Heterogeneous immunoreactivity of axonal spheroids in focal symmetrical encephalomalacia produced by Clostridium perfringens type D epsilon toxin in sheep

Since axonal injury (AI) is an important component of many veterinary neurologic disorders, we assessed the relative ability of a panel of antibodies (amyloid precursor protein, 3 subunits of neurofilament protein, protein gene product 9.5, ubiquitin, and synaptophysin) to detect axonal swellings or spheroids. Abundant axonal spheroids found in necrotic internal capsule foci produced in 4 sheep by chronic Clostridium perfringens type D epsilon neurotoxicity provided a model system in which to evaluate this important diagnostic tool. There was heterogeneous labeling of subsets of spheroids by the respective antibodies, suggesting that, in order to detect the complete spectrum of AI in diagnostic cases, a range of antibodies should be used, not only when spheroids are plentiful but also when they are few in number or incompletely developed. The application of insufficient markers in the latter cases can potentially lead to the contribution of AI to lesion pathogenesis being underappreciated.

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

mBio ◽

10.1128/mbio.00770-13 ◽

2013 ◽

Vol 4 (5) ◽

Cited By ~ 27

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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The production and evaluation of monoclonal antibodies to Clostridium perfringens type D epsilon toxin

Journal of Biological Standardization ◽

10.1016/0092-1157(88)90008-x ◽

1988 ◽

Vol 16 (3) ◽

pp. 207-218 ◽

Cited By ~ 7

Author(s):

C.D.H. Boarer ◽

M.G. Sojka ◽

V.J. White ◽

P.L. Roeder

Keyword(s):

Monoclonal Antibodies ◽

Clostridium Perfringens ◽

Type D ◽

Epsilon Toxin

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Outbreak of Clostridium perfringens type D enterotoxemia in sheep

Semina Ciências Agrárias ◽

10.5433/1679-0359.2019v40n6p2593 ◽

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

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638719900190 ◽

2020 ◽

Vol 32 (2) ◽

pp. 282-286 ◽

Cited By ~ 2

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.

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

Anaerobe ◽

10.1016/s1075-9964(03)00069-6 ◽

2003 ◽

Vol 9 (3) ◽

pp. 145-149 ◽

Cited By ~ 10

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

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Selection of a Clostridium perfringens type D epsilon toxin producer via dot-blot test

Archives of Microbiology ◽

10.1007/s00203-009-0510-y ◽

2009 ◽

Vol 191 (11) ◽

pp. 847-851 ◽

Cited By ~ 8

Author(s):

Luciana A. Gonçalves ◽

Zélia I. P. Lobato ◽

Rodrigo O. S. Silva ◽

Felipe M. Salvarani ◽

Prhiscylla S. Pires ◽

...

Keyword(s):

Clostridium Perfringens ◽

Dot Blot ◽

Type D ◽

Epsilon Toxin ◽

Selection Of

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NanI Sialidase, CcpA, and CodY Work Together To Regulate Epsilon Toxin Production by Clostridium perfringens Type D Strain CN3718

Journal of Bacteriology ◽

10.1128/jb.00349-15 ◽

2015 ◽

Vol 197 (20) ◽

pp. 3339-3353 ◽

Cited By ~ 14

Author(s):

Jihong Li ◽

John C. Freedman ◽

Bruce A. McClane

Keyword(s):

