A scanning electron microscope study of the effect of an enterotoxin from Clostridium perfringens 8-6 on mice of different ages

Intestinal damage to mice caused by an enterotoxin from a coatless spore mutant of Clostridium perfringens type A (8-6) was examined by scanning electron microscopy. Two distinct types of damage were observed, both of which could be correlated with animal age. Damage appeared to occur in a specific sequence similar to that found in previous studies in rabbits. We conclude that the type of ileal tissue damage reflects the mode of toxin incorporation from the gut, which is a function of animal age.     

Kinetics of spore coat protein synthesis byClostridium perfringens type A

Spore coat proteins of several strains ofClostridium perfringens were analyzed by immunoblotting with antisera against two major spore coat proteins, one [34-kilodalton (kDa)] from an enterotoxin-positive and one (19-kDa) from an enterotoxin-negative (ent^–) strain of this organism. The results indicated that spore coat proteins from many strains ofC. perfringens were immunologically related regardless of their ability to produce enterotoxin, but were not immunologically related to enterotoxin. The kinetics of synthesis and deposition of two major spore coat proteins differed depending upon the strain. Coat protein synthesis was sporulationspecific, since coat protein was not detected in vegetative cell extracts. There was no similarity between the amino acid composition of either coat protein and enterotoxin. These results suggest that, contrary to previous reports (W.R. Frieben and C.L. Duncan, Eur J Biochem 39:393–410, 1973), enterotoxin synthesis is not closely related to spore coat protein synthesis in this organism.     

Localization of the azoreductase ofClostridium perfringens by immuno-electron microscopy

An antibody againstClostridium perfringens azoreductase was used with protein A (gold-labeled) to locate the site of synthesis of extracellular azoreductase in this bacterium. Electron microscopy of immunogold-stained thin sections ofC. perfringens cells showed an average of 134 gold particles per cell, distributed throughout the cytoplasm and not associated with any organized structures.     

Cloning and sequencing of the Clostridium perfringens enterotoxin gene

Several gene banks of Clostridium perfringens in E. coli were constructed. Using a mixture of synthetic 29-mer DNA probes clones were selected containing inserts from the C. perfringens gene coding for the enterotoxin. This has allowed sequencing of the complete gene and its flanking regions. The decuded amino acid sequence (320 a.a.) was found to differ at several sites from the sequence published previously by others. Two 40-mer DNA-probes were used to detect the toxin gene in C. perfringens strains isolated from the faeces of different non-symptomatic animals. Only 6% of the strains were found to possess the gene.     

Identification of the product oftetP gene: A possible mechanistic basis for tetracycline resistance inClostridium perfringens

Using cloning andin vitro protein synthesis we identified the polypeptide product of thetetP gene ofClostridium perfringens which is responsible for conferring resistance to tetracycline. TwoEcoRI fragments invariably share the resistance determinant in all of theClostridium perfringens isolates that we studied. Likewise, two proteins of 10 and 20 kDa were found to be conserved in all of the recombinant clones. The 10 kDa protein appears to be responsible for the constitution of the expression oftetP gene inC. perfringens.     

Nosocomial Diarrhoea in the Elderly Due to Enterotoxigenic Clostridium perfringens

To diagnose sporadic diarrhoea due to Clostridium perfringens infection, faecal specimens from elderly patients were examined directly for C. perfringens enterotoxin using reverse passive latex agglutination assay, and then cultured for this organism. C. perfringens isolates from those samples were grouped by slide agglutination and by pulsed-field gel electrophoresis (PFGE). Fifty of the 60 isolates agglutinated with newly raised antiserum WX2 and 38 shared the same genomic PFGE pattern. Characteristics of the epidemics and experimental data suggest that the diarrhoea was caused by a nosocomial spread of C. perfringens, and not by a food-borne outbreak.     

