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Encephalitozoon Cuniculi, in an Experimental Model V INFECTION AND IMMUNITY, JUlY 1991, p. 2225-2231 Vol. 59, No. 7 0019-9567/91/072225-07$02.00/0 Copyright © 1991, American Society for Microbiology Enteric Infection with an Obligate Intracellular Parasite, Encephalitozoon cuniculi, in an Experimental Model V. WICHER,1* R. E. BAUGHN,2 C. FUENTEALBA,3 J. A. SHADDUCK,3 F. ABBRUSCATO,' AND K. WICHER' Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201-05091; Departments of Microbiology and Immunology and ofDermatology, Baylor College of Medicine, and Syphilis Research Laboratory, Veterans Administration Medical Center, Houston, Texas 772112; and College of Veterinary Medicine, Texas A&M University, College Station, Texas 7784344613 Received 20 December 1990/Accepted 4 April 1991 Downloaded from Rabbits were intrarectally infected with 3 doses (5 x 103 5 x 105, and 5 x 107) of an obligate intracellular parasite, Encephalitozoon cuniculi, with or without prior colonic lavages. Although chronic administration of enemas seems to interfere to some degree with the intestinal translocation of the parasite, systemic infection was observed in both manipulated and nonmanipulated animals. The animals responded with antibodies of immunoglobulin A (IgA) and IgG isotypes, reflecting the route of infection. They also produced significant amounts of circulating immune complexes composed of IgA and IgG antibodies and E. cuniculi antigens. Lesions compatible with encephalitozoonosis were seen in the liver, kidney, lung, and brain. In all instances, nonmanipulated animals had more severe lesions than manipulated rabbits given the same dose of parasites. http://iai.asm.org/ Levels of serum antibodies, circulating immune complexes, and histopathologic changes were associated with the infection dose. The presented data suggest that human microsporidiosis may also be transmitted via the rectal route. It is, therefore, of clinical relevance in view of several reports of microsporidian infections in patients with acquired immunodeficiency. Systemic infections with enteric microorganisms (proto- cuniculi is a microsporidian parasite that has a wide range of zoa, bacteria, and viruses) in homosexual and bisexual men hosts including rodents, lagomorphs, carnivores, and pri- were reported long before AIDS was recognized (10, 16, 20, mates (6) and the potential to affect multiple organs including 38). They are frequently associated with malabsorption, the brain, kidneys, liver, heart, and lungs (36). E. cuniculi on September 21, 2018 by guest diarrhea, weight loss, and anemia (18). Infection with com- infection has been extensively explored in rabbits (26), in monly recognized agents of sexually transmitted diseases which under normal immunological conditions it produces and enteric pathogens are traditionally associated with the only chronic asymptomatic brain and kidney lesions (37). rectal-oral route of transmission. Moreover, findings regard- The choice of this parasite is not capricious. There is ing several pathological processes affecting the liver of increasing evidence that members of the order Microsporida homosexual men with or without AIDS (7, 13) have stressed (genera Nosema, Enterocytozoon, Pleistophora, and En- the vulnerability of this organ as one of the major targets of cephalitozoon) may become important pathogens for immu- enteric infections. nosuppressed individuals, including AIDS patients (for re- Although it is questionable whether deterioration of the views, see references 5 and 35). immune system is the cause or consequence of infection with the human immunodeficiency virus, it is undisputable that homosexual men are the most affected by the disease (17, MATERIALS AND METHODS 29). Results of previous experimental studies suggested that Organisms. E. cuniculi was grown in primary rabbit em- long-term colonic lavages (43, 44) and intrarectal (i.r.) insem- bryo fibroblast cultures in minimal essential medium and 5% ination (31, 44, 45) may selectively predispose the host to fetal calf serum as previously described (37). The organisms immunosuppression. Chronic colonic lavages promoted an used for inoculation of rabbits were freshly collected from increase in the humoral response to soluble (seminal fluid culture supernatants after centrifugation at 400 x g for 30 and bovine serum albumin) and particulate (spermatozoa min. After two washes in sterile phosphate-buffered saline and sonicated sheep erythrocytes) antigens and the produc- (PBS), the organisms were resuspended in PBS, counted in tion of immune complexes (43-45). The implication is that a hemacytometer, and adjusted to the desired concentration. this kind of manipulation may increase the host susceptibil- Animal treatment and infection. Adult (5 to 6 months