A method for harvesting non-cultivable filamentous segmented microbes inhabiting the ileum of mice

A method by which non-cultivable filamentous segmented microbes colonising the ileal epithelium of mice can be harvested was devised. Intact epithelial cells with attached filamentous microbes were obtained. An ethanol-treated preparation of epithelial material was used to restore the filamentous segmented microbes to the normal microflora of mice maintained by gnotobiotic methodology.     

Host specificity of filamentous, segmented microorganisms adherent to the small bowel epithelium in mice and rats

Germfree rats and mice were given by gavage samples of ileal homogenates prepared from conventional rats and mice. Filamentous, segmented procaryotes adhered to the small bowel epithelium in the ex-germfree mice only when the homogenate was made from mouse bowel and in the ex-germfree rats only when the inoculum came from rats. Thus, the filamentous microorganisms are host animal specific.     

Host specificity of filamentous, segmented microorganisms adherent to the small bowel epithelium in mice and rats.

Germfree rats and mice were given by gavage samples of ileal homogenates prepared from conventional rats and mice. Filamentous, segmented procaryotes adhered to the small bowel epithelium in the ex-germfree mice only when the homogenate was made from mouse bowel and in the ex-germfree rats only when the inoculum came from rats. Thus, the filamentous microorganisms are host animal specific.     

Evidence for a complex life cycle and endospore formation in the attached, filamentous, segmented bacterium from murine ileum.

Light and electron microscope observations showed that the filamentous, segmented bacterium commonly found attached to the ileal epithelium of rats and mice undergoes a complex life cycle. Filaments comprising up to 90 segments were attached to the microvillous border of absorptive epithelial cells by a specialized terminal holdfast segment. Starting at the free end of the filament and progressing toward the attached end, undifferentiated segments were converted into reproductive or mother segments. Within each mother cell two new holdfast segments developed. As the holdfasts matured, their mother cells degenerated and released them into the intervillar space where they attached, grew, and divided to produce new segmented filaments. Alternately, in some filaments, newly formed but not yet released holdfasts were converted into endospores, which were released in the same manner as holdfasts, presumably to spread the bacterial colony to other members of the rodent population.     

Scanning electron microscopy of the gut microflora of two earthworms: Lumbricus terrestris and Octolasion cyaneum

Scanning electron microscopy was used to investigate the presence of microorganisms, probably bacteria, on the gut surface of earthworms. The washed surfaces of the intestines of two earthworms, Lumbricus terrestris and Octolasion cyaneum, were examined. Numerous organisms resembling bacteria were observed throughout the gut, some in situations suggesting attachment. Compared with similar investigations in other invertebrates, there were fewer bacteria, showing less morphological diversity, on the earthworm gut surface. The majority of organisms viewed were coccoid, some were filamentous, and a few rod-shaped cells were observed. Cocci, often in chains, were seen in the foregut of both species. Although cocci were also numerous in the midgut region, particularly in the typhlosole, in O. cyaneum tufts of segmented, filamentous organisms were also seen with some segments resembling spores. Fewer organisms were found in the hindgut, but in L. terrestris there were segmented, filamentous organisms, attached to the epithelium by way of a socket-like structure, similar to that by which segmented, filamentous bacteria (SFBs) are attached to the ileum of rats and mice. Transmission electron microscopy of the hindgut of L. terrestris was undertaken to explore the structure and attachment of SFBs to the gut epithelium. However, although a few rod-shaped bacteria were observed, no SFBs were located. The observations reported here provide evidence that earthworms have an attached gut microflora of filamentous microorganisms which are probably indigenous, and as far as we are aware this is the first published report of such findings in these invertebrates. Offprint requests to: J.M. Jolly.     

Interactions between gut-associated lymphoid tissue and colonization levels of indigenous, segmented, filamentous bacteria in the small intestine of mice.

