R espiratory tract cryptosporidiosis in immunosuppressed 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 D10 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 ryptosporidiose pulmonaire du rat immunodéprimé 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, etJ14 après l'infestation. A J1 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, Cryptosporidium parvum. rat. appareil respiratoire. C ryptosporidium parvum is a protozoa respon- sible for opportunistic chronic infection in immunocompromised hosts with an intestinal tropism (Current and Garcia, 1991). Extra digestive and extra biliary manifestations were uncommonly observed in man, and most of them involve the respi- ratory tract (Brady et al ., 1984; Forsacs et al., 1983; Goodstein et al., 1989; Hojyling and Jensen, 1988; Kibbler et al., 1987; Kocoshis et al., 1984; Ma et al., 1984; Travis et al., 1990; Weitz et al., 1986). In birds, C. baileyi is responsible for respiratory infections (Blagburn et al., 1987). Respiratory cryptosporidiosis * Laboratoires d’Histologie, ** Laboratoire de Parasitologie Expérimentale, *** Laboratoire d'Anatomie Pathologique B, Groupe de recherches ERPUR, CHU de Rouen F-76000. **** Laboratoire d'immunologie et d'Immunopathologie, CHU de Caen, F- 14033. Correspondance : Philippe Brasseur, Laboratoire de Parasitologie, Hôpital Charles Nicolle, Centre Hospitalier Universitaire, F-76031 Rouen cédex. Tél. : 35 08 80 15 - Fax : 35 08 80 17. was not often reported in mammal models (Meulbroek et al., 1991). In this study, we document the lesions observed in a immunosuppressed rat model of respiratory crypto- sporidiosis. Sprague Dawley rats weighting between 300 and 350 g and free of C. parvum infection were immuno- suppressed as previously described (Brasseur et al., 1988). Briefly, rats were fed a low protein (7%) diet (exclusively white bread) and given a regimen of 25 mg of hydrocortisone acetate injected subcuta- neously twice weekly, five weeks before and two weeks after C. parvum challenge (i.e. from D35 to D14). Sulfamethoxazole (30 mg/kg/day) and trime- thoprim (6mg/kg/day) (Eusaprim®, Wellcome, France) were given in drinking water from D35 to D3 to avoid Pneumocystis carinii infection. Animals were challenged at D0 with human C. parvum sporo- zoites. Oocysts were obtained from the stools of a Parasite, 1995, 2, 85-87 N ote de recherche 85 Article available at http://www.parasite-journal.org or http://dx.doi.org/10.1051/parasite/1995021085
3
Embed
Respiratory tract cryptosporidiosis in immunosuppressed ... · Respiratory tract cryptosporidiosis in immunosuppressed RAT IS ASSOCIATED WITH AN EPITHELIAL METAPLASIA. ROUSSEL F.*,
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
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
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
Parasite, 1995, 2, 85-87 Note de recherche 8 5
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.
cryptosporidiosis in acquired immune deficiency syndrome. Jou rn a l o f the A m erican M edical A ssociation, 1984, 252, 89-90.
B rasseur P., Lem eteil D. & B allet J.J. Rat model for human cryptosporidiosis. Jou rnal o f Clinical Microbiology, 1988, 26, 1037-1039.
B u ra u d M., F o r g e t E., F a v en n e c L., B iz e t J . , D e lu o l A.M. & G o b e r t J .G . S e x u a l s ta 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 aC O2 c e ll lin e . Infection an d Immunity, 1991, 59 , 4610-4613.
C u r r e n t W.L. & G a rc ia L.S. Cryptosporidiosis. C linical Microbiology Reviews, 1991, 4, 325-58.
F o r s a c s P . , T a r s h is A ., M a P . , F e d e r m a n M ., M e l e L., S ilverman M .L. & Shea J.A. Intestinal and bronchial cryptosporidiosis in an immunodeficient homosexual man. Annals o f Internal Medicine, 1983, 99, 793-794.
G o o d ste in R.F., C o lo m b o C .S ., Illfelder M.A. & S k ag gs R.E. Bronchial and gastrointestinal cryptosporidiosis in AIDS. Jou rn al o f the American Osteopathic Association, 1989, 89, 195-197.
H ein e J. Eine einfache nachweismethode für Kryptospo- ridien im Kot. Zentralblatt fü r Veterinaermedizin, 1982, 29, 324-327.
H o jylin g N. & J ensen B.N. Respiratory cryptosporidiosis in HIV-positive patients. The L an cet , 1988, i i , 590-591.
K ib b l e r C.C., S m ith A ., H a m ilto n - D u t o it S .J . , M ilbu rn H .,
P a ttin so n J .K . & P ren tice H .G . Pulmonary cryptosporidiosis occuring in a bone marrow transplant patient. Scandinavian Jou rn a l o f Infectious Disease, 1987, 19, 581-584.
K o c o sh is S.A., C ibull M.L., D avis T.E., H into n J.T., Se ip M. & B anwell J.G. Intestinal and pulmonary cryptosporidiosis in an infant with severe combined immune deficiency. Jo u rn a l o f P ed iatric G astroenterology a n d Nutrition, 1984 , 3, 149-157.
M a P., V il l a n u e v T . G . , K a u f m a n D. & G il l o o l e y J.F . Respiratory cryptosporidiosis in the AIDS. Journal o f the American Medical Association, 1984, 252, 1298-1301.
M eu lbr o ek J.A., Novilla M .N . & C urrent W.C. An immunosuppressed rat model of respiratory cryptosporidiosis. Jou rnal o f Protozoology, 1991, 38, 113S-115S.
R ig g s M.W. & P erryman L.E. : Inactivation and neutralization of Cryptosporidium parvum sporozoïtes. Infection an d Immunity, 1987, 55, 2081-2087.
T r a v is W.D., S c h m id t K., M a c L o w r y J.D ., M a s u r H., Co n d ro n K.S. & Fojo A.T. Respiratory cryptosporidiosis in a patient with malignant lymphoma. A rchives o f Pathology an d Laboratory Medicine, 1990, 114, 519-522.
W eitz J.C., T assara R., M u n o z P., M erca do R. & A ttias R. : Cryptosporidiosis del aparato respiratorio. Revista Medica de Chile, 1986, I 14, 691-692.
Accepté le 13 octobre 1994
REFERENCESB la g bu r n B.L., L in d sa y D.S., G ia m b r o n e J.J., S un d erm a n n
C.A. & H o er r F.J. Experimental cryptosporidiosis in broiler chickens. Poultry. Science, 1987, 66, 442-449.
B rady E .M ., M arg olis M .L. & K o rz en io w sk i O .M . Pulmonary