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A new PCR followed by MboI digestion for the detection of all variants of the Clostridium perfringens cpb2 gene

Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, P.O. Box 80.158, 3508 TD Utrecht, The Netherlands.
Veterinary Microbiology (Impact Factor: 2.51). 04/2008; 127(3-4):412-6. DOI: 10.1016/j.vetmic.2007.08.035
Source: PubMed

ABSTRACT

Clostridium perfringens which is a causative agent of several diseases in animals and humans is capable of producing a variety of toxins. Isolates are typed into five types on the basis of the presence of one or more of the four major toxins genes, i.e. cpa, cpb, etx, and iap. A decade ago another toxin termed beta2 (beta2) and its gene (cpb2) were identified. Two alleles of cpb2 are known and a possible link between differences in gene expression and allelic variation has been reported. A correlation between the level of expression and the origin of the isolates has also been suggested. The demonstration and typing of the cpb2 gene in the genome of isolates can be seen as a vital part of research on the role of the beta2 toxin in the pathogenesis of disease. This study describes a PCR with a single primer set which in contrast to published primer sets recognizes both alleles. Subsequent restriction enzyme analysis of the PCR product enables typing of the alleles. Applying this protocol on a total of 102 isolates, a sub-variant was found which occurred only in C. perfringens isolates from pigs and appeared to be the predominant variant found in C. perfringens isolates from this species.

