Validation of Multiplex PCR and Serology Detecting Helicobacter Species in Mice

Abstract

High-throughput multiplexed assays are needed to simplify detection of Helicobacter species in experimental infection and routine health monitoring of laboratory mice. Therefore, fluorescent bead-based hybridization assays for Helicobacter sp. DNA and serology were developed. Multiplex PCR amplicons (H. hepaticus, H. bilis, H. typhlonius, H. pylori, H. muridarum, H. pullorum, H. cinaedi, H. heilmanii, C. jejuni) and antibodies against H. pylori, H. hepaticus, H. bilis were assessed in naturally and experimentally infected mice, and results compared to conventional PCR. Species-specific and sensitive detection of seven Helicobacter spp. <100 copies/PCR, and of two species <1000 copies/PCR was successfully established in the Helicobacter multiplex DNA finder. The novel assay was highly comparable with conventional PCR (kappa = 0.98, 95%CI: 0.94–1.00). Antibody detection of H. hepaticus and H. bilis showed low sensitivity (71% and 62%, respectively) and cross-reactivity in H. typhlonius-infected mice. Infection experiments showed that antibodies develop earliest two weeks after DNA detection in feces. In conclusion, detection of Helicobacter antibodies showed low sensitivity depending on the timing relative to infection. However, Helicobacter multiplex DNA finder is a sensitive and specific high-throughput assay applicable in routine health monitoring for laboratory animals.

Helicobacter hepaticus and Helicobacter bilis in liver and biliary cancers from ATBC and PLCO

“…Previous studies have reported that cholesterol gallstone formation in mice was detected after infection with urease‐positive species H. hepaticus and H. bilis , but not with urease‐negative species Helicobacter rodentium or Helicobacter cinaedi 3 and further that the urease capacity of these strains may contribute to gallstone formation via precipitation of calcium 31 . Helicobacter bilis antigen, HRAG01818, predicted to be a cytolethal distending toxin may contribute to the virulence of the bacterial infection, 21,32 and P167D is both highly immunogenic and highly specific to H. bilis but as yet has no known function 33 . Similarly, H. hepaticus antigen, HH0435, is also highly immunogenic 34 .…”

“…For each H. hepaticus and H. bilis antigen, protein homology was compared to known H. pylori proteins (Table S1). 21 Helicobacter pylori positivity was defined as individuals who had an antibody response to ≥4 antigens as previously described 20 . Associations between H. pylori and hepatobiliary cancers in the ATBC and PLCO nested studies have been published previously 8,25 .…”

“…Antibody responses to H. pylori, H. hepaticus, and H. bilis proteins were determined by multiplex serology at the German Cancer Research Center (DKFZ) in Heidelberg, Germany, as described previously. 20,21 Briefly, recombinantly expressed glutathione-S-transferase (GST)-tag fusion proteins were affinity-purified on fluorescently labeled polystyrene beads (Luminex Corp., Austin, TX, USA). Mixing of the antigenloaded bead sets resulted in a suspension array that was incubated with serum (final serum dilution 1:1000).…”