Atomic force microscopy of pollen grains,cellulose microfibrils,and protoplasts

Summary Atomic force microscopy (AFM) holds unique prospects for biological microscopy, such as nanometer resolution and the possibility of measuring samples in (physiological) solutions. This article reports the results of an examination of various types of plant material with the AFM. AFM images of the surface of pollen grains ofKalanchoe blossfeldiana andZea mays were compared with field emission scanning electron microscope (FESEM) images. AFM reached the same resolutions as FESEM but did not provide an overall view of the pollen grains. Using AFM in torsion mode, however, it was possible to reveal differences in friction forces of the surface of the pollen grains. Cellulose microfibrils in the cell wall of root hairs ofRaphanus sativus andZ. mays were imaged using AFM and transmission electron microscopy (TEM). Imaging was performed on specimens from which the wall matrix had been extracted. The cell wall texture of the root hairs was depicted clearly with AFM and was similar to the texture known from TEM. It was not possible to resolve substructures in a single microfibril. Because the scanning tip damaged the fragile cells, it was not possible to obtain images of living protoplasts ofZ. mays, but images of fixed and dried protoplasts are shown. We demonstrate that AFM of plant cells reaches resolutions as obtained with FESEM and TEM, but obstacles still have to be overcome before imaging of living protoplasts in physiological conditions can be realized.Abbreviations AFM atomic force microscope - FESEM field emission scanning electron microscope - PyMS pyrolysis mass spectrometry - TEM transmission electron microscope     

The effect of a Clostridium perfringens 8-6 enterotoxin on viability and macromolecular synthesis in Vero cells

Clostridium perfringens type A 8-6 enterotoxin causes gross morphological damage to Vero cells grown in tissue culture. Damage was observed to occur after only 30 minutes exposure at concentrations as low as 20 ng/ml, and within 60 minutes 95% of the cells had detached. Concentrations as low as 0.01 ng were able to cause detectable inhibition of plating efficiency. The enterotoxin inhibited DNA, RNA and protein synthesis and caused the reversal of glucose transport. Heat inactivated enterotoxin had no effect on cell function or morphology.     

Preparation of chromosome spreads for electron (TEM,SEM, STEM), light and confocal microscopy

In the past, ultrastructural studies on chromosome morphology have been carried out using light microscopy, scanning electron microscopy and transmission electron microscopy of whole mounted or sectioned samples. Until now, however, it has not been possible to use all of these techniques on the same specimen. In this paper we describe a specimen preparation method that allows one to study the same chromosomes by transmission, scanning-transmission and scanning electron microscopy, as well as by standard light microscopy and confocal microscopy. Chromosome plates are obtained on a carbon coated glass slide. The carbon film carrying the chromosomes is then transferred to electron microscopy grids, subjected to various treatments and observed. The results show a consistent morphological correspondence between the different methods. This method could be very useful and important because it makes possible a direct comparison between the various techniques used in chromosome studies such as banding, in situ hybridization, fluorescent probe localization, ultrastructural analysis, and colloidal gold cytochemical reactionsAbbreviations CLSM confocal laser scanning microscope - EM electron microscopy - kV kilovolt(s) - LM light microscope - SEM scanning electron microscope - STEM scanning-transmission electron microscope - TEM transmission electron microscope     

Morphology of the Leather Defect Light Flecks and Spots

The skin histology and the scanning electron microscope morphology of the hide defect light flecks and spots after tanning were studied in 11 steers infested with biting lice (Damalinia bovis). Nine steers from herds free of lice were used as controls. Skin biopsies from 6 of the animals in the lice infested group showed mild to moderate hyperkeratosis and moderate perivascular to diffuse dermatitis with infiltration of mainly mononuclear cells and some eosinophilic granulocytes. The steers were slaughtered at an age of 18 to 23 months. Light flecks and spots occurred on all examined hides from the infested group after tanning. No examined hides from the control group demonstrated similar damage. Both light microscopic examination of sections of tanned hide with light flecks and spots and scanning electron microscopy of the same defects showed superficial grain loss and craters with a irregular fibre base encircled by smooth and intact grain.     

Intracellular proteases during sporulation and enterotoxin formation byClostridium perfringens type A

Intracellular proteolytic activity was detected in cell-free extracts ofClostridium perfringens NCTC 10239 and NCTC 8798. The kinetics of protease, enterotoxin, and spore formation as well as growth of the wild type at elevated temperature and the use of sporulation mutants indicated that most protease activity was related to sporulation. Intracellular protease activity was inhibited by a mixture of tetrasodium ethylenediaminetetraacetic acid and phenylmethylsulfonyl fluoride; this indicated the presence of an alkaline serine protease and a neutral metallo-protease. Stage 0 sporulation mutants produced only metallo-sensitive proteases; this indicated that only the serine protease was sporulation-specific.     

