THE INFLUENCE OF POTATO CYST NEMATODE G. ROSTOCHIENSIS INFESTATION ON DIFFERENT POTATO CULTIVARS

The potato cyst nematode Globodera rostochiensis is one of the most serious pests of potato in Slovenia. Precise nematode identification and knowledge about potato cultivars, which are most suitable for growing in the Slovenian climate conditions and most resistant to G. rostochiensis, are necessary to develop an effective integrated pest control. Here we report the results of the influence of G. rostochiensis pathotype Ro1/4 on the yield of different potato cultivars: the susceptible cultivar Desiree, the resistant cultivars White Lady, Miranda, Aladin, Sante and Adora, and the clone KIS 94-1/5-14. The yield of cv. White Lady was the highest and that of susceptible cv. Desiree the lowest. The influence of several resistant and one susceptible potato cultivars on population dynamics of G. rostochiensis was also determined. The total number of cysts/100 cm3 and the number of eggs and juveniles per cyst increased in the susceptible cv. Desiree and decreased in the resistant cultivars White Lady, Sante and Adora

Variations in TcdB Activity and the Hypervirulence of Emerging Strains of Clostridium difficile

Hypervirulent strains of Clostridium difficile have emerged over the past decade, increasing the morbidity and mortality of patients infected by this opportunistic pathogen. Recent work suggested the major C. difficile virulence factor, TcdB, from hypervirulent strains (TcdBHV) was more cytotoxic in vitro than TcdB from historical strains (TcdBHIST). The current study investigated the in vivo impact of altered TcdB tropism, and the underlying mechanism responsible for the differences in activity between the two forms of this toxin. A combination of protein sequence analyses, in vivo studies using a Danio rerio model system, and cell entry combined with fluorescence assays were used to define the critical differences between TcdBHV and TcdBHIST. Sequence analysis found that TcdB was the most variable protein expressed from the pathogenicity locus of C. difficile. In line with these sequence differences, the in vivo effects of TcdBHV were found to be substantially broader and more pronounced than those caused by TcdBHIST. The increased toxicity of TcdBHV was related to the toxin's ability to enter cells more rapidly and at an earlier stage in endocytosis than TcdBHIST. The underlying biochemical mechanism for more rapid cell entry was identified in experiments demonstrating that TcdBHV undergoes acid-induced conformational changes at a pH much higher than that of TcdBHIST. Such pH-related conformational changes are known to be the inciting step in membrane insertion and translocation for TcdB. These data provide insight into a critical change in TcdB activity that contributes to the emerging hypervirulence of C. difficile

Comparative genomic analysis of toxin-negative strains of Clostridium difficile from humans and animals with symptoms of gastrointestinal disease

Background: Clostridium difficile infections (CDI) are a significant health problem to humans and food animals. Clostridial toxins ToxA and ToxB encoded by genes tcdA and tcdB are located on a pathogenicity locus known as the PaLoc and are the major virulence factors of C. difficile. While toxin-negative strains of C. difficile are often isolated from faeces of animals and patients suffering from CDI, they are not considered to play a role in disease. Toxin-negative strains of C. difficile have been used successfully to treat recurring CDI but their propensity to acquire the PaLoc via lateral gene transfer and express clinically relevant levels of toxins has reinforced the need to characterise them genetically. In addition, further studies that examine the pathogenic potential of toxin-negative strains of C. difficile and the frequency by which toxin-negative strains may acquire the PaLoc are needed. Results: We undertook a comparative genomic analysis of five Australian toxin-negative isolates of C. difficile that lack tcdA, tcdB and both binary toxin genes cdtA and cdtB that were recovered from humans and farm animals with symptoms of gastrointestinal disease. Our analyses show that the five C. difficile isolates cluster closely with virulent toxigenic strains of C. difficile belonging to the same sequence type (ST) and have virulence gene profiles akin to those in toxigenic strains. Furthermore, phage acquisition appears to have played a key role in the evolution of C. difficile. Conclusions: Our results are consistent with the C. difficile global population structure comprising six clades each containing both toxin-positive and toxin-negative strains. Our data also suggests that toxin-negative strains of C. difficile encode a repertoire of putative virulence factors that are similar to those found in toxigenic strains of C. difficile, raising the possibility that acquisition of PaLoc by toxin-negative strains poses a threat to human health. Studies in appropriate animal models are needed to examine the pathogenic potential of toxin-negative strains of C. difficile and to determine the frequency by which toxin-negative strains may acquire the PaLoc