Unlike most other indigenous bacteria, segmented filamentous bacteria (SFB) are potent activators of the mucosal immune system. SFB are strongly anchored to the epithelial cells of the small intestine where they have a preference for mucosal lymphoid epithelium. Since SFB are only present in high numbers shortly after weaning, it was investigated whether an SFB-induced immune reaction results in the removal of these bacteria from the small intestine. A correlation was found between age and colonization levels in the small intestines of SFB monoassociated Swiss mice. Five-week-old athymic BALB/c (nu/nu) mice showed lower colonization levels than their heterozygous littermates, but the opposite was found at the age of 12 weeks. However, SFB inoculation of germfree Swiss mice resulted in higher colonization levels in 5-week-old mice when compared with 4-month-old mice. We conclude that SFB colonization levels in the small intestine are likely influenced by the activity of the mucosal immune system. However, an additional age-dependent factor that modulates SFB colonization levels cannot be excluded.     

Segmented filamentous bacteria in the rodent small intestine: Their colonization of growing animals and possible role in host resistance toSalmonella

The establishment and proliferation of a model population of autochthonous surface-associated microorganisms in the small bowel of growing rats (2–12 weeks of age) was studied. Segmented filamentous bacteria on the distal ileal villi were examined by scanning electron microscopy (SEM) and countedin situ by transect line analysis. In young animals, these bacteria first colonized the villous base, but occupied all areas on the villus by adult age. Their distribution on Peyer's patches was also noted.In growing animals, colonization of the ileal villi by filamentous bacteria was significantly correlated to the development of host resistance to fatal infection by orally-dosedSalmonella enteritidis. In animals givenSalmonella and examined by SEM and transmission EM (TEM), the pathogen was seen only on ileal tissue surfaces, predominantly the villous base, from which the autochthonous population was absent. Conversely, in animals with filamentous bacteria,Salmonella surface colonization was not observed. The results suggest a possible protective role for the surface flora in the small bowel.     

Effects of hypothermia on the survival and cryopreservation of minipig ileal cells and Chinese hamster ovary cells.

Temperature of culture can be used to modulate cellular metabolism for improving small intestinal cell culture and cryopreservation. An hypothermia pretreatment (2 days at 25°C and 3 hours recovery at 37°C) improved hamster cell survival to freeze-thaw damage (p < 0.01) but decreased the survival of 2 immortal pig ileal cell lines even though epithelioid IPI-2I cells were more tolerant to hypothermia than IPI-I fibroblasts. Epithelioid cells survived 3 days at 25°C with unaltered expression of cytokeratin-18 whereas colonies of fibroblasts did not survive more than a day at 25°C (p < 0.001). These results suggest that hypothermia-tolerance of pig ileal cell lines might differ according to cell lineage calling for further experiments on small intestinal primary cell culture.     

Pathogenesis of Helicosporidium sp. (Chlorophyta: Trebouxiophyceae) in susceptible noctuid larvae

Helicosporidium sp. is a unique, achlorophyllous green alga that has been reported to infect various insect orders, including Lepidoptera, Diptera, and Coleoptera. The infectious cyst stage is ingested by the host, ruptures in the midgut lumen, and releases a filamentous cell. Histopathological examinations using larvae of a susceptible noctuid host, Spodoptera exigua, showed both cysts and filamentous cells affiliated with the microvillar lining of the midgut epithelium. A considerable proportion of the ingested cysts (22-39%) were recovered in feces collected 24 h after ingestion. A small number of filamentous cells passed the midgut epithelium and entered the hemocoel within 4-24 h after cyst ingestion. After 48 h, vegetative cell stages were detected in the hemolymph, followed by a 4- to 5-day period of increasing multiplication. Cyst differentiation in the colonized hemolymph began 6-7 days after the treatment.     

Electron microscopic studies on the macronuclear development in the ciliate Chilodonella uncinata.

The development of macronuclear anlage in the ciliate Chilodonella uncinata has been studied through electron microscopy. The ultrastructure of macro- and micronuclei is described for comparison. During the first stage of development, when the DNA content of the macronuclear anage increases from 2 C to 32 C, chromosomes are visible as distinct osmiophilic bodies inside the anlage. At the end of the initial polyploidization phase, the chromosomes despiralize, giving rise to long filamentous structures 25 to 50 nm in diameter. The latter show a singular banding pattern, with dense bands 12 to 25 nm thick alternating regularly with less dense interbands. Such an organization has not yet been observed in any other type of nucleus. These filamentous structures have been interpreted as highly despiralized oligotenic chromosomes. During the final stage of macronuclear development, these structures condense into thin chromatin strands and small dense granules; the number of granules increases progressively as the chromatin strands disappear. These small granules very likely fuse amongst themselves to form the chromatin granules of the vegetative macronucleus. No evidence has yet been found for a fragmentation of chromatin in this ciliate, but this problem needs further study. The old macronucleus maintains a normal ultrastructure until a late stage of development of the macronuclear anlage, becoming pycnotic only towards the very end of the latter process.     

