Establishment of Noninvasive Methods for the Detection of Helicobacter pylori in Mongolian Gerbils and Application of Main Laboratory Gerbil Populations in China

Identifying Helicobacter pylori (H. pylori, Hp) infection in animals before and after artificial infection influences the subsequent experiment. We established effective and noninvasive detection methods, including the gastric fluid nested polymerase chain reaction (PCR) method and the 13C-urea breath test, which can detect Hp before modeling Hp infection in Mongolian gerbils. We designed a gas collection equipment for gerbils. Hp nested PCR was also performed on gastric fluid, gastric mucosa, duodenal contents, and faeces of gerbils challenged with Hp. Conventional Hp detection methods, including rapid urease assay and immunohistochemistry, were compared. Moreover, we assessed the natural infection of Hp in 135 gerbils that had never been exposed to Hp artificially from the major laboratory gerbil groups in China. In 10 Hp infected gerbils, the positive detection results were 100%, 100%, 90%, and 10% in gastric fluid, gastric mucosa, duodenal contents, and faeces with nested PCR, respectively. A rapid urease test performed on gastric mucosa showed that all animals were infected with Hp. Immunohistochemical detection and bacteria culture of gastric mucosa samples that were positive by the nested PCR method also confirmed the presence of Hp. 9% (3/35) and 6% (2/31) natural infection rates were found in conventional gerbil groups from the Capital Medical University and Zhejiang Laboratory Animal Center. In conclusion, we established two noninvasive Hp detection methods that can be performed before modelingHp infection, including the gastric fluid nested PCR method and the 13C-urea breath test.


Introduction
Helicobacter pylori (H. pylori, Hp) is a gram-negative bacterium that infects more than half of the world's people and is associated with a variety of severe gastric diseases, including gastritis, peptic ulcer, and even adenocarcinoma [1]. Further understanding of the specific mechanism of H. pyloriinduced gastric cancer is needed.
Researchers have tried to replicate H. pylori infection model in animals to study the pathogenesis of H. pylori infection in vivo. The colonization efficiency is unsatisfactory in large animals such as monkeys, cats, and dogs [2]. In mice, H. pylori infection results in development of gastric adenocarcinoma with some genetically engineered mice or with mice that had been induced with chemical carcinogens in advance [3,4]. H. pylori infection in rats only induces inflammation with preinjury of the gastric mucosa [5]. In 1998, Watanabe and Honda found that Mongolian gerbils can develop gastric adenocarcinoma after long-term H. pylori infection without additional chemical carcinogens [6,7]. Mongolian gerbils are currently recognized as an efficient, cost-effective, and robust rodent model of H. pylori infection.
The gerbil model is often used to study the inflammatory response, pathogenic mechanism and therapeutic strategies of H. pylori infection. The cancer progression associated with H. pylori in gerbils is characterized by erosion, inflammation with neutrophil infiltration, chronic superficial gastritis, atrophic gastritis, intestinal metaplasia, and finally dysplasia and adenocarcinoma, which are similar to those of humans [8,9]. Nevertheless, in many studies, H. pylorifree gerbils were not used or gerbils were not evaluated for natural infection before infection with H. pylori. It impacts the reliability and consistency of previous studies [10,11].
A greater variety of H. pylori detection methods have been applied to detect H. pylori in human and experimental animals [12]. Bacterial culture was considered the most specific method for the detection of H. pylori [12]. However, animals need to be sacrificed first. Immunohistochemistry also requires the animals to be sacrificed. Serum antibody detection and real-time PCR have been widely used clinically [13]. Nested PCR is often performed in gastric mucosa, gastric contents and duodenum to detect H. pylori infection, whereas gastric tissue and duodenum can only be obtained after the animals were euthanized. Hence, we developed a gastric fluid nested PCR method that can perform noninvasive Hp detection on live gerbils. 13 C-urea breath test is one of the most reliable noninvasive methods for detecting H. pylori infection in humans. H. pylori produces urease enzyme and then hydrolyses urea to release CO 2 and NH 3 . 13 C-urea breath test can detect the urease production and has been used for the clinical detection of bacteria in antral biopsies [14,15]. Therefore, we designed a 13 C-urea breath test equipment for gerbils that are suitable for the size of animals and the volume of expired gas. The 13 C-urea breath test can detect Hp before modelling Hp infection in Mongolian gerbils.
In this study, we established two noninvasive H. pylori detection methods including the gastric fluid nested PCR method and the 13 C-urea breath test. We compared these two new methods with conventional Hp detection methods and assessed the natural infection of H. pylori in gerbils from the major laboratory gerbil groups in China. and plated onto Columbia agar (Difco) supplemented with 5% sheep blood and Dent antibiotic supplement (Oxoid, Basingstoke, UK). Incubating the plates at 37°C in a microaerophilic condition for 72 h. Organisms were identified as H. pylori by modified Gram staining, oxidase, catalase, and urease reactions and kept frozen at -80°C.

