scholarly journals Clostridium perfringens type D epsilon toxin produces a rapid and dose-dependent cytotoxic effect on cerebral microvascular endothelial cells in vitro

2019 ◽  
Vol 32 (2) ◽  
pp. 277-281 ◽  
Author(s):  
Kimberley A. Mander ◽  
Francisco A. Uzal ◽  
Ruth Williams ◽  
John W. Finnie
Keyword(s):  
Endothelial Cells ◽  
Clostridium Perfringens ◽  
Vasogenic Edema ◽  
Ultrastructural Studies ◽  
Type D ◽  
Epsilon Toxin ◽  
Capillary Endothelial Cells ◽  
Dose Dependent ◽  
Ruminant Livestock

Clostridium perfringens type D epsilon toxin (ETX) is responsible for a severe and frequently fatal neurologic disorder in ruminant livestock. Light microscopic, immunohistochemical, and ultrastructural studies have suggested that ETX injury to the cerebral microvasculature, with subsequent severe, generalized vasogenic edema and increased intracranial pressure, is critically important in producing neurologic dysfunction. However, the effect of ETX on brain capillary endothelial cells in vitro has not been examined previously, to our knowledge. We exposed a well-characterized human blood–brain barrier cell line to increasing concentrations of ETX, and demonstrated a direct and dose-dependent endotheliotoxic effect. Our findings are concordant with the primacy of vasculocentric brain lesions in the diagnosis of acute epsilon toxin enterotoxemia in ruminant livestock.

scholarly journals Pathogenesis and diagnostic features of brain and ophthalmic damage produced by Clostridium perfringens type D epsilon toxin

2020 ◽  
Vol 32 (2) ◽  
pp. 282-286 ◽  
Author(s):  
John W. Finnie ◽  
Mauricio A. Navarro ◽  
Francisco A. Uzal
Keyword(s):  
Clostridium Perfringens ◽  
Vasogenic Edema ◽  
Differential Susceptibility ◽  
Brain Regions ◽  
Cerebral Lesions ◽  
Diagnostic Features ◽  
Microvascular Damage ◽  
Neurologic Disorder ◽  
Type D ◽  
Epsilon Toxin

Clostridium perfringens type D epsilon toxin (EXT) causes an important neurologic disorder of sheep, goats and, rarely, cattle. The disease can occur in peracute, acute, subacute, and chronic forms. High circulating levels of ETX produce vasculocentric brain lesions, in which microvascular endothelial injury results in diagnostically useful perivascular and intramural extravasations of plasma protein, especially in sheep, and less frequently in goats. With lower toxin doses, a more protracted clinical course tends to occur, particularly in sheep, leading to focal, bilaterally symmetrical, necrotic foci in certain brain regions. Although these morphologic features usually permit the diagnostic pathologist to make a definitive etiologic diagnosis, there are many aspects of the pathogenesis of these cerebral lesions that are not completely understood. ETX has also been shown to produce microvascular damage in the retina of rats, resulting in severe, diffuse vasogenic edema, similar to that found in brains exposed to this neurotoxin. The pathoclisis and vascular theories offer alternative explanations of the differential susceptibility of different brain regions to the same neurotoxic insult.

scholarly journals Development and Application of an Oral Challenge Mouse Model for Studying Clostridium perfringens Type D Infection

2007 ◽  
Vol 75 (9) ◽  
pp. 4282-4288 ◽  
Author(s):  
Mariano E. Fernandez-Miyakawa ◽  
Sameera Sayeed ◽  
Derek J. Fisher ◽  
Rachael Poon ◽  
Vicki Adams ◽  
...  
Keyword(s):  
Mouse Model ◽  
Clostridium Perfringens ◽  
Clinical Signs ◽  
Passive Immunization ◽  
Small Animal ◽  
Toxin Production ◽  
Oral Challenge ◽  
Type D ◽  
Epsilon Toxin

ABSTRACT Clostridium perfringens type D isolates cause enterotoxemia in sheep, goats, and probably cattle. While the major disease signs and lesions of type D animal disease are usually attributed to epsilon toxin, a class B select agent, these bacteria typically produce several lethal toxins. Understanding of disease pathogenesis and development of improved vaccines are hindered by the lack of a small-animal model mimicking natural disease caused by type D isolates. Addressing this need, we developed an oral challenge mouse model of C. perfringens type D enterotoxemia. When BALB/c mice with a sealed anus were inoculated by intragastric gavage with type D isolates, 7 of 10 type D isolates were lethal, as defined by spontaneous death or severe clinical signs necessitating euthanasia. The lethalities of the seven type D isolates varied between 14 and 100%. Clinical signs in the lethally challenged mice included seizures, convulsions, hyperexcitability, and/or depression. Mild intestinal gas distention and brain edema were observed at necropsy in a few mice, while histology showed multifocal acute tubular necrosis of the kidney and edema in the lungs of most challenged mice that developed a clinical response. When the lethality of type D isolates in this model was compared with in vitro toxin production, only a limited correlation was observed. However, mice could be protected against lethality by intravenous passive immunization with an epsilon toxin antibody prior to oral challenge. This study provides an economical new model for studying the pathogenesis of C. perfringens type D infections.

