cheally, and it has been show n that sporozoites per os do not in fect the ileum (R iggs and Perrym an, 1987). The presence o f som e contaminating oocysts in sporozoite inocula cannot be excluded however.

The time interval before the appearance o f parasites in the feces was longer than after per os challenge, w hich is also consistent with the ingestion o f oocysts from the trach ea and further d ev elo p m en t o f the parasite in the gut.

Altogether data confirm that the respiratory tract is a fully perm issive area for C. p a r v u m . In our m odel, the permissive territory consists more specifically o f ciliated respiratory epithelium. Persisting C ryptospori­

d iu m infestation was associated with the progressive ap p earance o f an im m ature m alpighian m etaplasia (D 10, D 14). Such lesions w ere not present in normal and im m unosuppressed rats (Brasseur et al., 1988).

M alpighian m etaplasia is generally consid ered as a response to local irritations and it is remarkable that in in fe c te d rats, m e ta p la stic are as e x h ib ite d th e h igh est p arasite density. Thus m etaplastic p rocess does not seem to contribute to protection.

In our model, the developm ent o f the bronchic infec­

tio n s e e m e d s lo w e r th a n in th e rat m o d e l o f M eu lb ro e k e t a l . ( 1 9 9 1 ). T h is w as p resu m ab ly a co n se q u e n ce o f the u se o f sp o ro zo ites instead o f oocysts. Our protocol yields a low mortality rate, and m odelizes late and/or chron ic stages o f respiratory cryptosporidiosis. In both models, data indicate that it is possible to mimick features o f the human respira­

tory cryptosporidiosis in experim ental animals.

ACKNOWLEDGEMENTS

W

e thank Mrs J . E lou ard fo r h er skilfu l technical assistance. The work was sup­

ported by grants from ANRS (1993) and a subvention from Lions’ Club, Deauville-Trouville and Fondation Luc, Deauville, France.

cryptosporidiosis in acquired immune deficiency syn­

drome. Jo u r n a l o f the A m erican M edical A ssociation , 1984, 252, 89-90.

Bra sseu r P., Le m eteil D. & BalletJ.J. Rat model for human cryptosporidiosis. Jou rn a l o f Clinical Microbiology, 1988, 26, 1037-1039.

B u r a u d M., F o r g e t E., F a v e n n e c L., B i z e t J . , D e l u o l A.M. &

G o b e r t J . G . S e x u a l s t a g e d e v e lo p m e n t o f C ry p to sp o rid ia in th e C a C O

2

Cu r r e n t W.L. & Ga r c ia L.S. Cryptosporidiosis. C lin ical Microbiology Reviews, 1991, 4, 325-58.

Fo r s a c s P ., Ta r s h i s A ., Ma P ., Fe d e r m a n M ., Me l e L.,

Silverman M .L . & Sh eaJ.A. Intestinal and bronchial cryp­

tosporidiosis in an immunodeficient homosexual man.

Annals o f Internal M edicine, 1983, 99, 793-794.

Go o d s t e in R.F., Co l o m b o C .S ., IllfelderM.A. & Sk a g g s R.E.

Bronchial and gastrointestinal cryptosporidiosis in AIDS.

Jo u rn a l o f the A m erican Osteopathic Association, 1989, 89, 195-197.

He in e J. Eine einfache nachweismethode für Kryptospo- ridien im Kot. Zentralblatt fü r Veterinaermedizin, 1982, 29, 324-327.

Ho jy l in g N. & Jen sen B.N. Respiratory cryptosporidiosis in HIV-positive patients. The L a n cet, 1988, i i , 590-591.

Kib b l e r C.C., Sm it h A ., Ha m il t o n- Du t o it S .J ., Mil bu r n H ., Pa t t in so n J .K . & Pr e n t ic e H .G . Pulmonary cryptospori­

diosis occuring in a bone marrow transplant patient.

S can din avian Jo u rn a l o f Infectiou s Disease, 1987, 19, 581-584.

Ko c o s h is S.A., CibullM.L., DavisT.E., Hin to nJ.T., Se ipM. &

BanwellJ.G. Intestinal and pulmonary cryptosporidiosis in an infant with severe combined immune deficiency.

J o u r n a l o f P ed ia tric G astroenterology a n d Nutrition, 1984 , 3, 149-157.

Ma P., Vil l a n u e v T . G . , Ka u f m a n D. & Gi l l o o l e y J.F . Respiratory cryptosporidiosis in the AIDS. Jou rn al o f the American M edical Association, 1984, 252, 1298-1301.

Me u l br o e kJ.A., Novilla M .N . & Cu rr en t W.C. An immuno­

suppressed rat model of respiratory cryptosporidiosis.

Jou rn a l o f Protozoology, 1991, 38, 113S-115S.

Rig g sM.W. & Perrym an L.E. : Inactivation and neutralization of Cryptosporidium parvum sporozoïtes. Infection a n d Immunity, 1987, 55, 2081-2087.

Tr a v i s W .D., Sc h m i d t K., Ma cLo w r y J.D ., Ma s u r H.,

Co n d r o n K.S. & Fojo A.T. Respiratory cryptosporidiosis in a patient with malignant lymphoma. A rchives o f Pathology a n d Laboratory Medicine, 1990, 114, 519-522.

We it zJ.C., Tassara R., Mu n o z P., Me r c a d o R. & At tia s R. : Cryptosporidiosis del aparato respiratorio. Revista M edica de Chile, 1986, I 14, 691-692.

Accepté le 13 octobre 1994

REFERENCES

Bl a g b u r n B.L., Lin d s a y D.S., Gia m b r o n e J.J., Su n d er m a n n

C.A. & Ho e r rF.J. Experimental cryptosporidiosis in broi­

ler chickens. Poultry. Science, 1987, 66, 442-449.

Br a d y E .M ., Ma r g o lis M .L . & Ko r z e n io w s k i O .M . Pulmonary

Parasite, 1995, 2, 8 5-87

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