Implementation of a Risk-Orientated Hygiene Analysis for the Control of Salmonella JAVA in the Broiler Production.

This field study aimed to establish a risk-orientated hygiene analysis on two broiler farms in Northwestern Germany for the practical use in broiler housing to evaluate the success of disinfection procedures for eradicating S. Java. The risk-orientated hygiene analysis enables fast, reproducible and cost-effective testing of broiler farms and in turn helps minimize the public health risk ensuing from S. Java. Farms were tested before and after cleaning as well as after disinfection according to a risk-orientated hygiene analysis for the presence of Salmonella DNA with qPCR. Positive PCR samples were confirmed by classical microbiology. Before cleaning, all checkpoints were tested positive for Salmonella DNA. Salmonella reduction of ca 66% of the sampled points could be achieved by intensive cleaning. A first disinfection on farm A and B failed to completely eradicate S. Java. A second disinfection followed and finally achieved a Salmonella-free status of the barns. During nine rearing periods, farms were tested weekly with boot swabs for Salmonella and at slaughter carcasses were tested for Salmonella status. No Salmonella were detected in these examinations. The two studied broiler farms have, to date, remained free of Salmonella.

Cytotoxicity of the Bacillus cereus Nhe enterotoxin requires specific binding order of its three exoprotein components.

This study focuses on the interaction of the three components of the Bacillus cereus Nhe enterotoxin with particular emphasis on the functional roles of NheB and NheC. The results demonstrated that both NheB and NheC were able to bind to Vero cells directly while NheA lacked this ability. It was also shown that Nhe-induced cytotoxicity required a specific binding order of the individual components whereby the presence of NheC in the priming step as well as the presence of NheA in the final incubation step was mandatory. Priming of cells with NheB alone and addition of NheA plus NheC in the second step failed to induce toxic effects. Furthermore, in solution, excess NheC inhibited binding of NheB to Vero cells, whereas priming of cells with excess NheC resulted in full toxicity if unbound NheC was removed before addition of NheB. By using mutated NheC proteins where the two cysteine residues in the predicted beta-tongue were replaced with glycine (NheCcys-) or where the entire hydrophobic stretch was deleted (NheChr-), the predicted hydrophobic beta-tongue of NheC was found essential for binding to cell membranes but not for interaction with NheB in solution. All data presented here are compatible with the following model. The first step in the mode of action of Nhe is associated with binding of NheC and NheB to the cell surface and probably accompanied by conformational changes. These events allow subsequent binding of NheA, leading to cell lysis.

Research Note: First evidence of infectious bronchitis virus Middle-East GI-23 lineage (Var2-like) in Germany.

The infectious bronchitis virus Middle-East GI-23 lineage (Var2-like) was observed on a German broiler farm, for the first time. The animals suffered from respiratory and nephropathogenic disease. Gross lesions observed during necropsy included tracheitis, aerosacculitis, and nephritis. Tracheal swabs were tested positive for infectious bronchitis virus Middle-East GI-23 lineage (Var2-like) by PCR. Furthermore, sequence analysis of the S1 spike protein showed close relationship to the commercially available vaccine TAbic IBVAR206 and polish isolates.

Induction of MAPK-dependent transcription factors by deoxynivalenol in human cell lines.