scholarly journals In vitro potency test for evaluation of Clostridium perfringens type D epsilon toxoid

2013 ◽  
Vol 80 (4) ◽  
pp. 450-452 ◽  
Author(s):  
Felipe Masiero Salvarini ◽  
Zelia Ines Portela Lobato ◽  
Prhiscylla Sadana Pires ◽  
Rodrigo Otavio Silveira Silva ◽  
Guilherme Guerra Alves ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Neutralizing Antibodies ◽  
Mdck Cells ◽  
In Vitro Test ◽  
Type D ◽  
Epsilon Toxin ◽  
Vitro Test ◽  
Darby Canine Kidney

The control of enterotoxemia caused by the epsilon toxin, produced by Clostridium perfringens type D, is based on vaccination with epsilon toxoid. The potency test for this immunogen is conducted using seroneutralization in mice. Here, an in vitro test for detection of neutralizing antibodies with Madin-Darby Canine Kidney (MDCK) cells was standardized in order to study alternative methodologies for the potency test. Titers observed in the in vivo and in vitro seroneutralization tests had a correlation of 99.73%.

Ultrastructural studies on the interaction of haemophilus influenzae with human endothelial cells in vitro

1990 ◽  
Vol 48 (3) ◽  
pp. 636-637
Author(s):  
D.J.P. Ferguson ◽  
M. Virji ◽  
H. Kayhty ◽  
E.R. Moxon
Keyword(s):  
Endothelial Cells ◽  
Haemophilus Influenzae ◽  
Intercellular Junctions ◽  
Blood Stream ◽  
Ultrastructural Studies ◽  
Endothelial Barriers ◽  
Serotype B ◽  
Meningitis In Children ◽  
Respiratory Epithelial

Haemophilus influenzae is a human pathogen which causes meningitis in children. Systemic H. influenzae infection is largely confined to encapsulated serotype b organisms and is a major cause of meningitis in the U.K. and elsewhere. However, the pathogenesis of the disease is still poorly understood. Studies in the infant rat model, in which intranasal challenge results in bacteraemia, have shown that H. influenzae enters submucosal tissues and disseminates to the blood stream within minutes. The rapidity of these events suggests that H. influenzae penetrates both respiratory epithelial and endothelial barriers with great efficiency. It is not known whether the bacteria penetrate via the intercellular junctions, are translocated within the cells or carried across the cellular barrier in 'trojan horse' fashion within phagocytes. In the present studies, we have challenged cultured human umbilical cord_vein endothelial cells (HUVECs) with both capsulated (b+) and capsule-deficient (b-) isogenic variants of one strain of H. influenzae in order to investigate the interaction between the bacteria and HUVEC and the effect of the capsule.

scholarly journals Age influences susceptibility of brain capillary endothelial cells to La Crosse virus infection and cell death

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Rahul Basu ◽  
Vinod Nair ◽  
Clayton W. Winkler ◽  
Tyson A. Woods ◽  
Iain D. C. Fraser ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Death ◽  
Virus Infection ◽  
La Crosse Virus ◽  
Brain Capillary ◽  
Adult Mice ◽  
Age Related ◽  
Capillary Endothelial Cells ◽  
The Brain

Abstract Background A key factor in the development of viral encephalitis is a virus crossing the blood-brain barrier (BBB). We have previously shown that age-related susceptibility of mice to the La Crosse virus (LACV), the leading cause of pediatric arbovirus encephalitis in the USA, was associated with the ability of the virus to cross the BBB. LACV infection in weanling mice (aged around 3 weeks) results in vascular leakage in the olfactory bulb/tract (OB/OT) region of the brain, which is not observed in adult mice aged > 6–8 weeks. Thus, we studied age-specific differences in the response of brain capillary endothelial cells (BCECs) to LACV infection. Methods To examine mechanisms of LACV-induced BBB breakdown and infection of the CNS, we analyzed BCECs directly isolated from weanling and adult mice as well as established a model where these cells were infected in vitro and cultured for a short period to determine susceptibility to virus infection and cell death. Additionally, we utilized correlative light electron microscopy (CLEM) to examine whether changes in cell morphology and function were also observed in BCECs in vivo. Results BCECs from weanling, but not adult mice, had detectable infection after several days in culture when taken ex vivo from infected mice suggesting that these cells could be infected in vitro. Further analysis of BCECs from uninfected mice, infected in vitro, showed that weanling BCECs were more susceptible to virus infection than adult BCECs, with higher levels of infected cells, released virus as well as cytopathic effects (CPE) and cell death. Although direct LACV infection is not detected in the weanling BCECs, CLEM analysis of brain tissue from weanling mice indicated that LACV infection induced significant cerebrovascular damage which allowed virus-sized particles to enter the brain parenchyma. Conclusions These findings indicate that BCECs isolated from adult and weanling mice have differential viral load, infectivity, and susceptibility to LACV. These age-related differences in susceptibility may strongly influence LACV-induced BBB leakage and neurovascular damage allowing virus invasion of the CNS and the development of neurological disease.