Regional variation in ontogeny of class II antigens in enterocytes of mouse small intestine.

The ontogeny of major histocompatibility class II antigens in small intestine enterocytes of postnatal C3H/He mice was investigated. Cryosections of duodenal, jejunal, and ileal segments from 7-, 14-, 16-, 20-, 21-, 23-, 25-, 27-, 28-day-old and 7-week-old mice were stained for the class II antigens with MRC OX6 monoclonal antibodies by peroxidase-antiperoxidase labelling. In adults, the duodenum exhibited least expression of class II antigens that increased progressively towards the ileum. The expression in the villous epithelium was first seen in the duodenum and jejunum 21 days after birth but the ileal enterocytes did not exhibit any class II antigens. The earliest appearance (21 days postnatal) of class II antigens in the enterocytes coincides with the age of weaning which suggests that immunologic stimulation by ingested antigens after weaning may influence expression of these antigens. At day 28 after birth, the duodenum and jejunum expressed levels comparable to those in the adults. The first expression of the antigens seen in the ileum was at day 28 postpartum. Crypt epithelium of the three regions of the small intestine showed expression similar to that of corresponding regional villous enterocytes. We conclude that there is an age-dependent regional variation in the expression of class II antigens in enterocytes, and the expression increases with age. The variation in expression of the class II antigens in enterocytes of postnatal mice is attributed to the developmental status of the tissue. The nature of postnatal expression of the antigens is important since an early appearance of these antigens may have implications in autoimmunity.     

Transfer of carbonic anhydrase-positive particles from the follicle-associated epithelium to lymphocytes of Peyer's patches in foetal sheep and lambs

Summary Carbonic anhydrase cytochemistry of the ileal Peyer's patch in foetal and neonatal lambs has indicated secretion from the follicle-associated epithelium to the follicles. Reaction for carbonic anhydrase in the follicle-associated epithelium was found in the luminal plasma membrane, in cytoplasmic vesicles, and in vacuoles containing 50-nm membrane-bounded particles that seemed to be shed to the intercellular space. The lateral plasma membrane was negative for carbonic anhydrase, indicating that formation of carbonic anhydrase-positive particles was restricted to vacuoles. Administration of ferritin to ileal loops of sheep foetuses showed ferritin localized in vesicles and vacuoles of the follicle-associated epithelium followed by exocytosis, together with carbonic anhydrase-positive particles, into the indentations of the lateral cell border. The carbonic anhydrase-positive particles seemed to be transported to the centres of lymphoid follicles where many were attached to the plasma membrane of lymphocytes. Carbonic anhydrase-positive particles were also seen in vesicles and sometimes free in the cytoplasm of the lymphocytes or attached to their nuclear envelope. Light microscopically, carbonic anhydrase reactivity of the follicle-associated epithelium was associated with the early formation of the ileal Peyer's patch at about 100 days gestation. At this time the follicle-associated epithelium showed a strong luminal but at most a week lateral staining. With further foetal development there was a progressive increase in the amount of carbonic anhydrase-positive reaction product in extracellular particles, both along the lateral cell borders of the follicle-associated epithelium and among the lymphocytes of the follicle centres.     

Absorption of horseradish peroxidase by the small intestinal epithelium in postnatal developing rats.