Preliminary results from a microscopic examination on the effects of aluminium on the root tips of wheat

Root tips from aluminium (Al) tolerant (Waalt) and Al sensitive (Warigal) wheat (Triticum aestivum (L). Thell.) cultivars exposed to low concentrations of Al (10 M) for 10, 24 and 72 hours were examined under the light and electron microscope. After fixing and embedding, longitudinal and transverse thin and ultrathin sections were cut. There was no evidence of Al damage to the root tips of the Al tolerant cultivar under both the light and electron microscope. For the Al sensitive cultivar, Al had no observable effect on the root tips 10 hours after Al addition when examined under the light microscope. When examined under an electron microscope, electron dense globular deposits were observed between the cell wall and cell membrane of the epidermal cells. There was not obvious damage to the cell cytoplasm. Two or 3 days after Al addition, light microscopy showed that the cells in the root tips had become swollen and extensively vacuolated. The tissues appeared disorganised and degenerate, particularly in the epidermis and outer cortical cells. The electron microscope also revealed a thickening of the cell wall. The cell wall was broken down, particularly in the epidermis in the region 4–6 mm from the root tip. The tissue in the meristematic area was largely intact.     

Morphological changes in the rat kidney following long-term diabetes

The morphological basis of diabetic nephropathy has been studied using light and electron microscopy. Kidneys of streptozotocin-induced diabetic rats were examined on the light microscope at 4 weeks and 8 months after induction of diabetes mellitus. In addition, the 8-month diabetic kidneys were examined with the electron microscope. Renal hypertrophy was evidenced by the increase in the weight of kidneys of diabetic rats. Whilst the diabetic kidneys were approximately twice as large after 4 weeks they were only 30% larger compared to age-matched controls after 8 months of induction of diabetes. After 4 weeks, light microscopy revealed dilated tubules within the cortex of the diabetic kidneys. Light microscopy showed a significant amount of destruction of the distal convoluted tubules while electron microscopy revealed a spectrum of damage that included basement membrane thickening, loss of podocytic foot processes, disruption of tubular basal infoldings and their related mitochondria and fibrosis of the tubules 8 months after induction of diabetes. It is concluded that renal hypertrophy persists after a prolonged occurrence of diabetes but the extensive damage and loss of renal tissue including the loss of the foot processes of podocytes might be partly responsible for the clinical presentation of diabetic nephropathy.     

Three-dimensional structure of living chloroplasts as visualized by confocal scanning laser microscopy

Summary The newly developed confocal scanning laser microscope, together with image processing by computer, has been used to obtain three-dimensional information on the organization of grana in chloroplasts in living plant tissue. Chloroplasts are ideally suited for such studies because their pigments show bright autofluorescence. The high-resolution stereo images bridge a gap between classic light microscopy and electron microscopy. Our preliminary observations on several plant species resemble most the early observations of Strugger (1951: Die Strukturordnung im Chloroplasten. Ber Deutsch Bot Ges 64: 69–83) and suggest that the 3-D technique might well be suitable to solve discrepancies in the interpretation of classical light microscopic and electron microscopic observations.Abbreviations 3-D three dimensional - CSLM confocal scanning laser microscopy - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - DNA deoxyribonucleic acid     

The enteric toxins of Clostridium perfringens

The Gram-positive pathogen Clostridium perfringens is a major cause of human and veterinary enteric disease largely because this bacterium can produce several toxins when present inside the gastrointestinal tract. The enteric toxins of C. perfringens share two common features: (1) they are all single polypeptides of modest (~25–35 kDa) size, although lacking in sequence homology, and (2) they generally act by forming pores or channels in plasma membranes of host cells. These enteric toxins include C. perfringens enterotoxin (CPE), which is responsible for the symptoms of a common human food poisoning and acts by forming pores after interacting with intestinal tight junction proteins. Two other C. perfringens enteric toxins, -toxin (a bioterrorism select agent) and -toxin, cause veterinary enterotoxemias when absorbed from the intestines; - and -toxins then apparently act by forming oligomeric pores in intestinal or extra-intestinal target tissues. The action of a newly discovered C. perfringens enteric toxin, 2 toxin, has not yet been defined but precedent suggests it might also be a pore-former. Experience with other clostridial toxins certainly warrants continued research on these C. perfringens enteric toxins to develop their potential as therapeutic agents and tools for cellular biology.

