Assessment of Shiga Toxin-Producing Escherichia coli Isolates from Wildlife Meat as Potential Pathogens for Humans

A total of 140 Shiga toxin-producing Escherichia coli (STEC) strains from wildlife meat (deer, wild boar, and hare) isolated in Germany between 1998 and 2006 were characterized with respect to their serotypes and virulence markers associated with human pathogenicity. The strains grouped into 38 serotypes, but eight O groups (21, 146, 128, 113, 22, 88, 6, and 91) and four H types (21, 28, 2, and 8) accounted for 71.4% and 75.7% of all STEC strains from game, respectively. Eighteen of the serotypes, including enterohemorrhagic E. coli (EHEC) O26:[H11] and O103:H2, were previously found to be associated with human illness. Genes linked to high-level virulence for humans (stx₂, stx_2d, and eae) were present in 46 (32.8%) STEC strains from game. Fifty-four STEC isolates from game belonged to serotypes which are frequently found in human patients (O103:H2, O26:H11, O113:H21, O91:H21, O128:H2, O146:H21, and O146:H28). These 54 STEC isolates were compared with 101 STEC isolates belonging to the same serotypes isolated from farm animals, from their food products, and from human patients. Within a given serotype, most STEC strains were similar with respect to their stx genotypes and other virulence attributes, regardless of origin. The 155 STEC strains were analyzed for genetic similarity by XbaI pulsed-field gel electrophoresis. O103:H2, O26:H11, O113:H21, O128:H2, and O146:H28 STEC isolates from game were 85 to 100% similar to STEC isolates of the same strains from human patients. By multilocus sequence typing, game EHEC O103:H2 strains were attributed to a clonal lineage associated with hemorrhagic diseases in humans. The results from our study indicate that game animals represent a reservoir for and a potential source of human pathogenic STEC and EHEC strains.Shiga toxin-producing Escherichia coli (STEC) strains represent an important emerging group of food-borne zoonotic pathogens causing diarrhea, hemorrhagic colitis (HC), and the life-threatening hemolytic uremic syndrome (HUS) in humans (30). Production of potent cytotoxins, which are called Shiga toxins (Stx) or Vero toxins (VT) and are encoded on the genomes of temperate lambdoid bacteriophages, is the major virulence determinant of STEC strains. Additional virulence factors, such as genes encoding the attaching and effacing function and virulence plasmid-encoding genes, contribute to the pathogenicity of STEC strains. These virulence genes are closely associated with a subgroup of STEC strains that are frequently isolated from patients with hemorrhagic diseases (HC and HUS) and were therefore designated enterohemorrhagic E. coli (EHEC) strains. Strains belonging to serogroups O157, O26, O103, O111, and O145 are the EHEC types most frequently isolated from humans with HC and HUS (33).STEC strains are part of the gut flora of different animal species, and ruminants, particularly cattle, have been identified as a major reservoir of STEC strains that are highly virulent to humans (27). Today, it is evident that STEC strains can be transmitted from their animal reservoirs to humans via ingestion of contaminated food and water or by contact with STEC-excreting animals or the environment (9).Recent reports indicate that wildlife animals play an important role as carriers and transmitters of STEC strains in nature. EHEC O157 strains (13, 32, 36, 40, 46) and other STEC strains were isolated from feces of different ruminant deer species at different geographic locations (2, 20, 28, 34, 36, 42). Deer have been suggested to play a role as transmitters of EHEC O157 strains to cattle by fecal contamination of farmland (43). Wild migrating birds have been identified as STEC excretors and participate in the spread of EHEC O157 and other STEC strains over long distances (17, 37, 47). To date, only a few reports have been published on the contamination of raw game meat and other game products with STEC strains. A study conducted in Belgium indicated that about half of meat samples from wildlife ruminants contained STEC strains (38). Deer meat and jerky were identified as sources of EHEC O157 infections in humans in the United States (31, 39). In Germany, different types of STEC strains were isolated from venison samples (34), and surveys performed in the Federal Institute for Risk Assessment revealed a contamination rate of wild meat samples with STEC strains of 9.0% to 14.8% between 2005 and 2006. In this time period, the proportion of STEC-contaminated samples from game was considerably higher than that found with beef samples (1.3% to 4.5% STEC positives) (23, 24).Current data suggest that wild-living animals and their meat products are underestimated as natural reservoirs for STEC strains and as possible sources for human infections. Game meat is popular in Germany, since it is considered to be a high-quality product, and per capita consumption is rising steadily (report from the Federal Institute for Risk Assessment [http://www.bfr.bund.de/cd/7134]). To meet the demand for game meat, a total of 36,126 tons of wild animals were hunted from 2005 to 2006. These were divided into 19,000 tons of wild boar (n = 461,881 animals), 11,300 tons of roe deer (n = 905,387), and about 4,000 tons of red deer (n = 60,664) (Deutscher Jagdschutz-Verband [http://www.jagd-online.de]). Taking these data as a basis for estimation, the average amount of annual wild meat consumption is about 0.45 kg/person and accounts for 0.8% of the total meat consumption in Germany (22).About 62% of retailed game meat originates from animals hunted in the wild in Germany. Only 3% of the meat is from animals that are grown in captivity, with fallow deer the most frequently grown captive game animal. Imported game accounts for 35% of retailed meat (26). In compliance with the legal regulations, hunters are educated in meat inspection, and hygiene rules request evisceration of hunted game immediately after killing (C. Commichau [http://www.tiho-hannover.de/einricht/lmmikro/wild1.doc]). Inspected and acceptable carcasses are allowed to proceed to immediate sale to individuals, restaurants, and food handlers. For safety reasons, processing of game meat must occur separately from processing of other meat; when processing of game meat is conducted on a larger scale, it is performed in special meat-processing plants. Only a small portion of hunted game meat is inspected by official meat inspection authorities (26).At present, little is known about the characteristics of STEC strains other than O157 strains from wildlife meat. In order to provide data for estimating the impact of game as a potential source of human pathogenic STEC types, we characterized 140 STEC strains found in meat isolates from deer, wild boar, and hare. The strains were examined for their serotypes, for properties related to virulence of E. coli for humans, and for their genetic relationship to STEC isolates from farm animals, from their food products, and from human patients. The aim was to determine the similarities between STEC strains from wildlife meat and those from other sources, including humans. Our data indicate that game is a natural reservoir for and a potential source of human pathogenic EHEC and STEC types.     

