Detection of coronaviruses in children with acute gastroenteritis in Maddina, Saudi Arabia.

BACKGROUND: The role of coronaviruses in paediatric gastro-enteritis is not well defined. We investigated the detection rate and epidemiological features of infection with coronavirus in children receiving hospital care for acute gastro-enteritis in Maddina, Saudi Arabia. METHODS: Stool specimens were collected from children less than 5 years of age who were either hospitalised in Maddina or given oral rehydration therapy as outpatients between April 2004 and April 2005. Coronaviruses were detected by electron microscopy. RESULTS: Coronaviruses were detected in 63 (6%) of 984 children with acute gastro-enteritis and were more commonly detected in outpatients (47/423, 11%) than in inpatients (16/561, 3%). The median age (range) of children with coronavirus infection was 42 months (10-60). Coronaviruses were detected throughout the year with the highest detection rate at the end of the winter season. CONCLUSIONS: Coronaviruses were commonly identified in children with diarrhoea in Saudi Arabia. Their role in paediatric gastro-enteritis warrants further evaluation.

Carriage of potentially pathogenic Escherichia coli in chickens.

DNA-DNA hybridization, cultured cell lines, and transmission electron microscopy were used to study pathogenicity traits of 64 Escherichia coli isolated from apparently healthy chickens from 18 small-scale farms in Thika District, Kenya. A total of 39 (60.9%) isolates hybridized with the eae gene probe for enteropathogenic E. coli (EPEC) whereas another 16 (25%) hybridized with the lt and st gene probes and were categorized as enterotoxigenic E. coli. Electron microscopic examination of the eae probe-positive E. coli cultures with the HT-2919A cell line confirmed that they were able to attach intimately and produced effacement typical of EPEC. In addition, negative stain electron microscopy showed that the EPEC strains produced pili that have previously been associated with increased virulence of E. coli infections in chickens. This study has also demonstrated that apparently healthy chickens may carry enteropathogenic E. coli strains.

beta-catenin expression in primary and metastatic colorectal carcinoma.

beta-catenin plays a fundamental role in the regulation of the E-cadherin-catenin cell adhesion complex. It also functions in growth signalling events, independently of the cadherin-catenin complex, and these signalling pathways are disturbed in colorectal cancer. Mutations in either the APC or beta-catenin genes in colorectal cancer cells result in up-regulation of protein expression and subsequent cytoplasmic and nuclear distribution of beta-catenin. In this study, we examined beta-catenin expression in 47 primary colorectal tumors and the corresponding liver metastases. Immunohistochemical studies demonstrated loss of membranous beta-catenin expression in 26% of primary tumors and 60% of liver metastases and a concomitant increase in cytoplasmic and nuclear staining. Widespread nuclear expression of beta-catenin was found in 64% of primary tumors and 21% of liver metastases. No associations were found between any form of beta-catenin expression and either tumor stage or tumor grade. Cellular distribution of beta-catenin was also examined by detergent extraction and Western blot analysis in 16 primary tumors and 23 liver metastases. This analysis showed that most tumors demonstrated reduced beta-catenin in the cytoskeletal fraction and increased beta-catenin in the cytosolic fraction. Furthermore, 3 liver metastases were found to contain a truncated beta-catenin protein of approximately M(r) 80,000. Immunoprecipitation studies showed that the truncated beta-catenin proteins only bound weakly to E-cadherin and beta-catenin compared with non-truncated beta-catenin. These results demonstrate gross alterations in the cellular distribution of beta-catenin in primary colorectal cancers with metastatic potential, as well as in the metastatic tumors. These changes may be the consequence of APC or beta-catenin gene mutations, or possibly result from a post-translational modification of the E-cadherin-catenin complex.

Characterization of intestinal morphologic, biochemical, and ultrastructural features in gluten-sensitive Irish Setters during controlled oral gluten challenge exposure after weaning.