After an intraluminal injection of horseradish peroxidase into the small intestine, the localization of peroxidase was studied in neonatal developing and adult rats by means of electron microscopy. Until around the 14th day of the neonatal period absorbed peroxidase granules in the duodenal and jejunal epithelium were abundant in the microvillous membrane, the apical tubulo-vacuolar system, and the Golgi apparatus, and on the lateral cell and basal membranes, and the luminal surfaces of the capillary cells. At the weaning period the tubulo-vacuolar system was absent in the duodenal and jejunal epithelial cells, and at that point absorbed peroxidase was observed in the same sites as in the adult rats: the microvillous membrane, the lateral cell and basal membranes, the Golgi apparatus, and the vesicles and vacuoles of the cytoplasm. During the suckling period, in the ileal epithelial cells exogenous peroxidase was found on the microvilli, in the tubulo-vacuolar system, in the supranuclear vacuole, in the Golgi apparatus, on the lateral cell and basal membranes, and also on the luminal surface of the endothelial cells of blood capillaries. When the tubulo-vacuolar system and the supranuclear vacuole were lost from the ileal cells at the weaning period, no exogenous peroxidase uptake was observed in the absorptive cell of the ileal epithelium.     

'Normalization' of germfree mice after direct and indirect contact with mice having a 'normal' intestinal microflora

Germfree mice were associated via direct and indirect contact with a 'normal' microflora by placing 'normal' mice in an isolator with germfree mice. Relative caecal weights, the ratio of secondary to primary bile acids, the presence of filamentous segmented bacteria in the small intestine and faecal beta-aspartylglycine were normal 5 days after direct contact and 15 days after indirect contact. Enterobacteriaceae were demonstrated by the third day after direct contact and the fourth day after indirect contact. Volatile and non-volatile fatty acids in the caecal contents were variable and appeared to be unrelated to the 'normalization' process of germfree mice after association with a microflora.     

Comparison of urine cytology between the ileal conduit and Indiana pouch

OBJECTIVE: To compare the cytomorphologic features of urine obtained from two different kinds of urinary diversions constructed after total bladder resection. STUDY DESIGN: The smears of urine from 11 ileal conduits and 6 Indiana pouches were evaluated. All patients underwent total bladder resection due to transitional cell carcinoma (TCC) or other kinds of cancer before urine diversion. RESULTS: The cytologic features of Indiana pouch urine include degenerated, small, round cells without columnar cells derived from intestinal epithelium. In ileal conduit urine, well-preserved columnar cells and degenerated, small, round cells were frequently observed. The columnar cells in ileal conduit urine exhibited cytologic features that should be distinguished from TCC cells. CONCLUSION: The method of reconstructing the urinary tract is important in urine cytology from urine diversions because the cytomorphologic features of urine are different between the two kinds of urinary diversions. Since columnar cells in ileal conduit urine might lead to misdiagnosis as TCC, special consideration is required to examine ileal conduit urine.     

Epithelial differentiation in the fetal rat colon: I. Plasma membrane phosphatase activities

During the last week of gestation of the fetal rat, the epithelium of the colon is rapidly remodeled. At 16 days a primitive stratified epithelium surrounds a small central lumen. Over the next 3 days, the main lumen extends narrow clefts down to the basal cell layer and small secondary lumina appear within the stratified epithelium between these clefts. At 19 and 20 days, secondary lumina enlarge but remain discrete; an infusion of cationic ferritin into the main lumen does not enter secondary lumina. During the 2 days prior to birth (21–22), the secondary lumina join the main lumen as superficial cells are sloughed, and the epithelium becomes simple columnar. Freeze-fracture replicas indicate that luminal and nonluminal membrane domains of epithelial cell plasma membranes are separated by continuous tight junctions throughout the conversion process. Cytochemical analysis of tissue slices from 16- to 22-day fetal colon demonstrated the appearance and segregation of two phosphatases on apical and basolateral membrane domains during epithelial conversion. Cysteine-sensitive pH 9.0 (alkaline) phosphatase activity was first detected along the luminal membranes of cells bordering both primary and secondary lumina at 18 days gestation and increased to a maximum at 20–21 days; weaker activity was present on basolateral membranes. Phosphatase activity at pH 8.0 also appeared at 18 days and increased thereafter, but was localized primarily on nonluminal membranes. At pH 8.0, reaction product appeared on both inner and outer sides of the membrane, and was only partially abolished by omission of K⁺ or addition of ouabain; thus the reaction may be only partially due to K⁺-dependent ATPase activity. Biochemical analysis of the cytochemical media confirmed the appearance of phosphatase activities at 18 days. Thus, plasma membrane phosphatase activities appear while the epithelium is still stratified, but are segregated to luminal and nonluminal membrane domains at the onset of activity. Segregation is maintained throughout the process of conversion of a simple columnar epithelium.     

