J. Stephen

Growth of Clostridium difficile and production of toxins A and B in complex and defined media.

The ability of several strains of Clostridium difficile to grow and to produce toxins A and B in complex and defined culture media has been studied with special reference to the amino-acid composition of the medium. The production of these toxins varied with the strain used and with the composition of the growth medium. Toxin A production was not inextricably linked to production of toxin B since conditions were found in which only one or other toxin was produced.

Interaction of Salmonella choleraesuis, Salmonella dublin and Salmonella typhimurium with porcine and bovine terminal ileum in vivo

Quantitative experiments on the interaction of Salmonella choleraesuis and Salmonella dublin with porcine and bovine intestinal epithelia yielded no evidence to suggest that host restriction of S. choleraesuis and S. dublin for pigs and calves respectively could be explained in terms of the patterns of intestinal invasion observed in ligated ileal loops in vivo, at 3 h after challenge. No evidence was found to support the idea that Peyers patches, or specifically M cells, are the major route of entry for these serotypes in vivo. Three hours after loop inoculation, each serotype was recovered in comparable numbers from either absorptive or Peyers patch mucosae present in the same ileal loop, indicating that both types of tissue are involved in the early stages of the enteropathogenic process induced by both serotypes. More detailed transmission electron microscopic (TEM) analyses of follicle-associated epithelia (FAE) challenged with S. choleraesuis showed that in the same region of FAE, organisms invaded both M cells and enterocytes directly; comparable detailed TEM studies with S. dublin could not be carried out because of the tissue-destructive properties of this serotype. S. dublin was clearly more histotoxic than S. choleraesuis as had previously been found in rabbits: this difference is almost certainly due to a tissue-damaging toxin which is neither host nor gut-tissue specific. The tissue-destructive potential of S. dublin has profound implications for the measurement of and the assignment of significance to the invasiveness of S. dublin. S. dublin was nearly always seen entering gut cells in micro-colonies whereas S. choleraesuis entered mainly as single organisms or small groups of two or three.

An electron microscopic investigation of time-related changes in the intestine of neonatal mice infected with murine rotavirus

Seven-day-old mice were infected orally with murine rotavirus (EDIM) and regions of the gut examined at 24 h intervals up to 7 days by electron microscopy. Structural changes were correlated with data on viral antigen production, thymidine kinase activity, and clinical signs of diarrhea. No pathological changes were detected in the colon. Infection and structural damage were confined to the small intestine, with middle regions showing the most pronounced changes. Constriction of villus bases, edema of the lamina propria, and vacuolation of enterocytes occurred at 24 h postinfection (PI), i.e., before evidence of major virus replication. Transient villus atrophy occurred at 48 h PI. Recovery of villus length was evident by 72 h PI accompanied by evidence of marked enterocyte replication at villus bases. Many enterocytes were damaged with little evidence for the presence of virus particles. By 96 h PI, villi had almost recovered from infection although some enterocytes were still damaged; no virus particles were detected in these cells. A second phase of villus damage and edema of the lamina propria occurred at 120 h PI; the pathology resembled that at 24-48 h PI. By 144 to 168 h PI, recovery of the mucosa from infection was virtually complete. We suggest that many of the pathological features following rotavirus infection result from rotavirus-induced ischemia of villi and that diarrhea results from malabsorption of fluid by damaged villi and hypersecretion of ions released from increased numbers of dividing cells at villus-crypt borders.

The Nature and Role of Mucosal Damage in Relation to Salmonella Typhimurium-Induced Fluid Secretion in the Rabbit Ileumx

Summary The time course and nature of mucosal damage induced in rabbit ileal loops by two strains of Salmonella typhimurium (TML and W118) isolated from human infections was assessed by immunofluorescence microscopy and by scanning and transmission electronmicroscopy. Salmonella-induced fluid secretion occurred in the presence or absence of gross mucosal architectural damage. Neither strain caused mucosal ulceration. When damage did occur, the villi were shortened by loss of their tip regions with concomitant reforming of an intact mucosal surface. Immediately preceding the onset of fluid secretion, marked infiltration of the mucosa with polymorphonuclear leukocytes and occasional macrophages was seen. This revives an earlier suggestion that interaction between invading salmonellae and acute inflammatory cells may be an important factor in initiation of fluid secretion. Brush-border invasion by salmonellae cannot per se be the immediate cause of fluid secretion, because the latter occurred several hours after initial invasion.

Intestinal enzyme profiles in normal and rotavirus-infected mice

To investigate further the pathophysiology of rotavirus-induced diarrhea, changes in specific activities of eight relevant intestinal enzymes [alkaline phosphatase, thymidine kinase, lactase, maltase, sucrase, Na+,K+-adenosine triphosphatase (ATPase), adenylate and guanylate cyclases] were measured following infection of suckling mice with murine rotavirus (epizootic diarrhea of infant mouse strain) and compared with age-matched control mice. The concentration of lactose within the lumen of the gastrointestinal tract during infection was also measured. During the course of infection, activities of alkaline phosphatase and lactase decreased, whilst the activity of thymidine kinase increased. Precocious maturation profiles of sucrase and maltase enzymes were observed. No significant changes were detected in the activities of Na+,K+-ATPase or the adenylate and guanylate cyclases. These results are discussed in relation to existing and novel hypotheses on the pathogenesis of rotavirus-induced diarrhea.

