Rat Model for the Human Intesinal Microsporidian Enterocytozoon bieneusi

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Rat Model for the Human Intestinal Microsporidian Enrerocyrozoon bieneusi ISABELLE ACCOCEBERRY, JEAN CARRIER% MARC THELLIER, SYLVESTRJ? BILIGUI, MARTIN DANIS, &WICK DATRY. Service de Parasitologie-Mycologie.Unite lNSERM313,CHU Pihe-SalpBtriere, Paris, France. Fig. 1 : Intensity of Enterocytozoon bienezcri spores fecal shedding in It is always an emergency to develop a reliable animal model or an experimentally infected Spragpe-Dawley adult rats immunosuppressed(ISC) or in v i f r o model for the human intestinal microsporidian Enterocytozoon bieneusi in order to assess a large number of drugs not (NISC) by corticotherapy. for prophylaxis or therapy, to investigate immunological modulation 9 of this infection and to study biochemical and taxonomic 8 1 T C B' I I characteristics of the parasite. The objective of our study is to establish a convenient adult rodent model of persistent E. bieneusi infection to assess potential anti-microsporidian compounds. The validity of the "Sprague-Dawley adult rats immunosuppressed with corticosteroids model" is supported by : i) it is used for studies of Pneumocystis carinii or cryptosporidiosis infections [1,3], ii) spontaneous microsporidiosis infections occur in several species 0 . D c u . m g z + = s including small rodents [8,10], microsporidia of genus Encephalitozoon and Nosema infect both human and non human o l i g g g hosts [2], iii) it is easily adaptable in laboratory. Days post- inoculation MATEFWLS AND METHODS. Fresh stools tiom HIV-infected patients containing microsporidia spores were stored in 2.5% (v/v) Intensity scare. number ofspora p a 100 rmcroscopil: field X 1.ooO mapfication. potassium dichromate solution at 4 C and concentration of spores Considering those f i s t result, four other protocols have been was obtained by a new filtration-centrihgation technic. Species identification of microsporidia was confirmed by TEM examination. settled in order to optimize the developed model. In the protocol 2, Immunosuppression was induced by a regimen of 25 mg of the immunosuppression by hydrocortisone acetate was reintbrced by hydrocortisone acetate injected subcutaneousely twice a week, 5 an hypoprotidic diet [1,14], and an immunosuppression by weeks before and during 6 weeks after the microsporidia challenge dexamethasone (0.25 mg/kg/d), added to dnnking water from 10 [ 11. Pneumocystosis prophylaxis was assured by administration in days before infestation to the end of the experimentation was drinlung water of trimethoprim (50 mgkg) and sulfamethoxazole evaluated [9]. We studied the influence of the nature of the (250 m a g ) per day [4]. Four groups of 6 adult male Sprague- inoculum [7,12] (protocol 3), of the inSectivity and level of Dawley rats (Janvier, France) were constituted, 6 non infestation (protocol 4), and the interference of the age or the non challenged (NISNC), 6 non murine species with the sensibility to infection [5,11] (protocol 5). immunosuppressed immunosuppressed challenged (NISC), 6 immunosuppressed non In spite of all the optimization assays, the Sprague-Dawley rat challenged (ISNC) and 6 immunosuppressed challenged (ISC). model, that is immunosuppressed by corticotherapy, whatever its Microsporidia inoculation was performed at day 0, by gastric sex, age or clone, seems to be not adapted to the evaluation of gavage, with a single dose of 4. lo6 spores in 1 ml volume. Spores therapeutic agents but remains the first example of a chronic shedding was evaluated quantitatively by counting spores under infection to E. bieneusi in small rodents. 1.000 X magnification on 100 fields, on total stools from each rats collected at days 0, 1,2, 3 and then every 2-3 days until day 40 and 1. Brasseur P, Lerneteil D, Ballet J. J. Clin. Microbiol. (1988) 26:1037. stained using : tluorochrome Uvibio stain [13], Weber's modified 2. Didier ES, Varner PW, Didier PJ, '4ldra.s AM, Milfichamp NJ, MurphcyCorb M, Bohm R Shadduck JA Folia Parasitol. Praho. (1994) 41:l. trichrome stain [6]. For the intestinal idection detection procedure, with rats sacrificed at different times after microsporidia challenge, 3. Frenkel KK, Good TT, Schults J. Lab. Invesr. (1966) 15:1559. duodenal and jejunal histological sections and imprints were 4. Hughes W T, IMCNabb PC, Makres TD, Feldman S. Anrrmicrob. Agents (1974a) 5:289. performed and examined after hematoxylin-eosin and Weber's 5 . Chemorher. Klesius PH, Hinds SE. Infict. Immun. (1979) 2 6 : l l l l . modified trichrome stairmg. 6. Kokoskin E, Gyorkos TW,Camus A, Cedilotte L. Furtill T, Ward B. J. RESULTS AND DISCUSSION. Rats shed spores from days 2 Clin.iUicrobio1.(1994) 32:1974. after destation but only developed a chronic and asymptomatic 7. Moon HW, Bemrick WJ. Vef.Porhol. (1981) 18:248. parainfection with a weak excretion of spores in feces (Fig. 1). 8 . Perrin TL, Bethesda UD. .4rch. Paih. (1947) 43559. In imprints obtained from all the 6 challenged rats necropsied at 9. Rehg JE,Hancock UL, Woodmansee DB. J. Infect Dis. (1988) 158:1406. day 40 post-inoculation, a few spores of E. bieneusi were observed 10. Shadduck JA, Watson WT, Pakss SP, Cali A J. Paranfoi. (1979) in the duodenum (2 rats), proximal jejunum (3 rats) or in the 3 1 1 . 65(1):123. Sherwood DK, Angus W, Snodgass DR, Tzipori S . Infect. Immun. (1982) different parts of the small intestine (1 rat). In parallel, the study of 38:471. histological sections, showed, at the same levels, extensive 12. Tzipori SK, Angus W, Gray EW, Campbell I, Allan F. Am. J. Vet. Res. vacuolization in the apical pole of enterocytes. Neither parasites, (1981) 43:1400. nor histological lesions were observed in the small intestine of 13. van Goo1 T, Snijdsn F, Reiss P, Eeftinck Schattenkerk JKM,van der Bergh Weeman l L 4 , Bartelsrnan JFWM, Bruins JJM, Canning EU, unchallenged control-rats necropsied at day 40. Dankert J. J. Clin. Parhol. (1993) 36:694. 14. Walzer PD, Powzll RD, Yoneda K, Rutledge ME, Milder JE. Infect Immun.( 1980) 27:925.