Regional difference in the responsiveness to the inductive influence of the gizzard mesenchyme among the endoderms of various small intestinal segments of the chick embryo

The differentiation of the endoderms of duodenal, jejunal and ileal segments of the small intestine of 6 day old chick embryos cultured in recombination with the gizzard mesenchyme of 6 day chick embryos was examined. Only the duodenal endoderm differentiated in a mesenchyme-dependent fashion into gizzard-like mucous epithelium forming tubular glands that expressed no sucrase-antigen, while jejunal and ileal endoderms tended to become the sucrase-antigen-positive epithelium most likely according to their developmental fates. The analysis on the differentiation of the duodenal and gizzard endoderms in the presence of various digestive-tract mesenchymes confirmed that the duodenal endoderm had the tendency to differentiate into intestine-type and was different from the gizzard endoderm, which showed the differentiation tendency into gizzard-type. Thus, among the segments of small intestine, only the endoderm of duodenum that was situated next to the gizzard was found to have an ability to respond to the inductive influence of the gizzard mesenchyme and to change its developmental fate.     

Colonization and distribution of segmented filamentous bacteria (SFB) in chicken gastrointestinal tract and their relationship with host immunity

Uncultivable segmented filamentous bacteria (SFB) reside in the gastrointestinal (GI) tract of mammals and can boost the host immunity. Immunoglobulin A (IgA) from mother's milk has been previously shown to be a key factor in regulating SFB colonization. Because neonatal chicken cannot acquire IgA from maternal milk, they are a good model to examine the role of IgA in SFB colonization. Here, we used the fluorescent in situ hybridization (FISH) and quantitative PCR (qPCR) to monitor the colonization and distribution of SFB in chickens aged from 2-day-old to 6-week-old. Early SFB colonization, which primarily occurred in the ileal mucosa (< 13 days old), was IgA independent. From the age of 17-42 days, there was an increase in IgA in the gut mucosa, which was correlated with a decrease in SFB. To examine the effect of probiotics and immunosuppression on SFB colonization, we treated the chickens by feeding them Lactobacillus delbrueckii or giving them a subcutaneous injection of cyclophosphamide (CTX). Feeding lactobacilli at birth rendered SFB colonization occurring 4 days earlier, while CTX treatment increases the SFB colonization through reducing the other non-SFB bacteria. Altogether, our data suggest that early colonization of SFB in chicken occurs independently of IgA and the population of SFB in the GI tract of chicken may be manipulated from birth via probiotic or CTX treatment.     

RpoS controls the Vibrio cholerae mucosal escape response

Vibrio cholerae causes a severe diarrhoeal disease by secreting a toxin during colonization of the epithelium in the small intestine. Whereas the initial steps of the infectious process have been intensively studied, the last phases have received little attention. Confocal microscopy of V. cholerae O1-infected rabbit ileal loops captured a distinctive stage in the infectious process: 12 h post-inoculation, bacteria detach from the epithelial surface and move into the fluid-filled lumen. Designated the "mucosal escape response," this phenomenon requires RpoS, the stationary phase alternative sigma factor. Quantitative in vivo localization assays corroborated the rpoS phenotype and showed that it also requires HapR. Expression profiling of bacteria isolated from ileal loop fluid and mucus demonstrated a significant RpoS-dependent upregulation of many chemotaxis and motility genes coincident with the emigration of bacteria from the epithelial surface. In stationary phase cultures, RpoS was also required for upregulation of chemotaxis and motility genes, for production of flagella, and for movement of bacteria across low nutrient swarm plates. The hapR mutant produced near-normal numbers of flagellated cells, but was significantly less motile than the wild-type parent. During in vitro growth under virulence-inducing conditions, the rpoS mutant produced 10- to 100-fold more cholera toxin than the wild-type parent. Although the rpoS mutant caused only a small over-expression of the genes encoding cholera toxin in the ileal loop, it resulted in a 30% increase in fluid accumulation compared to the wild-type. Together, these results show that the mucosal escape response is orchestrated by an RpoS-dependent genetic program that activates chemotaxis and motility functions. This may furthermore coincide with reduced virulence gene expression, thus preparing the organism for the next stage in its life cycle.