Contamination of cereals with the trichothecene deoxynivalenol (DON) is a global problem in agriculture. DON-related cytotoxicity results from inhibition of translation preceded by a ribotoxic stress response, which is characterized by phosphorylation of the mitogen-activated protein kinases and activation of downstream transcription factors. In this study, we measured the expression of AP-1 associated transcription factor mRNA levels in six human cell lines (Hep-G2, A549, U937, A204, Jurkat, and CaCo-2) by using real-time RT-PCR. Previous work suggested that transcription factors mRNA and protein levels are affected by DON in Hep-G2 cells. In this study, we found significant cell-specific differences in mRNA levels for the transcription factors JUN, JUND, FOS, FOSL2, ATF3, and EGR1. After exposure to 1 µmol/l DON for 3 h, the induction of the transcription factor JUN was highest in the Jurkat (342-fold) and Hep-G2 (84-fold) cell lines. JUND expression was mainly affected in the immunocompetent cell-lines U937 (11-fold) and Jurkat (12-fold), whereas significant FOSL2 induction (5-fold) was only found in Jurkat cells. DON-induced expression of FOS was mainly observed in Hep-G2 (96-fold) and U937 cells (59-fold). In contrast, response of A549 cells to DON was characterized by a distinct induction of ATF3 (44-fold) and EGR1 (92-fold). Time- and concentration-dependent induction of the transcription factors by DON was studied in detail for Hep-G2, A549, A204, and U937 cells. Considering the chronic dietary exposure of humans, broader investigations on DON influence on cell signaling pathways are required to understand the impact of this mycotoxin on human health.

Impact of DUSP1 on the apoptotic potential of deoxynivalenol in the epithelial cell line HepG2.

The trichothecene deoxynivalenol (DON) is the most common mycotoxin contaminant of cereal-based food products. Several studies revealed DON as a potent inducer of the three major mitogen-activated protein kinases (MAPKs). Until now, little is known about the role of negative regulators of MAPK pathway in the cellular response to DON. In this report we evaluated, for the first time, the impact of mitogen-activated protein kinase phosphatases (MKPs), particularly dual specific phosphatase 1 (DUSP1), on the toxic potential of DON in the epithelial cell line HepG2. Our results indicate that both low and high concentrations of DON trigger a strong and sustained DUSP1 mRNA and protein expression, mediated by the sustained activation of MEK/ERK pathway. Furthermore, the expression of DUSP1 protein correlates with the inactivation of JNK1/2, whereas a sustained activation of p38 and ERK1/2 was observed in the presence of DON. In contrast, treatment of DUSP1 knock-down cells with DON triggers a prolonged activation of JNK1/2, which leads to the induction of apoptosis. Taken together, we propose DUSP1 as a novel target gene of DON, which is essential for the prevention of DON induced apoptosis in the epithelial cell line HepG2.

Trichothecene-induced cytotoxicity on human cell lines.

Trichothecene cytotoxicity of type A (T-2 toxin and HT-2 toxin), type B (deoxynivalenol, DON, and nivalenol, NIV), and type D (satratoxins G and H) compounds was determined comparatively by using eight permanent human cell lines (Hep-G2, A549, CaCo-2, HEp-2, A204, U937, RPMI 8226, and Jurkat). Viability of cells was measured by a water-soluble tetrazolium (WST-1) reagent cell proliferation assay assessing mitochondrial metabolic activity. Toxicity was expressed as the toxin concentration inhibiting 50% of cell viability (IC50). Depending on the chemotype of the tested trichothecenes, relative cytotoxic activity differed by a factor of 100-1,000, and the corresponding IC50 values were in the range from 2.2 nmol/l (satratoxin H on Jurkat and U937 cells) to 4,900 nmol/l (deoxynivalenol on HEp-2 cells). In contrast, the specific toxicity of each individual mycotoxin towards different cell lines was within remarkable close limits, and between-cell line differences were much smaller than previously reported. For the cell lines tested, IC50 values were 4.4-10.8 nmol/l for T-2 toxin, 7.5-55.8 mol/l for HT-2 toxin, 600-4,900 nmol/l for DON, 300-2,600 nmol/l for NIV, and 2.2-18.3 nmol/l for satratoxins G/H. In addition, for the first time, the toxic activity of trichothecenes on primary cell culture of human endothelial cells (HUVEC) was tested. The susceptibility of this cell line was comparable to the other cell lines tested, with IC50 values ranging from 16.5 nmol/l (T-2 toxin) to 4,500 nmol/l (DON). The results suggest that the current focus of cytotoxicological studies on trichothecenes on lymphoid cell lines may lead to an underestimate of their potential on other target cell systems.