Comparison of Direct and Indirect Toxoplasma gondii Detection and Genotyping in Game: Relationship and Challenges.

The importance of game as a source of Toxoplasma gondii (T. gondii) infection in humans is largely unknown. New data on the presence of T. gondii in game hunted in the Federal State of Brandenburg, Germany, were obtained by direct and indirect detection (ELISA). DNA extracted either directly (5 g heart or foreleg muscle, DE) or after acid pepsin digestion (50 g heart, PD) or enriched by magnetic capture (50 g heart, MC) was examined by real-time PCR (qPCR). ELISA revealed seroprevalences of 20% in wild boar (Sus scrofa), 11% in roe deer (Capreolus capreolus) and 6% in red deer (Cervus elaphus). T. gondii DNA was detected by at least one direct detection method in 12% of wild boar, 6% of roe deer, 2% of fallow deer (Dama dama) and 2% of red deer. In both, positive wild boar and roe deer, T. gondii type II specific alleles were the most prevalent, as assessed by PCR-restriction fragment length polymorphism. The highest proportion of positive animals was detected by MC qPCR, followed by PD qPCR with a similar proportion of positive findings. Investigation of 50 g of heart muscle revealed a significantly higher proportion of positive qPCR results than analysis of 5 g (p = 0.048). An association between seropositivity and direct detection was evident in wild boar and roe deer (p < 0.001). Infectivity of T. gondii DNA-positive samples was confirmed by bioassay (4/4), providing evidence that game could represent a relevant source of viable T. gondii posing a risk for human infection.

Characterization of trh2 harbouring Vibrio parahaemolyticus strains isolated in Germany.

BACKGROUND: Vibrio parahaemolyticus is a recognized human enteropathogen. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) as well as the type III secretion system 2 (T3SS2) are considered as major virulence factors. As tdh positive strains are not detected in coastal waters of Germany, we focused on the characterization of trh positive strains, which were isolated from mussels, seawater and patients in Germany. RESULTS: Ten trh harbouring V. parahaemolyticus strains from Germany were compared to twenty-one trh positive strains from other countries. The complete trh sequences revealed clustering into three different types: trh1 and trh2 genes and a pseudogene Ψtrh. All German isolates possessed alleles of the trh2 gene. MLST analysis indicated a close relationship to Norwegian isolates suggesting that these strains belong to the autochthonous microflora of Northern Europe seawaters. Strains carrying the pseudogene Ψtrh were negative for T3SS2ß effector vopC. Transcription of trh and vopC genes was analyzed under different growth conditions. Trh2 gene expression was not altered by bile while trh1 genes were inducible. VopC could be induced by urea in trh2 bearing strains. Most trh1 carrying strains were hemolytic against sheep erythrocytes while all trh2 positive strains did not show any hemolytic activity. TRH variants were synthesized in a prokaryotic cell-free system and their hemolytic activity was analyzed. TRH1 was active against sheep erythrocytes while TRH2 variants were not active at all. CONCLUSION: Our study reveals a high diversity among trh positive V. parahaemolyticus strains. The function of TRH2 hemolysins and the role of the pseudogene Ψtrh as pathogenicity factors are questionable. To assess the pathogenic potential of V. parahaemolyticus strains a differentiation of trh variants and the detection of T3SS2ß components like vopC would improve the V. parahaemolyticus diagnostics and could lead to a refinement of the risk assessment in food analyses and clinical diagnostics.

Genotypic diversity and virulence characteristics of clinical and environmental Vibrio vulnificus isolates from the Baltic Sea region.

The genetic diversity of Vibrio vulnificus isolates from clinical and environmental sources originating from the Baltic Sea region was evaluated by multilocus sequence typing (MLST), and possible relationships between MLST clusters, potential genotypic and phenotypic traits associated with pathogenicity, and source of isolation were investigated. The studied traits included genotyping of polymorphic loci (16S rRNA, vcg, and pilF), presence/absence of potential virulence genes, including nanA, nab, and genes of pathogenicity regions, metabolic features, hemolytic activity, resistance to human serum, and cytotoxicity to human intestinal cells. MLST generated 35 (27 new) sequence types and divided the 53 isolates (including four reference strains) into two main clusters, with cluster I containing biotype 1 and 2 isolates of mainly environmental origin and cluster II containing biotype 1 isolates of mainly clinical origin. Cluster II isolates were further subdivided into two branches. Branch IIB included isolates from recent cases of wound infections that were acquired at the German Baltic Sea coastline between 2010 and 2011 and isolates from seawater samples of the same regions isolated between 1994 and 2010. Comparing the MLST data with the results of genotyping and phenotyping showed that strains of MLST cluster II possess a number of additional pathogenicity-associated traits compared to cluster I strains. Rapid microbiological methods such as matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry combined with typing of selected virulence-associated traits (e.g., serum resistance, mannitol fermentation, nanA, and pathogenicity region XII) could be used for risk assessment purposes regarding V. vulnificus strains isolated from the Baltic Sea region.

An essential role for tripeptidyl peptidase in the generation of an MHC class I epitope.

Most of the peptides presented by major histocompatibility complex (MHC) class I molecules require processing by proteasomes. Tripeptidyl peptidase II (TPPII), an aminopeptidase with endoproteolytic activity, may also have a role in antigen processing. Here, we analyzed the processing and presentation of the immunodominant human immunodeficiency virus epitope HIV-Nef(73-82) in human dendritic cells. We found that inhibition of proteasome activity did not impair Nef(73-82) epitope presentation. In contrast, specific inhibition of TPPII led to a reduction of Nef(73-82) epitope presentation. We propose that TPPII can act in combination with or independent of the proteasome system and can generate epitopes that evade generation by the proteasome-system.

Immunoproteomics of Helicobacter pylori infection and relation to gastric disease.

The Gram negative bacterium Helicobacter pylori is a human pathogen which infects the gastric mucosa and causes an inflammatory process leading to gastritis, ulceration and cancer. A systematic, proteome based approach was chosen to detect candidate antigens of H. pylori for diagnosis, therapy and vaccine development and to investigate potential associations between specific immune responses and manifestations of disease. Sera from patients with active H. pylori infection (n = 24), a control group with unrelated gastric disorders (n = 12) and from patients with gastric cancer (n = 6) were collected and analyzed for the reactivity against proteins of the strain HP 26695 separated by two-dimensional electrophoresis. Overall, 310 antigenic protein species were recognized by H. pylori positive sera representing about 17% of all spots separated. Out of the 32 antigens most frequently recognized by H. pylori positive sera, nine were newly identified and 23 were confirmed from other studies. Three newly identified antigens which belong to the 150 most abundant protein species of H. pylori, were specifically recognized by H. pylori positive sera: the predicted coding region HP0231, serine protease HtrA (HP1019) and Cag3 (HP0522). Other antigens were recognized differently by sera from gastritis and ulcer patients, which may identify them as candidate indicators for clinical manifestations. The data from these immunoproteomic analyses are added to our public database (http://www.mpiib-berlin.mpg.de/2D-PAGE). This platform enables one to compile many protein profiles and to integrate data from other studies, an approach which will greatly assist the search for more immunogenic proteins for diagnostic assays and vaccine design.