old) ity to sensitization with multiple enteric antigens and to male New York State giant Flemish rabbits from a closed infection with opportunistic and pathogenic microorganisms. colony raised at our center since 1935 were used for these Among other risk factors, enema/douche use before sex experiments. The animals were free of Coccidia, Pas- have been associated with human immunodeficiency virus teurella, and E. cuniculi infections. Three groups of eight infection in two cohort studies involving 2,507 (17) and rabbits each were submitted to five colonic enemas (30 ml of approximately 5,000 (22) homosexual men. Fleet formula) per week for a period of 3 months as previ- To further delineate the contributory role of the enteric ously reported (43). Three groups containing a similar num- route to systemic infection, we have chosen as a marker the ber of animals were not manipulated. After that period, obligate intracellular parasite Encephalitozoon cuniculi. E. manipulated (60 to 90 min after colonic lavage) and nonma- nipulated animals were inoculated i.r. with 5 x 103, 5 x 105, or 5 x 107 E. cuniculi. The various doses of the parasite were * Corresponding author. given in a single 1-ml volume deposited in the rectum with a 2225 2226 WICHER ET AL. INFECT. IMMUN. 3 1/2 Fr. Tom Cat catheter (Sherwood Medical, St. Louis, proteins were then electrophoretically transferred to nitro- Mo.) to a depth of approximately 5 cm. Colonic lavages were cellulose paper at 100 V and 220 mA for 1 h in 25 mM discontinued after infection. The rabbits were bled before Tris-192 mM glycine-20% methanol by using a Mini-Trans- and periodically after inoculation. Four animals in each blot (Bio-Rad Laboratories, Richmond, Calif.). After a experimental group and two age-matched uninfected con- blocking step with 1% bovine serum albumin (BSA) in PBS trols were sacrificed by intravenous (i.v.) injection of 1 ml of containing 0.02% Tween 20 (PBS-T) for 1 h, the blots were sodium pentobarbital (Somlethol; J. A. Webster, Inc., North reacted overnight (18 h) at room temperature with rabbit Billerica, Mass.) 2 months postinfection, and the remaining anti-E. cuniculi serum (dot-ELISA titer, 1:10,240) at a dilu- experimental animals and five noninfected controls were tion of 1:100. After several washes with PBS-T, the blots sacrificed approximately 4 months postinfection. were probed with the second antibody, horseradish peroxi- Animal procedures have been approved by the Institu- dase-conjugated goat anti-rabbit IgG, and finally developed tional Animal Care and Use Committee of the Wadsworth with H202 and 4-chloro-1-naphthol (HRP-Color Developer; Center for and Research. to the recommendation of the manufac- Laboratories Bio-Rad) according Downloaded from Histopathology. Tissue samples of the brain, liver, kidney, turer. Controls included PEG-precipitated material from lung, and intestine were fixed in 10% neutral buffered preinfection samples and normal rabbits. Molecular weight formalin. Tissue blocks were embedded in paraffin, and standards (Bio-Rad) were run in parallel, and the Mrs of the sections were stained with hematoxylin and eosin. Lesions polypeptides were calculated (42). were evaluated microscopically according to the following (vi) Identification of antibodies. Solubilized E. cuniculi grading system: ±, very mild; +, mild; + ±, mild to moder- polypeptides were obtained by a modification of the method ate; + +, moderate; + + ±, moderate to severe; and + + +, reported by Irby et al. (15). Briefly, three-times-washed severe. Gram stain was applied to tissue sections with spores (108) in PBS were spun down and the pellet was lesions to search for organisms. resuspended in 0.1 ml of 1% Triton X-100 and boiled for 10 Immunologic techniques. (i) Dot-ELISA. Serum samples min, after which 0.4 ml of sample buffer containing 2% SDS http://iai.asm.org/ and positive and negative controls were serially diluted and and 5% P-mercaptoethanol in 0.06% M Tris-HCl buffer, pH examined in duplicate in an enzyme-linked immunosorbent 6.8, was added and the mixture was boiled again for another assay (ELISA) (27), using dots of formalin-fixed E. cuniculi. 10 min. A volume of 135 pI of solubilized antigen (2.7 x 107 The dots were developed with horseradish peroxidase-con- spores) was added to individual preparative 10% SDS-PAGE jugated goat anti-rabbit immunoglobulin G (IgG) (Zymed slab minigels. Following electrophoresis, proteins were Laboratories, San Francisco, Calif.) and H202. The tech- transferred to nitrocellulose paper for identification of pos- nique has been described elsewhere in detail (3). Serum sible anti-E. cuniculi antibodies in DIC. Sheets were cut into dilutions causing the development of clearly defined dark separate strips, and duplicate strips
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