Enterotoxin production by Salmonella typhimurium strains of different virulence

Six strains of Salmonella typhimurium (TML, W118, LT7, SL1027, M206 and Thax-1) of known virulence and ability to induce fluid secretion when inoculated into the rabbit ileum were examined for enterotoxin production. Enterotoxic activity, assayed in the rabbit ileal-loop test, was detected in polymyxin-B extracts from all strains (with the possible exception of Thax-1) cultured for 6 h in casamino acid-yeast extract medium. The extracts were inactive in tissue-culture assays with CHO, Y-1 adrenal and Vero cells, and in the infant mouse assay for enterotoxin. There was no correlation between enterotoxigenicity in vitro and the ability of whole organisms to induce fluid secretion in vivo. The significance of these results in relation to salmonellosis is discussed.

Cytotoxic Effects on Hair Cells of Guinea Pig Cochlea Produced by Pneumolysin, the Thiol Activated Toxin of Streptococcus pneumoniae

The cytolytic toxin, pneumolysin, from the gram positive bacterium, Streptococcus pneumoniae, when perfused through the scala tympani of the guinea pig cochlea reduced the amplitude of both the compound action potential and the cochlear microphonic potential. When the surface of the organ of Corti was examined by scanning electron microscopy, both inner and outer hair cells and supporting cells were found to be damaged. Inner hair cells and outer hair cells of row 3 were the most susceptible to damage by pneumolysin, followed by row 2 and then by row 1 of the outer hair cells. Damage to hair cells included disruption and splaying of stereocilia, loss of stereocilia and complete dissolution of hair bundles. Apical surfaces of hair cells and supporting cells were torn, pitted and cratered with shrinkage and tearing of cell boundaries. Within the dose range perfused (0.05-1 micrograms/microliters in a 10 microliters aliquot), the magnitude of the physiological and anatomical lesions was concentration dependent. The cytotoxic effects of pneumolysin reported here may be clinically significant factors in deafness caused by meningitis and otitis media in humans.

Rotavirus-induced changes in the microcirculation of intestinal villi of neonatal mice in relation to the induction and persistence of diarrhea.

Using a histochemical peroxidase technique, under conditions that preferentially stain erythrocytes, we have shown changes in the microcirculation of villi of neonatal mice infected with murine rotavirus. Between 18 and 48 h postinfection (PI), throughout all areas of the small intestine there occurred, sequentially, a marked ischemia and atrophy of villi. By 72 h PI, villi had recovered their normal height and showed incipient hyperemic microcirculation. At 96 h PI, hyperemic microcirculation was most marked. Between 120 and 144 h PI, a second phase of villus atrophy occurred, which was more attenuated and confined to the upper and middle regions of the intestine. This phase was not accompanied by a widespread ischemia of villi: a minority of villi were short and ischemic but many appeared hyperemic. Recovery of villus microcirculation occurred at 168 h PI, which coincided with recovery from diarrhea. These changes in villus microcirculation are discussed in relation to the pathology and pathophysiology of rotavirus infection. We make two novel suggestions. First, the reduction in red cells flowing through villi in the early stages of the infection instigates hypoxia and hence atrophy of villi. The ensuing but ephemeral increase in rate of cell division, necessary for the reconstitution of villi, induces hypersecretion. Second, the increase in numbers of erythrocytes found in villi during their regrowth phase and throughout the remaining time course of the infection perturbs the countercurrent system, lowering the osmolality of the hyperosmotic zone located at villus tips, thereby impairing water absorption and prolonging diarrhea.

Disaccharidase activities in small intestine of rotavirus-infected suckling mice : a histochemical study

A histochemical study of the time course of the appearance and location of lactase and α-glucosidase (used to detect sucrase and maltase) activities was carried out on control and rotavirus-infected mice from 7 to 14 days old. The overall pattern of enzyme activity was in agreement with previous quantitative studies on the activities of these enzymes. No evidence was obtained to support the idea that lactase deficiency was the result of repopulation of villi (denuded of lactase-producing villus cells) with immature lactase-negative cells. Low lactase activity was more likely to reflect profound changes in metabolically crippled cells, and recovery of lactase activity with recovery of normal metabolic functions. The location of enzyme activity to brush border regions rather than the cytoplasm of villus enterocytes enhances the significance of previous quantitative studies on these enzymes. The timing and duration of diminished lactase activities were such that they were unlikely to cause the induction or perpetuation of diarrhea in murine rotavirus diarrhea. The appearance in infected animals of α-glucosidase 3 days earlier than normal indicates that, in addition to reversible changes seen with lactase, developmental changes were accelerated that affected both crypt and villus cells.

The effects of Clostridium difficile crude toxins and toxin A on ileal and colonic loops in immune and nonimmune rabbits

Rabbits were solidly immunised by parenteral injection of purified Clostridium difficile toxin A such that they resisted an intravenous challenge with a normally lethal dose of toxin A. Ileal and colonic loops constructed in non-immune and immune animals received challenge injections of crude culture filtrate or purified toxin A of C. difficile. Protection of ileum was manifest after sufficient initial mucosal damage resulted in release of high levels of antitoxin A into the loop lumen of immune animals. There was less fluid accumulation in ligated ileal loops of immune than of non-immune rabbits. Less protection was observed when loops were challenged with crude culture filtrate containing toxins A and B than when challenged with purified toxin A. In-vitro studies with Ussing chambers yielded no evidence for tissue-localised immunity as judged by electrical responses and histology of toxin-treated tissue from non-immune and immune animals. No differences were found in the degree of epithelial damage, or volume or composition of fluid accumulating in colonic loops of non-immune and immune rabbits challenged with toxin A or crude culture filtrate. However, in colonic loops of immune rabbits there was no overt tissue-localised haemorrhage, whereas in those of non-immune rabbits tissue-localised haemorrhage was marked. In contrast to our findings with ileal loops, fluid accumulating in colonic loops was watery and contained substantially less total protein and (in immune animals) antitoxin A.