g Helicobacter pylori colonizes half of the world's population, and infection can lead to ulcers, gastric cancer, and mucosa-associated lymphoid tissue (MALT) lymphoma. Serology is the only test applicable for large-scale, population-based screening, but current tests are hampered by a lack of sensitivity and/or specificity. Also, no serologic test allows the differentiation of type I and type II strains, which is important for predicting the clinical outcome. H. pylori virulence factors have been associated with disease, but direct assessment of virulence factors requires invasive methods to obtain gastric biopsy specimens. Our work aimed at the development of a highly sensitive and specific, noninvasive serologic test to detect immune responses to important H. pylori virulence factors. This line immunoassay system (recomLine) is based on recombinant proteins. For this assay, six highly immunogenic virulence factors (CagA, VacA, GroEL, gGT, HcpC, and UreA) were expressed in Escherichia coli, purified, and immobilized to nitrocellulose membranes to detect serological immune responses in patient's sera. For the validation of the line assay, a cohort of 500 patients was screened, of which 290 (58.0%) were H. pylori negative and 210 (42.0%) were positive by histology. The assay showed sensitivity and specificity of 97.6% and 96.2%, respectively, compared to histology. In direct comparison to lysate blotting and enzyme-linked immunosorbent assay (ELISA), the recomLine assay had increased discriminatory power. For the assessment of individual risk for gastrointestinal disease, the test must be validated in a larger and defined patient cohort. Taking the data together, the recomLine assay provides a valuable tool for the diagnosis of H. pylori infection.

SummaryThe high-pathogenicity island (HPI) encodes a highly efficient yersiniabactin system of iron acquisition responsible for mouse lethality in Yersinia . Although the HPI is widely disseminated among Enterobacteriaceae it lacks functions necessary for its replication and transmission. Therefore, the mechanism of its horizontal transfer and circulation is completely obscure. On the other hand, the HPI is a genetically active island in the bacterial cell. It encodes a functional recombinase and is able to transpose to new targets on the chromosome. Here we report on a possible mechanism of the HPI dissemination based on site-specific recombination of the excised HPI with the attB -presenting ( asn tRNA gene) RP4 promiscuous conjugative shuttle plasmid. The resulting cointegrate can be transferred by conjugation to a new host, where it dissociates, and the released HPI integrates into any unoccupied asn tRNA gene target in the genome. This mechanism has been proven both with the 'mini' island carrying only the attP recognition site and genes coding for recombination enzymes and with the complete HPI labelled with an antibiotic resistance marker. After acquisition of the mobilized complete form of the HPI, the ability of the HPI-cured Yersinia enterocolitica WA-TH -strain to produce yersiniabactin has been restored. Such 'trapping' of pathogenicity islands and subsequent shuffling to new hosts by a conjugative replicon carrying a suitable attB site could be applied to other functional integrative elements and explain wide dissemination of PAIs.

The 137,036-bp plasmid pG8786 from rhamnose-positive Yersinia pestis G8786 isolated from the high mountainous Caucasian plague focus in Georgia is an enlarged form of the pFra virulence-associated plasmid containing genes for synthesis of the antigen fraction 1 and phospholipase D. In addition to the completely conserved genes of the pFra backbone, pG8786 contains two large regions consisting of 4,642 and 32,617 bp, designated regions 1 and 2, respectively. Region 1 retains a larger part of Salmonella enterica serovar Typhi plasmid pHCM2 resembling the backbone of pFra replicons, while region 2 contains 25 open reading frames with high levels of similarity to the transfer genes of the F-like plasmids. Surprisingly, region 1 is also present in the pFra plasmid of avirulent Y. pestis strain 91001 isolated in Inner Mongolia, People's Republic of China. Despite the fact that some genes typically involved in conjugative transfer of the F-like replicons are missing in pG8786, we cannot exclude the possibility that pG8786 might be transmissive under certain conditions. pG8786 seems to be an ancient form of the pFra group of plasmids that were conserved due to the strict geographical isolation of rhamnose-positive Y. pestis strains in the high mountainous Caucasian plague locus.

The research on fast screening methods for antibodies against zoonotic pathogens in slaughter animals is important for food safety in farming and meat-processing industries. As a proof-of-concept study, antibodies against the emerging zoonotic pathogen hepatitis E virus (HEV) and enteropathogenic Yersinia spp. were analyzed in parallel using immobilized recombinant antigens (rAgs) of HEV genotypes 1 and 3 and Yersinia outer protein D (YopD) on a flow-through chemiluminescence immunochip. These rAgs are usually part of commercially available line immunoassays (LIAs) used for human diagnostics. In this study, sera from slaughtered pigs were tested on the microarray analysis platform MCR 3 to detect anti-HEV and anti-Yersinia IgG. The new method was characterized regarding signal reproducibility and specificity. The analytical performance was compared with in-house enzyme-linked immunosorbent assay (ELISA) and a LIA based on recomLine HEV (Mikrogen) or the ELISA test kit pigtype Yersinia Ab (Qiagen), respectively. The immunochip revealed the highest analytical sensitivity and was processed in 9 min automatically on the MCR 3. A comparative screening of swine serum samples from Bavarian slaughterhouses regarding anti-HEV and anti-Yersinia IgG seroprevalence was conducted. By using the LIA, 78% of the sera were tested positive for HEV antibodies. The immunochip and the ELISA identified anti-HEV IgG in 96% and 93% of the tested samples using the O2C-gt1 and O2C-gt3 rAg, respectively. The screening for anti-Yersinia IgG resulted in 86% positive findings using the immunochip and 57% and 48% for the ELISA methods, respectively, indicating a higher detection capability of the new method. Serum samples of slaughtered pigs could be analyzed faster and in an automated way on the microarray analysis platform MCR 3 which shows the great potential of the new immunochip assay format for multiplexed serum screening purposes.

Helicobacter pylori infection shows a worldwide prevalence of around 50%. However, only a minority of infected individuals develop clinical symptoms or diseases. The presence of H. pylori virulence factors, such as CagA and VacA, has been associated with disease development, but assessment of virulence factor presence requires gastric biopsies. Here, we evaluate the H. pylori recomLine test for risk stratification of infected patients by comparing the test score and immune recognition of type I or type II strains defined by the virulence factors CagA, VacA, GroEL, UreA, HcpC, and gGT with patient's disease status according to histology. Moreover, the immune responses of eradicated individuals from two different populations were analysed. Their immune response frequencies and intensities against all antigens except CagA declined below the detection limit. CagA was particularly long lasting in both independent populations. An isolated CagA band often represents past eradication with a likelihood of 88.7%. In addition, a high recomLine score was significantly associated with high-grade gastritis, atrophy, intestinal metaplasia, and gastric cancer. Thus, the recomLine is a sensitive and specific noninvasive test for detecting serum responses against H. pylori in actively infected and eradicated individuals. Moreover, it allows stratifying patients according to their disease state.

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