OBJECTIVE: To characterize histologic, biochemical, and ultrastructural changes in the intestine of Irish Setters susceptible to gluten-sensitive enteropathy (GSE) during controlled oral challenge exposure with gluten after weaning. ANIMALS: Six gluten-sensitive and 12 healthy Irish Setters and 3 healthy Greyhounds. PROCEDURE: Jejunal biopsy specimens were taken at 4 and 12 months of age from the 6 gluten-sensitive Irish Setters, which had been reared on a gluten-free diet to which a controlled dose of gluten (0.5 g/kg of body weight/d) was added. Control specimens were obtained at 4 (n = 5) and 12 (7) months of age from the healthy Irish Setters, which had been fed a conventional gluten-containing diet, and at 4 months of age from the healthy Greyhounds fed the controlled dose of gluten. The specimens were subjected to histologic and ultrastructural examinations and assay of brush border enzymes. RESULTS: Gluten-sensitive Irish Setters developed abnormalities characteristic of GSE at 4 months. Abnormalities were comparable to changes not seen previously until 12 months in dogs with GSE fed a conventional gluten-containing diet. In addition, microvilli were stunted and irregular, and a few were vesiculated and reduced in number; the glycocalyx was reduced or absent. By 12 months of age, there was improvement in morphologic and biochemical parameters, indicating partial recovery despite continued exposure to gluten. CONCLUSIONS: Relative early onset of intestinal damage, compared with that previously reported, and subsequent partial recovery suggestive of oral tolerance to gluten may be attributable to oral administration of gluten as a purified extract rather than in dietary cereal, but alternative explanations include differences in environment or genetic susceptibility to gluten.

Growth and propagation of normal rat intestinal epithelial cells.

A combination of mild proteolytic digestion and selective growth stimulation has been used to isolate and propagate adult rat intestinal epithelial cells with a finite life span. Growth of these cells on a variety of matrices and on mesenchymal cells has resulted in the expression of brush border enzymes including sucrase-isomaltase, aminopeptidase N, and alkaline phosphatase. Examination of the cells at the electron microscopic level has revealed that although these cells express key brush border enzymes, they do not have a fully formed brush border. These findings suggest that the expression of brush border enzymes and structural proteins represent distinct stages of enterocyte differentiation that are under separate transcriptional and temporal control.

Use of a nested PCR method for the detection of astrovirus serotype 1 in human faecal material.

In this paper we describe a reverse-transcription nested polymerase chain reaction method for detecting human astrovirus serotype 1. It has been evaluated on 56 UK diarrhoeal stool specimens and six non-UK specimens. The method has greater sensitivity than electron microscopy and may be a useful test in areas such as the UK where this serotype predominates.

Human cowpox 1969-93: a review based on 54 cases.

This survey of the clinical and epidemiological features of human cowpox, a rare but relatively severe zoonotic infection, is based on 54 cases, many unpublished, which we have studied since 1969. Patients present with painful, haemorrhagic pustules or black eschars, usually on the hand or face, accompanied by oedema, erythema, lymphadenopathy, and systemic involvement. Severe, occasionally fatal, cases occur in eczematous and immunosuppressed individuals, although cowpox has not yet been reported in anyone infected with the human immunodeficiency virus. Variations in the clinical features are described, and the differential clinical diagnosis of cowpox, parapox, herpes virus, and anthrax infections is discussed. The role of the laboratory in diagnosis is described, and the value of electron microscopy in providing rapid confirmation is emphasized. Care in taking a detailed history will assist in the initial clinical diagnosis, and a history of contact with domestic cats, particularly during July-October, is important. The possible influence of smallpox vaccination on the incidence and severity is discussed and discounted.

Effects of enteropathogenic Escherichia coli on microvillar membrane proteins during organ culture of rabbit intestinal mucosa.

