A new microsporidium is reported infesting the enterocytes of a Haitian patients with AIDS. The stages observed were diplokaryotic cells, sporogonial plasmodia, unikaryotic sporoblasts, and spores. Neither a sporophorous vesicle (pansporoblastic membrane) nor parasitophorous vacuole were differentiated around the developmental stages, which were in direct contact with the host cell cytoplasm. The polar tube (5-6 coils) was differentiated before fission of the sporogonial plasmodium. The mature spores measured 1.5 micron X 0.5 micron. The spore wall was very thin as the endospore was absent or poorly differentiated. The organism is named Enterocytozoon bieneusi n. g., n. sp. and is assigned to the suborder Apansporoblastina.

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An animal model for Clostridium butyricum necrotizing cecitis has been developed in axenic chickens inoculated orally between 2 and 50 days of life. Cecitis was obtained with two C. butyricum strains isolated from neonatal necrotizing enterocolitis and not with a Clostridium beijerinckii strain from dairy products; the rate of colonization of the intestinal tract by this strain was lower than that obtained with C. butyricum strains. The clinical findings showed a slow gain in body weight. The cecitis lesions were well developed 3 and 4 weeks after oral inoculation, including enlargement with an increase of the cecum weight-body weight ratio, a marked hyperplasia, congestion, inflammatory infiltrate and pneumatosis of the cecal wall and mesentery, hemorrhage in the lamina propria and submucosa, and ulcerations and necrotic areas in the mucosa. By immunofluorescence and electron microscopy, the bacterial cells were located in the cecal lumen and in necrotic areas of the mucosa. The presence of 4% lactose in the diet seemed to be a prerequisite for the development of cecitis in chickens. A gradual rise of fluorescent antibodies in the sera was observed. Howard et al. (6) reported the presence of Clostridium butyricum in the blood of 9 of 10 and in the stools of 6 of 10 newborns with neonatal necrotizing enterocolitis (NNE). The authors concluded that C. butyricum was probably the final step in the pathogenesis of NNE in this group of babies. Since this observation, C. butyricum was recovered from several outbreaks of NNE (10, 16, 25, 26) and also from fecal specimens of healthy babies (5, 25). Various microorganisms, including Escherichia coli, Klebsiella spp., Enterobacter spp., Pseudomonas spp., Salmonella spp., Clostridium perfringens, Clostridium difficile, coronavirus, rotavirus, and enterovirus, were also associated with NNE (8). Attempts to induce NNE with C. butyricum in animals have not been successful. Lawrence et al. (12) reported hemorrhagic enteritis in neonatal monoxenic rats, but failed to reproduce this previous result (11). In the cecum of gnotoxenic chickens monoassociated with various microorganisms, we observed that C. butyricum produced volatile fatty acids and that its establishment was ca. 108 cells per g of the content (29). The purpose of this study was to develop a experimental chicken model for C. butyricum cecitis wherein necrotic lesions and pneumatosis could be induced through oral inoculation with human strains.

Using axenic quails fed a diet containing lactose, we have investigated the potentially pathogenic roles of six Clostridium butyricum strains of human origin. Three strains (CB155-3, CB1002, and CB203-1) isolated from neonatal necrotizing enterocolitis patients and two of three strains (CB19-1 and CB25-2) isolated from healthy newborns led to cecal or crop lesions or both similar to those observed in human neonatal necrotizing enterocolitis: thickening of the cecal wall with gas cysts, hemorrhagic ulcerations, and necrotic areas. The lactose-negative strain (CB46-1) did not develop any lesions. The neuraminidase-producing strain (CB155-3) caused lesions in all monoassociated quails, whereas the other strains caused lesions in 28 to 85% of animals. Removal of dietary lactose suppressed all pathological incidence. These results show that lactose fermentation is a prerequisite in these pathological changes and stress the roles played by both the strain and the host in the expression of C. butyricum enteropathogenicity.Clostridium butyricum is frequently isolated from feces of healthy human newborns (6, 10, 29), but the role played by this strain in neonatal necrotizing enterocolitis (NNE) has been questioned by several authors (6,10,11,13,29,30). Nevertheless, no enteropathogenic toxin has ever been attributed to C. butyricum.Popoff et al. (21) were the first to reproduce, in the ceca of chickens monoassociated with C. butyricum strains, histopathological lesions (congestion, hemorrhage, necrosis, and pneumatosis) similar to those observed in human newborns suffering from NNE. However, because of their large size, chickens are not practical animals to use for further investigation in isolators.Ulcerative (18) and necrotic (3) enteritises have been observed in quails. Their small size and fast growth rate encouraged us to test them as an experimental model for the study of C. butyricum enteropathogenicity. We tested three strains isolated from NNE patients; one strain had already been tested in chickens (21). We have also tested three other strains isolated from healthy newborns. MATERIALS AND METHODS Bacterial strains. Of six C. butyricum strains studied, three were isolated from sick premature human newborns: strain CB1002 from a fatal NNE case, strain CB203-1 from a patient with hemorrhagic colitis, and strain CB155-3 from an NNE patient. The three remaining strains, CB19-1, CB25-2, and CB46-1, were isolated from stools of healthy premature newborns (19). Strain CB1002 was used in a previous study (21). CB155-3 was the sole neuraminidase-producing strain (19), and CB46-1 was the sole lactose-nonfermenting strain. Strains were grown in TY broth (Pasteur Production, Paris, France).Maintenance and inoculation of quails. Quails (Coturnix coturnix subsp. japonica) were obtained by the method of Reynier and Sackteder (23) with the following modifications * Corresponding author. for germfree animals. Eggs from a conventional flock kept in a laying battery were collected twice daily and immediately brushed with 0.3% quate...

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