Rabbit model for human intestinal microsporidia

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J . EUK. MICROBIOL., VOL. 44, NO. 6, NOVEMBER-DECEMBER 1997

Rabbit model for human intestinal microsporidia ISABELLE ACCOCEBERRY, PIERRE GREINER MARC THELLIER ABDERRAHIM ACHBAROU, SYLVESTRE BILIGUI, MARTIN DANIS, ANNICK DATRY. Service de Parasitologie-Mycologie,Unite I N S E M 313. CHU Pitre-SalpLtrrere, Paris. France.

Microsporidia are emerging as opportunistic pathogens in patients with AIDS. Enterocytozoon bieneusi and Encephalitozoon intestinalis have been implicated in enteric infections in AIDSpatients with chronic diarrhea, a wasting syndrome and malabsorption [2,5]. Many features of the infection are poorly defined and the lack of an effective therapy has prevented experimentation in humans. Thus, we try to establish the infection in immunosuppressed laboratory rodents. Adult immunosuppressed Sprague-Dawley rats acquire the infection which can last until day 40 post-inoculation but low excretion of spores make them insuitable for many research purposes. One explaination of this, should be the too short enterocyte life duration in rats (2 days). So, in this study, we investigate the infection in young immunosuppressed laboratoy rabbits : the same life duration (5 days) of human and rabbit enterocytes guide our choice, this rabbit turn-over enterocyte would be capable of doing better conditions for developing an E. bieneusi infection. MATERIALS AND METHODS. Four weeks old New-Zealand female rabbits (Charles hver, France) free of microsporidia spores at the start of the experiment were used. E. bieneusi spores were obtained from feces of HN-infected patients. Feces containing spores were collected and concentrated by a new filtrationcentrifugation technic. We obtained stock cultures of E. intestinalis grown in Rabbit Kidney Cells (RK13 cell line) from T. van Gool (Department of ficrobiology, Academic Medical Center, Amsterdam) [9]. Immunosuppression was induced by 4 m a g of dexamethasone acetate injected intramuscularly twice a week, 2 weeks before and during 6 weeks after microsporidia challenge [3,8]. Four groups of 3 New-Zealand rabbits were studied : group A immunosuppressed and challenged with E. intestinalis. group B immunosuppressed and challenged with E. bieneusi, group C immunosuppressed and challenged with E. bieneusi after an antibiotic digestive decontamination, group D non immunosuppressed one non challenged, one challenged with E. bieneusi and one with E. intestinalis. Spores inoculation was performed, after overnight starvation, by gastric gavage, with a single dose of 4.10' spores of E. intestinalis or lo7 spores of E. bieneusi in 2 ml volume. Total stools from each rabbit were collected at days 1, 2, 3 and then every 2-7 days until day 40. Quantitative E. bieneusi or E. intestinalis shedding was measured by counting spores under 1.000 X magnification on 100 fields using tluorochrome Uvibio and Weber's modified trichrome stain methods [4,lo]. For anti-parasites antibodies detection, sera collected from all rabbits at days 15 and 30 were tested on E. intestinalis spores by immunofluorescence assays and western blot analysis and on E. bieneusi spores by immunofluorescence assays [6,7]. immediately after death, tissue sections were taken from duodenum, jejunum, ileon, liver, spleen, and brain, then stained using the hematoxylin-eosin, trichrome, Giemsa, silver stain methods [ l l ] . Immunohistochemistry was performed on tissue sections using a mouse monoclonal anti serum to E. intestinalis and peroxydase coupled goat anti-mouse Ig (Dako, Trappes, France) [I]. RESULTS AND DISCUSSION The challenged animals began to shed spores 48 at 72 hours after inoculation. All the animals continued to shed spores until the end of the experiment at day 40, however from day 3 no animal shed more than 2 to 3 spores per 100 MF.

The mean value of shedding was a little higher in group B than in groups C and A, but the difference was not significant at each steps of the comparison. A western blot analysis was performed on sera from rabbits 1 and 12 challenged with E. intestinalis and immunosuppressed or not by corticotherapy. The antigenic reaction, negative at day 15 post-inoculation, revealed at day 30 one antigen or a group of antigens, which had a molecular mass range of 55-60 kDa. Rabbit anti-serum raised against E. intestinalis was the positive control and normal rabbit serum was the negative control. For the immunofluorescence assays : on E. intestinalis spores, all the E. intestinalis challenged rabbits had positive serologic test results at days I5 and 30, with a bright fluorescence of spores wall stronger at day 30. The E. bieneusi challenged rabbits had negative serologic test results. On E. bieneusi spores, all the E. intestinalis challenged rabbits had negative serologic test results at days 15 and 30. The serologic test results, for the E. bieneusi challenged rabbits, were uninterpretabie probably for lack of purification of spores used as antigen. At necropsy, macroscopic examination showed an hepatomegaly in all the immunosuppressed rabbits. Histological studies and immunohistochemistry analysis, never revealed the presence of microsporidia or infection sites. Even though the animals were infested at day 0 by E. bieneusi or E. intestinalis they only developed an asymptomatic chronic infection, which could be seen through a very small fecal excretion of microsporidia spores, from day 2 to day 40 post-inoculation. In E. bieneusi challenged rabbits, the infestation appeared softer than among Sprague-Dawley adult rats. The antibiotic digestive decontamination, performed 24 hours before challenge, did not further the spores implantation in the enterocytes. Serologic analysis of E. intestinalis challenged rabbit sera allowed us to observe a seroconversion for E. intestinalis. No cross reaction was revealed with the E. bieneusi challenged rabbit sera. 1. 2. 3. 4.

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