In vitro effects of Clostridium perfringens type D epsilon toxin on water and ion transport in ovine and caprine intestine

Anaerobe ◽  
2003 ◽  
Vol 9 (3) ◽  
pp. 145-149 ◽  
Author(s):  
M.E. Fernandez Miyakawa ◽  
C.A. Ibarra ◽  
F.A. Uzal
Keyword(s):  
Ion Transport ◽  
Clostridium Perfringens ◽  
Type D ◽  
Epsilon Toxin ◽  
In Vitro Effects ◽  
Water And Ion Transport

29. In vivo and in vitro effects of epsilon toxin mutants from Clostridium perfringens

Toxicon ◽  
2014 ◽  
Vol 91 ◽  
pp. 176
Author(s):  
J. Dorca-Arévalo ◽  
L. Díaz ◽  
M. Martín-Satué ◽  
J. Blasi
Keyword(s):  
Clostridium Perfringens ◽  
Epsilon Toxin ◽  
In Vitro Effects

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 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%.

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.

scholarly journals A Novel Panel of Rabbit Monoclonal Antibodies and Their Diverse Applications Including Inhibition of Clostridium perfringens Epsilon Toxin Oligomerization

Antibodies ◽  
2018 ◽  
Vol 7 (4) ◽  
pp. 37 ◽  
Author(s):  
Jennifer Linden ◽  
Kiel Telesford ◽  
Samantha Shetty ◽  
Paige Winokour ◽  
Sylvia Haigh ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Monoclonal Antibodies ◽  
Western Blot ◽  
Clostridium Perfringens ◽  
Specificity And Sensitivity ◽  
Epsilon Toxin ◽  
Blocking Antibodies

The pore-forming epsilon toxin (ETX) produced by Clostridium perfringens is among the most lethal bacterial toxins known. Sensitive antibody-based reagents are needed to detect toxin, distinguish mechanisms of cell death, and prevent ETX toxicity. Using B-cell immuno-panning and cloning techniques, seven ETX-specific monoclonal antibodies were generated from immunized rabbits. ETX specificity and sensitivity were evaluated via western blot, ELISA, immunocytochemistry (ICC), and flow cytometry. ETX-neutralizing function was evaluated both in vitro and in vivo. All antibodies recognized both purified ETX and epsilon protoxin via western blot with two capable of detecting the ETX-oligomer complex. Four antibodies detected ETX via ELISA and three detected ETX bound to cells via ICC or flow cytometry. Several antibodies prevented ETX-induced cell death by either preventing ETX binding or by blocking ETX oligomerization. Antibodies that blocked ETX oligomerization inhibited ETX endocytosis and cellular vacuolation. Importantly, one of the oligomerization-blocking antibodies was able to protect against ETX-induced death post-ETX exposure in vitro and in vivo. Here we describe the production of a panel of rabbit monoclonal anti-ETX antibodies and their use in various biological assays. Antibodies possessing differential specificity to ETX in particular conformations will aid in the mechanistic studies of ETX cytotoxicity, while those with ETX-neutralizing function may be useful in preventing ETX-mediated mortality.

scholarly journals Outbreak of Clostridium perfringens type D enterotoxemia in sheep

2019 ◽  
Vol 40 (6) ◽  
pp. 2593
Author(s):  
Felipe Masiero Salvarani ◽  
Mayane Faccin ◽  
Nayra Fernanda de Queiroz Ramos Freitas ◽  
Mônica Regina de Matos ◽  
Edismair Carvalho Garcia ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Clinical Signs ◽  
Necrotic Enteritis ◽  
Spastic Paralysis ◽  
Type D ◽  
Laboratory Confirmation ◽  
Intestinal Contents ◽  
Epsilon Toxin ◽  
History Of ◽  
Diagnostic Modalities

This work describes the first Brazilian laboratory-confirmed outbreak of enterotoxemia caused by Clostridium perfringens type D in sheep, which occurred in the state of Paraná. We address the epidemiological aspects involved, the diagnostic modalities employed, and the clinical signs and pathological findings observed. Eight healthy pregnant female sheep with no history of vaccination for clostridiosis presented with a history of abrupt feeding changes and neurological manifestations that quickly evolved to illness, coma and death. Four other females with clinical neurological signs were referred to the Veterinary Hospital of the Universidade Federal do Paraná, Palotina Sector. These animals presented with lethargy, motor incoordination, opisthotonus, pedal movements, muscle tremors, spastic paralysis, bruxism, mandibular trismus, sialorrhea, hyperexcitability and the inability to stand. They were examined and euthanized due to the seriousness of the clinical picture with an unfavorable prognosis. We performed gross anatomical and microscopic analyses of the organs and intestinal contents. We also performed bacterial isolation with molecular typing. From the intestinal contents, we detected toxins by means of the seroneutralization technique in mice. At necropsy, we noted pulmonary edema (2/4), necrotizing enteritis (4/4) and hyperemia of the leptomeninges (1/4). Microscopically, we observed lymphohistiocytic interstitial pneumonia, necrotic enteritis associated with the presence of rods, and nephrosis with interstitial lymphohistiocytic nephritis. No significant brain lesions were observed. Using serum neutralization, we identified epsilon toxin in the intestinal contents of all four animals. C. perfringens type D was identified. Based on the history, clinical signs, postmortem findings, and laboratory confirmation of the presence of epsilon toxin, we concluded that C. perfringens type D enterotoxemia caused this outbreak of sheep deaths.

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