This study examines the effects of an enteropathogenic Escherichia coli on microvillar membrane proteins during organ culture of rabbit ileal explants. Explants maintained with enteropathogenic E coli showed brush border effacement affecting approximately 50% of enterocytes, and where enteropathogenic E coli were closely adherent to the enterocyte surface microvilli were apparently being shed as vesicles. The microvillar membrane enzymes alkaline phosphatase, aminopeptidase N and alpha-glucosidase were released into the culture medium during organ culture, and this process was significantly enhanced by enteropathogenic E coli. This increased loss of microvillar membrane enzymes into the culture medium was associated with decreased tissue activities of microvillar membrane enzymes in enteropathogenic E coli infected ileal explants compared with control. For aminopeptidase N in particular, however, total enzyme activities in the tissue plus culture medium were increased comparing enteropathogenic E coli with control, suggesting that there might be an increase in the rate of synthesis of certain microvillar membrane proteins. Reorientating sucrose density gradient centrifugation of culture medium showed that alkaline phosphatase, aminopeptidase N and alpha-glucosidase were predominantly associated with particles of peak modal density 1.19 g/ml in both groups, confirming that enteropathogenic E coli accelerate release of microvillar membrane enzymes as vesicles. Analytical fractionation of ileal explants showed that enteropathogenic E coli resulted in a loss of microvillar membrane enzyme activities from the main brush border peak of modal density 1.21 g/ml present in controls. The density of the remaining smaller and lighter peak increased from 1.19 g/ml to 1.23 g/ml after homogenisation in digitonin, confirming association of these proteins with cholesterol containing membranes and not endoplasmic reticulum. These findings suggest that enteropathogenic E. coli accelerate the normal shedding of microvillar membrane proteins as vesicles, and may stimulate a compensatory increase in microvillar membrane protein synthesis.

Novel invasion determinant of enteropathogenic Escherichia coli plasmid pLV501 encodes the ability to invade intestinal epithelial cells and HEp-2 cells.

An Escherichia coli K-12 transformant carrying 96.5-kb plasmid pLV501 from enteropathogenic E. coli (EPEC) strain K798 is able to produce the same characteristic attaching-effacing lesions in a rabbit ileal biopsy explant model as its parent strain. Cloned EcoRI-SalI DNA restriction fragments from this plasmid failed to reproduce the attaching-effacing lesions, but one recombinant plasmid, pLV527, containing 4.5 kb of pLV501 DNA, conferred on E. coli DH1 transformants the ability to invade enterocytes in the rabbit explant model. DH1(pLV527) was also able to adhere to and invade HEp-2 cells. The relative invasive ability of DH1(pLV527) was quantified by recovery of internalized bacteria following gentamicin treatment of infected HEp-2 monolayers. DH1(pLV527) was 1,000-fold more invasive than DH1 carrying pBR322 or a recombinant plasmid which had no physiological effect on ileal biopsy explants but was less invasive than an enteroinvasive E. coli strain or a transformant carrying the cloned invasion genes of Shigella flexneri. Invasion by DH1(pLV501) could also be detected but occurred at a level 30 times lower than that by DH1(pLV527). Colony-hybridization of the pLV527 insert against a panel of 49 EPEC and related strains revealed that only 11 contained pLV527-hybridizing sequences; thus, the invasion determinant is not an essential component of the attachment-effacement pathogenic mechanism. One pLV527-hybridizing strain displayed both attachment-effacement and invasiveness in the rabbit ileal biopsy explant model. No significant hybridization was observed to non-EPEC invasive pathogenic enteric bacteria, indicating that the invasion determinant encoded on pLV527 is distinct from those used by these organisms.

Attaching effacement of the rabbit enterocyte brush border is encoded on a single 96.5-kilobase-pair plasmid in an enteropathogenic Escherichia coli O111 strain.

An enteropathogenic Escherichia coli (EPE) O111 serotype a,b,H- strain carried the following four plasmids: pLV501 (96.5 kilobase pairs [kbp]) specifying resistance to chloramphenicol, tetracycline, and kanamycin; pLV502 (8 kbp) specifying ampicillin resistance; pLV503 (1.9 kbp) specifying streptomycin resistance; and pLV504 (80 kbp) with no resistance markers. This EPEC attached to HEp-2 cells to produce localized clumps of bacteria (localized adhesion) and attached intimately to the enterocyte surface, leading to loss of the brush border (attaching effacement). Plasmid pLV501 was also found to specify the ability to produce localized adhesion on HEp-2 cells and attaching effacement in a rabbit ileal explant model system. Restriction maps showed considerable dissimilarities between pLV501 and pMAR-2, an EPEC plasmid carrying the EPEC adherence factor (EAF) genes. Furthermore, pLV501 did not hybridize with the EAF probe, whereas pLV504 did. There was sequence homology between pLV501 and large plasmids in all seven other well-characterized EPEC, only five of which hybridized with the EAF probe. These findings indicate that pLV501 carries at least one of the genes responsible for production of the brush border damage characteristic of EPEC.

