A toxic approach to beta2-toxigenic Clostridium perfringens

Clostridium perfringens is one of the most important causes of intestinal disease in animals and humans. Its virulence is attributed to the several toxins it can produce, including the beta2 toxin encoded by cpb2. In this thesis we studied the role of the beta2 toxin produced by C. perfringens in the development of intestinal disease. Results described in this thesis demonstrate that a possible role for beta2 toxin in the development of intestinal disease is still disputable. The development of a new primer set which recognizes both alleles of cpb2 and the development of a new test strategy for application of the newly developed PCR on fecal samples improved test sensitivity for the presence of cpb2. However, a relationship between cpb2-harbouring C. perfringens, beta2 toxin,and intestinal disease could not be proven in chickens and roe deer. Although cpb2-positive C. perfringens was abundantly present in the intestinal tract of roe deer, only two isolated strains were able to produce the beta2 toxin in vitro and the beta2 toxin was not demonstrated in the intestinal tract by immunohistochemistry in any of the animals. C. perfringens was isolated from both healthy and diseased laying hens with a newly described form of subclinical necrotic enteritis. No relationship could be determined between intestinal disease and the presence of beta2 toxin producing C. perfringens. However, focal necrotic lesions were only found in the intestinal tract of laying hens with a high watery intestinal content which may have played a predisposing factor in the development of disease providing substrate for beta2 toxin production by C. perfringens. A significant role for the beta2 toxin in the development of intestinal disease could not be established in an in vitro model in which the cytotoxic effect of supernatant of cpb2-positive C. perfringens cultures with and without beta2 toxin were compared. A proven role for the beta2 toxin in the development of intestinal disease or reproduction of the disease by other infection models has not been achieved yet. Therefore the role of the beta2 toxin in the development of intestinal disease remains at least questionable. Other virulence factors, the intestinal microbiota, and environmental circumstances might influence the significance of the intestinal presence of cpb2-harbouring C. perfringens in the development of intestinal disease. This hypothesis was supported by the finding of a reducing effect of Lactobacillus fermentum on beta2 toxin production by cpb2-harbouring C. perfringens. A lower transcription of cpb2 and a lower production of beta2 toxin were demonstrated with the presence of L. fermentum while the viability of C. perfringens was not affected. In this way, environmental influences might increase the role of cpb2-harbouring C. perfringens in the development of intestinal disease too. Articles on predisposing factors for the development of C. perfringens-associated intestinal disease in animals and human beings were reviewed and discussed. In vivo studies investigating both beta2 toxin and predisposing factors are necessary to definitely determine or exclude a potential role for beta2 toxin in the development of intestinal disease

Beta2 toxin is not involved in in vitro cell cytotoxicity caused by human and porcine cpb2-harbouring Clostridium perfringens

Clostridium perfringens is a common cause of intestinal disease in animals and humans. Its pathogenicity is attributed to the toxins it can produce, including the beta2 toxin. The presence of cpb2, the gene encoding the beta2 toxin, has been associated with diarrhoea in neonatal piglets and humans. However, the exact role of the beta2 toxin in the development of diarrhoea is still unknown. In this study we investigated the level of cytotoxicity to porcine IPI-21 and human Caco-2 cell-lines caused by porcine and human cpb2-harbouring C. perfringens and the significance of the beta2 toxin for the induction of cell cytotoxicity. Supernatants of porcine cpb2-harbouring C. perfringens strains were cytotoxic to both cell lines. Cell cytotoxicity caused by supernatant of human cpb2- harbouring C. perfringens strains was variable among strains. However, removal of the beta2 toxin by anti-beta2 toxin antibodies or degradation of the beta2 toxin by trypsin did not reduce the cytotoxic effect of any of the supernatants. These data suggest that beta2 toxin does not play a role in the development of cell cytotoxicity in in vitro experiments. In vivo studies are necessary to definitely define the role of beta2 toxin in the development of cell cytotoxicity and subsequent diarrhoea

Transmission of methicillin-resistant Staphylococcus aureus strains between different kinds of pig farms

The main objective of the present study was to investigate if different kinds of pig farms, like farrowing farms and rearing farms, play a role in the transmission of methicillin-resistant Staphylococcus aureus (MRSA) to Dutch finishing farms. Twelve farrowing farms, 11 finishing farms, 6 farrow-to finish farms, 1 rearing farm and 1 centre for artificial insemination were included. Screening of 310 pigs from these 31 farms showed 35 pigs (11%) to carry MRSA in their nares. On 7 of the 31 (23%) investigated farms colonized pigs were found, including 3 finishing farms, 3 farrowing farms and 1 farrow-to-finish farm. The use of standard antimicrobial medication of the pigs seemed to be a risk factor for MRSA carriage. Screening of the pigs on six farms supplying pigs for the MRSA positive farms revealed that the pigs on all but one farm were MRSA positive. Genotyping revealed that all MRSA strains were non-typeable by PFGE using the SmaI restriction enzyme and had multilocus sequence type (MLST) ST398. Different spa-types were found including t011, t108, t567, t899 and t1939, but the spa-types on epidemiologically related farms were identical indicating that MRSA are transmitted between farms through the purchase of colonized pigs. Two SCCmec types were found among the MRSA: type IV and type V. SCCmec type V was predominant. On two farms MRSA isolates with ST398, the same spa-type but with different SCCmec types (IV and V) were found, suggesting that different SCCmec elements have been inserted into MSSA with the same genotype. All MRSA strains were resistant to tetracycline, but additional resistances to erythromycin, lincomycin, kanamycin and gentamicin were also found. All MRSA isolates were negative for the exfoliative toxin genes (eta and etb), PVL toxin genes (lukF and lukS), toxic shock syndrome gene (tst-1), and the leukotoxin genes (lukE, lukD, lukM, lukF')

Implantable defibrillator therapy and mortality in patients with non-ischaemic dilated cardiomyopathy An updated meta-analysis and effect on Dutch clinical practice by the Task Force of the Dutch Society of Cardiology

Background Primary prophylactic implantable cardioverter-defibrillators (ICDs) in patients with non-ischaemic cardiomyopathy (NICMP) remains controversial. This study sought to assess the benefit of ICD therapy with or without cardiac resynchronisation therapy (CRT) in patients with NICMP. In addition, data were compared with real-world clinical data to perform a risk/benefit analysis. Methods Relevant randomised clinical trials (RCTs) published in meta-analyses since DANISH, and in PubMed, EMBASE and Cochrane databases from 2016 to 2020 were identified. The benefit of ICD therapy stratified by CRT use was assessed using random effects meta-analysis techniques. Results Six RCTs were included in the meta-analysis. Among patients without CRT, ICD use was associated with a 24% reduction in mortality (hazard ratio [HR]: 0.76; 95% confidence interval [CI]: 0.62-0.93; P = 0.008). In contrast, among patients with CRT, a CRT-defibrillator was not associated with reduced mortality (HR: 0.74, 95% CI 0.47-1.16; P = 0.19). For ICD therapy without CRT, absolute risk reduction at 3-years follow-up was 3.7% yielding a number needed to treat of 27. Conclusion ICD use significantly improved survival among patients with NICMP who are not eligible for CRT. Considering CRT, the addition of defibrillator therapy was not significantly associated with mortality benefit compared with CRT pacemaker