Interaction of Neutrophils with Endothelial Cells, Fibroblasts and Their Extracellular Matrices: Microscopic and Computerised Analysis

1988 ◽  
Vol 16 (1) ◽  
pp. 48-53
Author(s):  
Marina Ziche ◽  
Lucia Morbidelli ◽  
Annalisa Rubino ◽  
Piero Dolara ◽  
Stefano Bianchi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Image Analysis ◽  
Vascular Endothelial Cells ◽  
Extracellular Matrices ◽  
Polymorphonuclear Neutrophil ◽  
Initial Event ◽  
Computerised Image Analysis ◽  
Capillary Endothelial Cells ◽  
Vitro Model

Polymorphonuclear neutrophil (PMN) interaction with vascular endothelial cells is the initial event in the migration of neutrophils through blood vessel walls before reaching inflammation sites in tissues. The interaction between fibroblasts and endothelial cells and their extracellular matrices might be modulated by the activation of neutrophils that occurs at inflammatory reaction sites. We have used an in vitro model to study PMN function, measuring the adhesion of human PMNs to capillary endothelial cells and fibroblasts grown in culture and to their extracellular matrices. The interaction was measured in basal conditions and in the presence of the chemotactic effector, formyl-methionyl-leucyl-phenylalanine (FMLP at the concentration of 10 7M). Adhesion was expressed by the number of adherent PMNs/mm2 on a histological specimen. Moreover, we have adapted a program for image analysis to quantify neutrophil adhesion. Three times more PMNs adhered to matrices than to monolayers, and adherence could be increased by the presence of 10-7M FMLP, except in the case of fibroblast monolayers. We found a good correlation between microscopic observation and computerised image analysis measuring PMN adhesiveness to extracellular matrices.

scholarly journals CodY Is a Global Regulator of Virulence-Associated Properties for Clostridium perfringens Type D Strain CN3718

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Jihong Li ◽  
Menglin Ma ◽  
Mahfuzur R. Sarker ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Null Mutant ◽  
Intestinal Infection ◽  
Wild Type ◽  
Global Regulator ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Type D ◽  
Epsilon Toxin ◽  
Virulence Properties

ABSTRACT CodY is known to regulate various virulence properties in several Gram-positive bacteria but has not yet been studied in the important histotoxic and intestinal pathogen Clostridium perfringens. The present study prepared an isogenic codY-null mutant in C. perfringens type D strain CN3718 by insertional mutagenesis using the Targetron system. Western blot analysis indicated that, relative to wild-type CN3718 or a complementing strain, this isogenic codY mutant produces reduced levels of epsilon toxin (ETX). Using supernatants from cultures of the wild-type, codY-null mutant, and complementing strains, CodY regulation of ETX production was shown to have cytotoxic consequences for MDCK cells. The CodY regulatory effect on ETX production was specific, since the codY-null mutant still made wild-type levels of alpha-toxin and perfringolysin O. Sialidase activity measurements and sialidase Western blot analysis of supernatants from CN3718 and its isogenic derivatives showed that CodY represses overall exosialidase activity due to a reduced presence of NanH in culture supernatants. Inactivation of the codY gene significantly decreased the adherence of CN3718 vegetative cells or spores to host Caco-2 cells. Finally, the codY mutant showed increased spore formation under vegetative growth conditions, although germination of these spores was impaired. Overall, these results identify CodY as a global regulator of many C. perfringens virulence-associated properties. Furthermore, they establish that, via CodY, CN3718 coordinately regulates many virulence-associated properties likely needed for intestinal infection. IMPORTANCE Clostridium perfringens is a major human and livestock pathogen because it produces many potent toxins. C. perfringens type D strains cause intestinal infections by producing toxins, especially epsilon toxin (ETX). Previous studies identified CodY as a regulator of certain virulence properties in other Gram-positive bacteria. Our study now demonstrates that CodY is a global regulator of virulence-associated properties for type D strain CN3718. It promotes production of ETX, attachment of CN3718 vegetative cells or spores to host enterocyte-like Caco-2 cells, and spore germination; the last two effects may assist intestinal colonization. In contrast, CodY represses sporulation. These results provide the first evidence that CodY can function as a global regulator of C. perfringens virulence-associated properties and that this strain coordinately regulates its virulence-associated properties using CodY to increase ETX production, host cell attachment, and spore germination but to repress sporulation, as would be optimal during type D intestinal infection.

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