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Microbial populations associated with the surface of the brown algaAscophyllum nodosum

The microorganisms on the surface of the brown algaAscophyllum nodosum, collected from an intertidal area in Nahant, Massachusetts, were examined using scanning electron microscopy. Differences in the microbial populations on the holdfast, internodal regions of the stipe, and the apical tips were apparent. The populations ranged from a lawn of end-attached bacteria above the holdfast to microcolonies of yeast cells near the apical tips. The greatest diversity of microorganisms was noted in the internodal region representing the fourth year of growth where a dense lawn of end-attached bacteria was overlaid by filamentous bacteria, pennate diatoms, and filamentous blue-green algae. A simple procedure was developed to estimate the number of bacteria on the surface of the seaweed using the scanning electron microscope. The observed distribution of epiphytes may be explained in terms of the age of the algal surface, differences in light intensity, and the differential secretion of tannin by various parts ofAscophyllum.     

Conjugative Transfer of theEscherichia coli–Clostridium perfringensShuttle Vector pJIR1457 toClostridium botulinumType A Strains

An RP4-oriT shuttle vector pJIR1457 originally developed forClostridium perfringenswas successfully transferred by conjugation fromEscherichia colitoClostridium botulinumtype A strains and to a nontoxigenicC. botulinumtype A–transposon Tn916mutant strain lacking the entire toxin gene cluster. The light chain (LC) of botulinum toxin was highly expressed in the toxin deletion mutant strain from a pJIR1457 construct containing the recombinant botulinal gene for LC. This shuttle vector system will be valuable for genetic analysis ofC. botulinumand will enable genetic manipulation and recombinant expression studies of botulinum neurotoxins as pharmaceutical agents.     

Phospholipase C ofPseudomonas chlororaphis and its toxicity for insects

Phospholipase C (Ec 3.1.4.3) obtained fromPseudomonas chlororaphis was purified 70-fold and its activity toward various substrates was determined. The enzyme was found to have no toxic effect either on larvae ofGalleria mellonella or on the rabbit. The molecular weight of the enzyme was approximately 54,000, its pH optimum 7.5 and the isoelectric point at pH 6.3. The biochemical and toxogenic properties of the enzyme were compared with those ofBacillus cereus and ofClostridium perfringens phospholipases.     

Clostridium perfringens type A cytotoxic-enterotoxin(s) as triggers for death in the sudden infant death syndrome: Development of a toxico-infection hypothesis

In our studies with the pathogenic bacteriumClostridium perfringens type A and its cytotoxic-enterotoxins (CTEs), we have obtained results that imply an involvement of this organism in the sudden infant death syndrome (SIDS). In fecal samples obtained from SIDS infants (n=164) and non-SIDS infants (n=57),C. perfringens type A was present in high numbers in >80% of SIDS and <2% of control non-SIDS cases respectively. Fecal samples from SIDS infants analyzed by ELISA forC. perfringens type A CTEs showed a very strong positive correlation with the presence of the organism. Histopathological examination of ileal tissue from SIDS infants showed remarkable similarity to tissue from animal models affected byC. perfringens type A CTEs, where the patterns of damage were positively correlated with the age of the animal. We propose that systemic distribution of the CTEs acts parasympathomimetically to trigger a biochemical cascade that alters cardiorespiratory control. Death may subsequently ensue in an immunologically vulnerable infant.Florida Agricultural Experiment Station Journal Series No. R-02419.     

A strategy for enrichment of claudins based on their affinity to Clostridium perfringens enterotoxin

Background  

Claudins, a family of protein localized in tight junctions, are essential for the control of paracellular permeation in epithelia and endothelia. The interaction of several claudins with Clostridium perfringens enterotoxin (CPE) has been exploited for an affinity-based enrichment of CPE-binding claudins from lysates of normal rat cholangiocytes.