WSES guidelines for management of Clostridium difficile infection in surgical patients

In the last two decades there have been dramatic changes in the epidemiology of Clostridium difficile infection (CDI), with increases in incidence and severity of disease in many countries worldwide. The incidence of CDI has also increased in surgical patients. Optimization of management of C difficile, has therefore become increasingly urgent. An international multidisciplinary panel of experts prepared evidenced-based World Society of Emergency Surgery (WSES) guidelines for management of CDI in surgical patients.Peer reviewe

The role of toxin A and toxin B in Clostridium difficile-associated disease: Past and present perspectives

Recently, we constructed and characterized isogenic tcdA and tcdB mutants of a virulent Clostridium difficile strain and used a hamster model of disease to demonstrate that toxin B, not toxin A, is essential for virulence of this emerging pathogen. Earlier studies had shown that purified toxin A alone was able to induce C. difficile disease pathology and that purified toxin B was not effective unless it was co-administered with toxin A, suggesting that the toxins act synergistically. In this addendum we discuss this paradigm-shifting conclusion in the context of current strain epidemiology, particularly with respect to naturally occurring toxin A-negative, toxin B-positive isolates and the NAP1/027 epidemic isolates. The role of toxin receptors and how variant toxins might exert their effects is also discussed in relation to the published data. We conclude that it is critical to use the natural infection process to dissect the role of toxins in disease, and that future studies are contingent on such work. The impact and importance of animal models of C. difficile virulence are therefore considered within this frame of reference

Membrane Translocation of Binary Actin-ADP-Ribosylating Toxins from Clostridium difficile and Clostridium perfringens Is Facilitated by Cyclophilin A and Hsp90 ▿

Some hypervirulent strains of Clostridium difficile produce the binary actin-ADP-ribosylating toxin C. difficile transferase (CDT) in addition to Rho-glucosylating toxins A and B. It has been suggested that the presence of CDT increases the severity of C. difficile-associated diseases, including pseudomembranous colitis. CDT contains a binding and translocation component, CDTb, that mediates the transport of the separate enzyme component CDTa into the cytosol of target cells, where CDTa modifies actin. Here we investigated the mechanism of cellular CDT uptake and found that bafilomycin A1 protects cultured epithelial cells from intoxication with CDT, implying that CDTa is translocated from acidified endosomal vesicles into the cytosol. Consistently, CDTa is translocated across the cytoplasmic membranes into the cytosol when cell-bound CDT is exposed to acidic medium. Radicicol and cyclosporine A, inhibitors of the heat shock protein Hsp90 and cyclophilins, respectively, protected cells from intoxication with CDT but not from intoxication with toxins A and B. Moreover, both inhibitors blocked the pH-dependent membrane translocation of CDTa, strongly suggesting that Hsp90 and cyclophilin are crucial for this process. In contrast, the inhibitors did not interfere with the ADP-ribosyltransferase activity, receptor binding, or endocytosis of the toxin. We obtained comparable results with the closely related iota-toxin from Clostridium perfringens. Moreover, CDTa and Ia, the enzyme component of iota-toxin, specifically bound to immobilized Hsp90 and cyclophilin A in vitro. In combination with our recently obtained data on the C2 toxin from C. botulinum, these results imply a common Hsp90/cyclophilin A-dependent translocation mechanism for the family of binary actin-ADP-ribosylating toxins