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      Association between avian necrotic enteritis and Clostridium perfringens strains expressing NetB toxin

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          Abstract

          A novel toxin, NetB, has recently been identified in virulent avian Clostridium perfringens isolates and shown to be an essential virulence factor in a clinical necrotic enteritis isolate. To assess whether NetB is more generally associated with avian necrotic enteritis isolates we have screened a range of C. perfringens strains from geographically diverse locations for both the presence and expression of the netB gene. Forty-four isolates were derived from necrotic enteritis disease cases from Australia, Belgium, Denmark and Canada and 55 isolates from healthy chickens from Australia and Belgium. The majority of strains isolated from necrotic enteritis-affected birds were netB positive (70%) and there was an absolute correlation between the presence of netB and in vitro expression of the NetB protein. Only two of the C. perfringens isolates from healthy chickens carried netB. Sequencing of the netB gene from 23 positive isolates showed that NetB is highly conserved, with only one predicted amino acid (A168T) difference, in six isolates, compared to the published sequence. This change did not alter the in vitro activity of the NetB toxin. The gene encoding the recently discovered TpeL toxin was also screened using PCR and only found in a small proportion of NetB-positive isolates from diseased birds. A selection of NetB-negative isolates, originating from diseased birds, was unable to cause disease in a necrotic enteritis induction model. This study provides further evidence that NetB is important in pathogenesis and advances our current understanding of C. perfringens virulence factors in avian necrotic enteritis.

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          Most cited references18

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          Clostridial enteric diseases of domestic animals.

          J Songer (1996)
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            Virulence genes of Clostridium perfringens.

            J Rood (1997)
            Clostridium perfringens causes human gas gangrene and food poisoning as well as several enterotoxemic diseases of animals. The organism is characterized by its ability to produce numerous extracellular toxins including alpha-toxin or phospholipase C, theta-toxin or perfringolysin O, kappa-toxin or collagenase, as well as a sporulation-associated enterotoxin. Although the genes encoding the alpha-toxin and theta-toxin are located on the chromosome, the genes encoding many of the other extracellular toxins are located on large plasmids. The enterotoxin gene can be either chromosomal or plasmid determined. Several of these toxin genes are associated with insertion sequences. The production of many of the extracellular toxins is regulated at the transcriptional level by the products of the virR and virS genes, which together comprise a two-component signal transduction system.
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              Construction and analysis of chromosomal Clostridium difficile mutants.

              Clostridium difficile is an emerging nosocomial pathogen of increasing importance and virulence but our ability to study the molecular mechanisms underlying the pathogenesis of C. difficile-associated disease has been limited because of a lack of tools for its genetic manipulation. We have now developed a reproducible method for the targeted insertional inactivation of chromosomal C. difficile genes. The approach relies on the observation that an Escherichia coli-Clostridium perfringens shuttle vector is unstable in C. difficile and can be used as a form of conditional lethal vector to deliver gene constructs to the chromosome. We have used this methodology to insertionally inactivate two putative response regulator genes, rgaR and rgbR, which encode proteins with similarity to the toxin gene regulator, VirR, from C. perfringens. Transcriptomic analysis demonstrated that the C. difficile RgaR protein positively regulated four genes, including a putative agrBD operon. The RgaR protein was also purified and shown to bind specifically to sites that contained two consensus VirR boxes located just upstream of the putative promoters of these genes. The development of this methodology will significantly enhance our ability to use molecular approaches to develop a greater understanding of the ability of C. difficile to cause disease.
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                Author and article information

                Journal
                Vet Res
                vetres
                Veterinary Research
                EDP Sciences
                0928-4249
                1297-9716
                25 November 2009
                Mar-Apr 2010
                : 41
                : 2 ( publisher-idID: vetres/2010/02 )
                : 21
                Affiliations
                [1 ] CSIRO Livestock Industries, Australian Animal Health Laboratory Geelong Victoria 3220 Australia
                [2 ] Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Department of Microbiology, Monash University Clayton Victoria 3800 Australia
                [3 ] Australian Poultry Cooperative Research Centre Armidale New South Wales 2315 Australia
                [4 ] Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University Salisburylaan 133 B-9890 Merelbeke Belgium
                Author notes
                [* ]Corresponding author: rob.moore@ 123456csiro.au
                Article
                v09517 10.1051/vetres/2009069
                10.1051/vetres/2009069
                2797654
                19931005
                0334de95-9a0b-4d03-b5b8-72761ac71414
                © INRA, EDP Sciences, 2009
                History
                : 15 September 2009
                : 20 November 2009
                Page count
                Figures: 2, Tables: 1, Equations: 0, References: 23, Pages: 8
                Categories
                Original Article

                Veterinary medicine
                netb toxin,necrotic enteritis,tpel,clostridium perfringens,virulence factor
                Veterinary medicine
                netb toxin, necrotic enteritis, tpel, clostridium perfringens, virulence factor

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