Spheroids of granulosa cells provide an in vitro model for programmed cell death coupled to steroidogenesis

We describe the use of rotary cultures (72 rpm) as an excellent method for generating spheroids from dispersed bovine granulosa cells (GC). The GC spheroids were symmetrical (diameter between 100 and 200 μm), easily accessible, and could be obtained at high yields. On day one, the spheroids showed a two-layered outer zone of cells that stained lighter than the inner zone in semi-thin sections. Bromodeoxyuridine (BrdU) uptake was frequent and randomly distributed. By day two, a striking decrease in BrdU uptake was noted. Apoptotic bodies appeared up to day four, as did TUNEL and propidium iodide labelled dead cells. At that time, the inner zone contained cells with large-sized vacuoles and the core was amorphous. The large-sized vacuoles were identified at the ultrastructural level and represented autophagosomes and autophagolysosomes that were in different stages of development. Surprisingly, conspicuous signs of cell death were accompanied by an increase in spontaneous luteinization compared to conventional stationary cultures. We detected high levels of progesterone (immunoassay) accompanied by high levels of the proteins and enzymes relevant for steroidogenesis (StAR, P450scc, 3β-HSD by immunoblot and immunohistochemistry, respectively).     

How to go extinct by mating too much: population consequences of male mate choice and efficiency in a sexual–asexual species complex