Sialic Acid ◽

Clostridium Perfringens ◽

Host Cells ◽

Start Codon ◽

Null Mutant ◽

Wild Type ◽

Mobility Shift ◽

Content Type ◽

Type D ◽

Epsilon Toxin

ABSTRACTClostridium perfringenstype D strains are usually associated with diseases of livestock, and their virulence requires the production of epsilon toxin (ETX). We previously showed (J. Li, S. Sayeed, S. Robertson, J. Chen, and B. A. McClane, PLoS Pathog 7:e1002429, 2011,http://dx.doi.org/10.1371/journal.ppat.1002429) that BMC202, ananInull mutant of type D strain CN3718, produces less ETX than wild-type CN3718 does. The current study proved that the lower ETX production by strain BMC202 is due tonanIgene disruption, since both genetic and physical (NanI or sialic acid) complementation increased ETX production by BMC202. Furthermore, a sialidase inhibitor that interfered with NanI activity also reduced ETX production by wild-type CN3718. The NanI effect on ETX production was shown to involve reductions incodYandccpAgene transcription levels in BMC202 versus wild-type CN3718. Similar to CodY, CcpA was found to positively control ETX production. A doublecodYccpAnull mutant produced even less ETX than acodYorccpAsingle null mutant. CcpA bound directly to sequences upstream of theetxorcodYstart codon, and bioinformatics identified putative CcpA-bindingcresites immediately upstream of both thecodYandetxstart codons, suggesting possible direct CcpA regulatory effects. AccpAmutation also decreasedcodYtranscription, suggesting that CcpA effects on ETX production can be both direct and indirect, including effects oncodYtranscription. Collectively, these results suggest that NanI, CcpA, and CodY work together to regulate ETX production, with NanI-generated sialic acid from the intestines possibly signaling type D strains to upregulate their ETX production and induce disease.IMPORTANCEClostridium perfringensNanI was previously shown to increase ETX binding to, and cytotoxicity for, MDCK host cells. The current study demonstrates that NanI also regulates ETX production via increased transcription of genes encoding the CodY and CcpA global regulators. Results obtained using singleccpAorcodYnull mutants and accpAcodYdouble null mutant showed thatcodYandccpAregulate ETX production independently of one another but thatccpAalso affectscodYtranscription. Electrophoretic mobility shift assays and bioinformatic analyses suggest that both CodY and CcpA may directly regulateetxtranscription. Collectively, results of this study suggest that sialic acid generated by NanI from intestinal sources signals ETX-producingC. perfringensstrains, via CcpA and CodY, to upregulate ETX production and cause disease.

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Epsilon-Toxin Plasmids of Clostridium perfringens Type D Are Conjugative

Journal of Bacteriology ◽

10.1128/jb.00767-07 ◽

2007 ◽

Vol 189 (21) ◽

pp. 7531-7538 ◽

Cited By ~ 53

Author(s):

Meredith L. Hughes ◽

Rachael Poon ◽

Vicki Adams ◽

Sameera Sayeed ◽

Juliann Saputo ◽

...

Keyword(s):

Clostridium Perfringens ◽

Mdck Cells ◽

Type A ◽

Department Of Agriculture ◽

Chloramphenicol Resistance ◽

Cytopathic Effects ◽

Type D ◽

Class B ◽

Epsilon Toxin ◽

Domestic Livestock

ABSTRACT Isolates of Clostridium perfringens type D produce the potent epsilon-toxin (a CDC/U.S. Department of Agriculture overlap class B select agent) and are responsible for several economically significant enterotoxemias of domestic livestock. It is well established that the epsilon-toxin structural gene, etx, occurs on large plasmids. We show here that at least two of these plasmids are conjugative. The etx gene on these plasmids was insertionally inactivated using a chloramphenicol resistance cassette to phenotypically tag the plasmid. High-frequency conjugative transfer of the tagged plasmids into the C. perfringens type A strain JIR325 was demonstrated, and the resultant transconjugants were shown to act as donors in subsequent mating experiments. We also demonstrated the transfer of “unmarked” native ε-toxin plasmids into strain JIR325 by exploiting the high transfer frequency. The transconjugants isolated in these experiments expressed functional ε-toxin since their supernatants had cytopathic effects on MDCK cells and were toxic in mice. Using the widely accepted multiplex PCR approach for toxin genotyping, these type A-derived transconjugants were genotypically type D. These findings have significant implications for the C. perfringens typing system since it is based on the toxin profile of each strain. Our study demonstrated the fluid nature of the toxinotypes and their dependence upon the presence or absence of toxin plasmids, some of which have for the first time been shown to be conjugative.

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