Development of an easy-to-use urease kit for detecting Helicobacter pylori in canine gastric mucosa

Abstract

Background and Aim: Helicobacter pylori is an important pathogen in humans and animals involved in chronic gastritis, leading to the development of gastric cancer. Urease produced by H. pylori is an enzyme that promotes bacterial colonization and can be used clinically as a biomarker of H. pylori infection as part of a rapid urease test (RUT). A test with high specificity (95-100%) would be more convenient and faster than histopathology, bacterial culture, and polymerase chain reaction (PCR). The aim of this study was to develop a simple, cheap, and fast kit for detecting H. pylori infection in the gastric mucosa of canines, which can be used in clinical practice for diagnosing infection with this bacterium. Materials and Methods: The RUT assays developed were prepared using 1% agar, 1% sodium phosphate monobasic, and 1% urea followed by the addition of 3% methyl red indicator. The cutoff value of sensitivity of the RUT assay was established using the urease of H. pylori ATCC 43504 and color change was monitored for 24 h. Comparisons of the sensitivity to H. pylori ATCC 43504 were made between the developed RUT assays and the Hp Fast™ commercial kit. Then, the limit of detection for H. pylori ATCC 43504 number was analyzed by the SYBR Green real-time PCR assay to measure the copy number of the ureC gene. Gastric biopsy samples from the antrum, body, and fundus of the stomach were collected from eight canines presenting with vomiting and gastroenteritis. Analyses were performed on fresh samples using the developed RUT assays and the Hp Fast™ commercial kit, which were read within 24 h; then, the results were confirmed with SYBR Green real-time PCR. The specificity of the RUT assays was tested with a number of different bacteria, including Staphylococcus pseudintermedius, Proteus spp., Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterococcus spp., Escherichia coli, and Salmonella spp.; H. pylori ATCC 43504 was used as a positive control. Results: The results showed that the developed assays were sensitive to the urease enzyme at 0.1 mg/mL. The lowest detection limit of this assay for H. pylori ATCC 43504 was found to be 102 copies at 30 min. The sensitivity of detection of H. pylori in gastric biopsies of canines occurred in a minimum of 30 min. The RUT showed similar results to the Hp Fast™ commercial kit. In the developed RUT, the color change of the test from red to yellow could be clearly distinguished between the color of the positive test and the negative one; however, in the commercial Hp Fast™, it was difficult to observe the gel color change in the negative pH range of 5.8 and the positive pH of 6.5. The developed RUT was specific for H. pylori and did not detect any of the other tested bacteria. The test kit can also be stored for 6 months at 4°C. Conclusion: The sensitivity of the developed assays allowed the detection of urease enzyme at a minimum concentration of 0.1 mg/mL. Our RUT could also detect H. pylori from one in eight canine specimens at a minimum of 102 copies within 30 min. This RUT is specific to H. pylori as it did not detect any of the other tested bacteria.

Potentialities of Dandelion (Taraxacum Mongolicum Hand.‐Mazz.) Flower Extracts on Gastric Protection against Helicobacter Pylori and Characterization of its Bioactive Constituents

“…A change in the testing kits from yellow to purple indicated the presence of H. pylori (Figure 2A). [14] After 2 weeks of EEDF administration, the scores of the EEDFL and EEDFH groups decreased by 56.25 % and 62.5 % compared with the GM group ( P <0.01) (Figure 2B). The score of the PM group decreased by 75 % compared with that of the GM group.…”

Treatment and Determination of the Presence of Helicobacter in Shelter dogs by Faecal Antigen Testing and Enzyme–Linked Immunosorbent Assay

“…Attention should be paid to the zoonotic importance of NHPHS due to the increased risk of occurrence in people who are in close and intense contact with animals [3]. Since Helicobacter pathogens can be excreted through defecation, expectorating, and vomiting, they present faecal-oral, oral-oral and gastro-oral transmission routes [4]. Dogs are the natural hosts of NHPH and harbour these bacteria in their gastric mucosa, intestines and oral cavities; therefore, gastric fluid, saliva and faeces are possible sources of transmission of these bacteria to humans [2].…”

Investigation with Real-Time PCR and Histopathology on the presence of H. felis, H. heilmannii and H. pylori in dogs

“…Diagnosis of Helicobacter species is made with tests like rapid urease test, histopathology, culture, Polymerase Chain Reaction (PCR) from tissue samples or non-invasive tests such as urea breath test, blood tests, and serological tests [3,13,15,26]. The aim of this current study was to reveal the presence of H. felis, H. heilmannii, and H. pylori in the stomach and liver tissues taken during necropsy from owned, stray dogs and dogs kept in shelters, which died from different causes in the first stage, by Real-time PCR and histopathological examination.…”