Selection acting on individuals is not predicted to maximize population persistence, yet examples that explicitly quantify conflicts between individual and population level benefits are scarce. One such conflict occurs over sexual reproduction because of the cost of sex: sexual populations that suffer the cost of producing males have only half the growth rate compared to asexuals. Male behaviour can additionally impact population dynamics in a variety of ways, and here we study an example where the impact is unusually clear: the riddle of persistence of sperm‐dependent sexual–asexual species complexes. Here, a sexually reproducing host species coexists with an ameiotically reproducing all‐female sperm parasite. Sexual–asexual coexistence should not be stable because the proportion of asexually reproducing females will rapidly increase and the relative abundance of the sexually reproducing host species will decline. A severe shortage of males will lead to sperm limitation for sexual and asexual females and the system collapses. Male mate choice could reduce the reproductive potential of the asexual species and thus potentially prevent the collapse. In the gynogenetic (sperm‐dependent parthenogenetic) Amazon molly Poecilia formosa and its host (P. latipinna or P. mexicana), males discriminate against asexual females to some extent. Using a population‐dynamical model, we examine the population dynamics of this species complex with varying strengths of male discrimination ability and efficiency with which they locate females and produce sperm. The sexual species would benefit from stronger discrimination, thus preventing being displaced by the asexual females. However, males would be required to evolve preferences that are probably too strong to be purely based upon selection acting on individuals. We conclude that male behaviour does not fully prevent but delays extinction, yet this is highly relevant because low local extinction rates strongly promote coexistence as a metapopulation.     

Susceptibility of rheumatoid arthritis synovial fibroblasts to FasL- and TRAIL-induced apoptosis is cell cycle-dependent

Introduction  

The rheumatoid arthritis (RA) synovium is characterised by the presence of an aggressive population of activated synovial fibroblasts (RASFs) that are prominently involved in the destruction of articular cartilage and bone. Accumulating evidence suggests that RASFs are relatively resistant to Fas-ligand (FasL)-induced apoptosis, but the data concerning tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) have been conflicting. Here, we hypothesise that the susceptibility of RASFs to receptor-mediated apoptosis depends on the proliferation status of these cells and therefore analysed the cell cycle dependency of FasL- and TRAIL-induced programmed cell death of RASFs in vitro.     

Maximizing the productivity of catalytic biofilms on solid supports in membrane aerated reactors

A new solid support membrane aerated biofilm reactor was designed for the synthesis of enantiopure (S)‐styrene oxide utilizing Pseudomonas sp. strain VLB120ΔC growing in a biofilm as biocatalyst. In analogy to traditional packed bed systems, maximizing the volumetric oxygen mass transfer capability (k_La) was identified as the most critical issue enabling a consistent productivity, as this parameter was shown to directly influence biofilm growth and biotransformation performance. A microporous ceramic unit was identified as an ideal microenvironment for biofilm growth and for efficient oxygen transfer. A uniform and dense biofilm developed on this matrix. Due to this dual function, the reactor configuration could be significantly simplified by eliminating additional packing materials, as used in traditional packed bed reactors. Up to now, a maximum productivity of 28 g L day^?1 was achieved by integrating an in situ substrate feed and an in situ product recovery technique based on a silicone membrane. The system was stable for more than 30 days before it was actively terminated. Biotechnol. Bioeng. 2010;106: 516–527. © 2010 Wiley Periodicals, Inc.     

The role of successional stage and small‐scale disturbance for establishment of pioneer grass Corynephorus canescens

Question: Which mechanisms promote the maintenance of the protected pioneer grass Corynephorus canescens in a mosaic landscape? Which are the interactive effects of small‐scale disturbances, successional stage and year‐to‐year variation on early establishment probabilities of C. canescens? Location: Brandenburg, NE Germany. Methods: We measured emergence and survival rates over 3 yr in a sowing‐experiment conducted in three successional stages (C. canescens‐dominated site, ruderal forb site and pioneer forest) under two different regimes of mechanical ground disturbance (disturbed versus undisturbed control). Results: Overall, disturbance led to higher emergence in a humid year and to lower emergence in a very dry year. Apparently, when soil moisture was sufficient, the main factor limiting C. canescens' establishment was competition, while in the dry year, water became the limiting factor. Survival rates were not affected by disturbance. In humid years, C. canescens emerged in higher numbers in open successional stages while in the dry year, emergence rates were higher in late stages, suggesting an important role of late successional stages for the persistence of C. canescens. Conclusions: Our results suggest that small‐scale disturbances can promote germination of C. canescens. However, disturbances should be carefully planned. The optimal strategy for promoting C. canescens is to apply disturbances just before seed dispersal and not during dry years. At the landscape scale, a mosaic of different vegetation types is beneficial for the protected pioneer grass as facilitation by late‐successional species may be an important mechanism for the persistence of C. canescens, especially in dry years.