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    • "However, the role of beta2 toxin in SNE might be variant dependent since all cpb2-carrying C. perfringens isolates from SNE birds carried the atypical allele, and the cpb2-carrying C. perfringens isolate from the SPF bird carried the consensus three allele. The role of the variants of beta2 toxin in intestinal disorders in various animal species is still under discussion (Jost et al., 2005;Lebrun et al., 2007;van Asten et al., 2008). The difference in Subclinical necrotic enteritis in laying hens 545 expression of cpb2 between the various atypical cpb2 allele-harbouring C. perfringens isolates from the same bird is in line with what has been demonstrated in another study (Jost et al., 2005). "
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    ABSTRACT: Since 2006 increasing numbers of laying hen flocks with decreased production have been reported in the Netherlands. At necropsy, birds from affected flocks showed multifocal areas of necrosis in the duodenum. Histologically the duodenum had moderate to marked villus atrophy and fusion with crypt hyperplasia and a mixed inflammatory infiltrate within the lamina propria underlying focal areas of degenerative epithelium. Multifocally, free within the intestinal lumen and associated with epithelial necrosis, were marked numbers of large rod-shaped bacteria. Anaerobic culturing and subsequent toxin typing revealed, in 19 out of 73 affected birds, the presence of Clostridium perfringens strains, either type A or type C harbouring the atypical allele of cpb2 and netB. Eighteen out of these 19 birds carried C. perfringens strains capable of producing beta2 toxin in vitro and all of these birds harboured C. perfringens strains capable of producing NetB toxin in vitro. In contrast, specific pathogen free (SPF) birds lacked gross or histological lesions in their duodenum, and C. perfringens type C was isolated from four out of 15 SPF birds tested. One of these isolates harboured the consensus three allele of cpb2 that produced beta2 toxin in vitro. None of the C. perfringens isolates originating from SPF birds harboured netB. These findings might indicate that the NetB toxin produced by C. perfringens is associated with subclinical necrotic enteritis in layers, whereas the involvement of beta2 toxin in subclinical necrotic enteritis, if any, might be variant dependent.
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    ABSTRACT: Zentraler Bestandteil der Bekämpfung C. perfringens-bedingter Durchfallerkrankungen von Ferkeln ist die Muttertierschutzimpfung mit Toxoidvakzinen. Da die zugelassenen Impfstoffe kein Beta2-Toxoid enthalten sind Neuentwicklungen unabdingbar. Eine Schlüsselrolle kommt der Herstellung der Toxine Beta und Beta2 zu, die für Wirksamkeitsnachweise, Pathogenitäts- und Immunisierungsstudien benötigt werden. Auf Grund ihrer besonderen Empfindlichkeit und der vielen Major- und Minortoxine, die C. perfringens-Wildstämme bilden, ist die heterologe, rekombinante Expression und affinitätschromatographische Aufreinigung zweckmäßig. Das Beta2-Toxin konnte mit dem pASK-IBA-Vektorsystem in E. coli TOP10F´ exprimiert und in SDS-PAGE, ELISA, Western und Immunblot nachgewiesen werden. Ein C-terminal mit einem Strep-Tag® II versehenes Beta2-Toxin aus dem Periplasma induzierter Klone wurde zur Gewinnung poly- und monoklonaler Antikörper genutzt, die natives Beta2-Toxin im Kulturüberstand von C. perfringens detektierten. Es besaß jedoch keine Aktivität im Mausmodell oder im Zellkulturversuch mit Caco-2- und IPEC-J2-Zellen. Daher wurden weitere Expressionsklone erzeugt. Doch auch durch Variation der Position des Strep-Tag® II Affinitätsanhängsels (C- bzw. N-terminal), des Expressions¬kompartimentes (Zyto- und Periplasma) und des Expressionswirtes (TOP10F´ und Rosetta-gami™ 2) gelang es in E. coli nicht, ein Beta2-Toxin mit toxischer Wirkung im Zell- oder Tierversuch zu gewinnen. In einem weiteren Ansatz wurde das Beta2-Toxingen einschließlich seines Signalpeptids mittels Plasmidvektor pHT01 auf den phylogenetisch näher mit C. perfringens verwandten Expressionsstamm B. subtilis WB800N übertragen. Die Sequenz des rekombinanten Beta2-Toxins entsprach exakt der des Wildtyp-Toxins aus C. perfringens. Die Expressionsbedingungen waren auf Produktschonung des temperatur- und sauerstoffempfindlichen Produktes ausgerichtet. Dennoch waren Beta2-Toxin-haltige Konzentrate des Kulturüberstandes für Caco-2 und IPEC-J2-Zellen atoxisch. Das Beta-Toxin-Gen cpb wurde mit Vektor pASK-IBA5plus ligiert und das resultierende Plasmid pHIT-VIc-42 auf E. coli TOP10F´ übertragen. Nach Induktion des Expressionsklons konnte das rekombinante Beta-Toxin mit N-terminalem Strep-Tag® II aus Ganzzelllysaten affinitätschromatographisch aufgereinigt und in SDS-PAGE, ELISA und Western Blot dargestellt werden. Nach intraperitonealer Applikation war es toxisch für BALB/c- und NMRI-Mäuse. Für letztere wurde eine halbletale Dosis von 78 µg/kg bzw. 1,56 µg/Tier ermittelt. Die letale Wirkung war damit im Vergleich mit einem aus C. perfringens-Kulturüberständen gewonnenen Toxin reduziert, was vermutlich auf das Affinitätsanhängsel zurückzuführen ist. Drei rekombinante Beta-Toxine anderer Arbeitsgruppen zeigten in Anwesenheit eines Affinitätsanhängsels keine Aktivität. Erstmalig wurde somit ein fusioniertes, biologisch aktives Beta-Toxin erzeugt, das für Neutralisationsversuche im Rahmen der Impfstoffzulassung zur Verfügung steht. Die gewonnenen Beta2-Toxin-Varianten besitzen keine biologische Aktivität obwohl sie sich sequentiell vom nativen Toxin aus C. perfringens nicht bzw. lediglich durch das angefügte Affinitätsanhängsels unterscheiden. Sie können zur Herstellung von Antikörpern und als Impfantigen genutzt werden. A central element for prevention and control of C. perfringens associated diarrhoea in piglets is the vaccination of pregnant sows with toxoid vaccines. Because none of the commercial vaccines contains Beta2 toxoid new developments are indispensable. The production of the two toxins Beta and Beta2, which are needed for efficacy testings, pathogenicity and immunization studies, is of great importance. Because of their high sensitivity and the numerous major and minor toxins which are produced by C. perfringens wild-type strains, the toxins were to be overexpressed in E. coli and to be purified by affinity chromatography. The Beta2 toxin was expressed in E. coli TOP10F´ using the pASK-IBA vector system. Carrying a C-termial Strep-Tag® II, it was purified from the periplasm of induced clones. The recombinant Beta2 was detected in SDS-PAGE, ELISA, Western and Immunoblot and used to produce poly- and monoclonal antibodies. Unfortunately, it did not show any activity in the mouse challenge or in cell culture assays with Caco-2 and IPEC-J2 cells. Therefore, further expression clones were generated. But although the position of the Strep-Tag® II (C- and N-terminal), the expression compartment (cyto- and periplasm) and the expression host (TOP10F´ und Rosetta-gami™ 2) were varied, a Beta2 product with toxic effect in cell culture or animal trial could not be obtained. In another approach the Beta2 toxin gene with its original signal peptide was introduced into the B. subtilis expression strain WB800N, which is phylogenetically more closely related to C. perfringens then E. coli, using plasmid vector pHT01. The sequence of the recombinant Beta2 toxin was consistent with that of wild type toxin from C. perfringens. The terms of expression were adapted to preserve the temperature and oxygen sensitive product. Nevertheless, concentrates of culture supernatants containing the Beta2 toxin were non-toxic for Caco-2 and IPEC-J2 cells. The Beta toxin gene cpb was ligated with vector pASK-IBA5plus and the resulting plasmid pHIT-VIc-42 was introduced into E. coli TOP10F´. After induction of the expression clone, the recombinant Beta toxin with its N-terminal Strep-Tag® II was purified from whole cell lysates via affinity chromatography. Beta toxin was detectable in SDS-PAGE, ELISA and Western blot and showed toxic activity in BALB/c- and NMRI-mice after intraperitoneal application. For NMRI-mice a median lethal dose of 78 µg/kg or 1.56 µg/animal was calculated respectively. The lethal effect was reduced compared to toxin from C. perfringens culture supernatants, probably due to the affinity tag. Three Beta toxins from different work groups showed no activity in presence of an affinity tag, so that this is the first time that a fused, biologically active Beta toxin was generated, which is available for neutralization assays within the scope of vaccine approvals.
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