Viral gastro-enteritis in children in Malawi.

In a 2-month survey of 186 children with gastro-enteritis attending an out-patient clinic in Malawi, 42% were infected with rotavirus (HRV), 9% Cryptosporidium, 4.2% adenovirus, 1.2% Astrovirus and 0.6% Norwalk agent and small round featureless viruses. We believe this to be the first report of HRV in Malawi and the first of Astrovirus in Africa. Almost all the HRV infections were in children under 12 months old, 40% were in children under 6 months and 64% of children were being breastfed at presentation. Signs of respiratory tract infection were not unique to HRV gastro-enteritis. Polyacrylamide gel electrophoresis of HRV dsRNA from 25 of the faecal samples revealed that each had the same long electropherotype.

Varicella in an immunocompromised patient an electron microscopic study.

We report the electron-microscopic appearances of tissues from an immunocompromised patient who died of chickenpox. We observed structures consistent with some of the reported appearances of Varicella-Zoster virus in cultured cells, and tubular structures that have not to our knowledge been previously described.

Interaction of enteropathogenic Escherichia coli 0111 with rabbit intestinal mucosa in vitro.

A model system using rabbit intestinal mucosal explants has been developed to examine the characteristic ultrastructural damage to the brush border induced by enteropathogenic Escherichia coli 0111. In this model, as in others, bacterial adherence to the microvillous membranes occurred in two morphologically distinct stages. Initial attachment of enteropathogenic strains of E. coli to ileal mucosa appeared to be a goblet cells and the mucous layer covering the microvilli. The next stage involved binding of enteropathogenic strains of E. coli to the bases of the microvilli that became elongated and vesiculated. Eventually, large areas of brush border effacement occurred with close apposition between bacterial and enterocyte membranes, leading to cup and pedestal formation. With a relatively large inoculum of bacteria (10(8) cfu/ml) these changes occurred within 4 h, but even with much lower inocula (10(5) cfu/ml) localized areas of damage were seen within 8 h. Although the bacteriostatic antibiotic tetracycline (700 mg/L) inhibited bacterial replication, it did not prevent the characteristic damage produced by enteropathogenic strains of E. coli. Enteropathogenic strains of E. coli 0111 were able to produce attaching effacement to gastric, duodenal, jejunal, ileal, and colonic mucosa.

Lectin-induced damage to the enterocyte brush border. An electron-microscopic study in rabbits.

The ultrastructural effects of 11 lectins on the intestinal brush border were examined by means of an in vitro rabbit ileal mucosal explant culture system. Five of the lectins that bind to oligosaccharides containing either N-acetylglucosamine (phytohaemagglutinin, Euonymus europaeus lectin, pokeweed mitogen, and wheat germ agglutinin) or N-acetylneuraminic acid (Mycoplasma gallisepticum lectin) all had a specific effect on microvilli. The effects varied in accordance with the lectin and included lengthening, distortion, and vesiculation of the microvilli. In contrast, lectins binding specifically to galactose, glucose, mannose, and N-acetylgalactosamine had no effect. Incubation of mucosal fragments with the divalent cation ionophore A23187 did not mimic the effect of the lectins. This apparent relationship between lectin damage and receptor specificity may reflect either accessibility of appropriate binding sites or a differential response to binding.

Recognition of whole Cryptosporidium oocysts in feces by negative staining and electron microscopy.

Crytosporidium oocysts in feces were recognizable in the electron microscope when prepared by the negative staining technique used by virologists. Their size, shape, and surface markings were sufficiently characteristic for cryptosporidiosis to be diagnosed by this method if more suitable methods are not available.