Animals.
A total of 69 Mongolian gerbils (6-8 weeks old and 50-60 g) comprising 59 conventional gerbils and 10 specific pathogen-free males were included to establish the H. pylori detection methods. The H. pylori infection model was replicated in an additional 94 conventional gerbils (6-8 weeks old and 50-60 g). All rodents were from Capital Medical University and fed at a Level II Biosafety laboratory in the Chinese Center for Disease Control and Prevention. Microbiological status of the conventional gerbils and specific pathogen-free gerbils were strictly controlled according to local standards of Beijing. In order to establish H. pyloriinfected gerbil model, we used 2 × 10 9 CFU/mL H. pylori ATCC 43504 to gavage gerbils for 5 times, 0.5 mL each time. Oral gavage was performed at an interval of 48 h, and the gerbils were fasted for 12 h prior to challenge. The infection method is based on previous reports [6,9].
All gerbils are housed in individual ventilated cages (IVC; 461 × 274 × 229 mm; Tecniplast, Milan, Italy) on a 12 h light/dark cycle, with 2 to 3 gerbils in each cage. Females and males were separated and housed separately. The room temperature was maintained at 22~24°C, and the humidity was maintained at 60~70%. The animal experiments were conducted in accordance with the Guidelines of the CMU Animal Experiments and Experimental Animals Management Committee under a protocol approved by the Animal Experiments and Experimental Animal Welfare Committee of CMU (Permit number: AEEI-2016-152).

Sample Extraction and Bacterial Genome.
To collect gastric fluid, gerbils were gavaged with 0.5 mL distilled water after fasting for 12 h. A gavage needle was inserted into the stomach, and gastric fluid was extracted into a centrifuge tube within 1 min. The gastric fluid was centrifuged, and the genome was extracted using a microsample genomic DNA extraction kit (Tiangen, Beijing, China). DNA concentration and quality were verified by Nanodrop 2000c (Thermo, USA).
After 10 weeks of infection, the animals were anesthetized with isoflurane (induced with 3%, maintained with 2%, in 30% O 2 /70% N 2 O) and then euthanized. Samples of gastric tissue, gastric contents, duodenal contents, and colonic faeces (0.4 g/each) were collected into 1.5 mL sterile centrifuge tubes, respectively. Genomic DNA was extracted BioMed Research International by a microsample genomic DNA extraction kit containing centrifugal adsorption columns (Tiangen, Beijing, China). Bacterial DNA was extracted using a bacterial genomic DNA extraction kit (Tiangen, Beijing, China).

Nested PCR Primer Design, Amplification, and DNA
Sequencing. According to previous reports, we compared different H. pylori strains, and the conserved region of H. pylori (GenBank ID: NC-000915.1) genome Urea gene was selected.
Primers were designed by the primer design software Primer Premier 5. The primers for the first PCR were F1: 5 ′ -AGTAGGGCCATACATAGAAA-3 ′ and R1: 5 ′ -GACA AAACTCGTAACCGT-3′. Each reaction was performed in a 20 μL reaction volume containing 10 μL Dream Taq Green PCR Master Mix (Thermo Fisher Scientific, Massachusetts, MA), 10 pmol each primer, and 50 ng of the extracted template. The PCR protocol was as follows: 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, 51.3°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 5 min. The expected PCR product was 499 bp.
The primers for the second PCR were F2: 5 ′ -CATAGT TGTCATCGCTTTT-3 ′ and R2: 5 ′ -GCGTTGGTTGATAG GC-3 ′ . Each reaction was performed in a 20 μL reaction volume containing 10 μL Dream Taq Green PCR Master Mix (Thermo Fisher Scientific, Massachusetts, MA), 10 pmol of each primer, and 50 ng of the extracted template. The PCR was performed at 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, 51.3°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 5 min. The expected PCR product was 100 bp.
After matching primers 1 and primers 2 of nested PCR with Helicobacter species (taxid: 209) on the GenBank database, we confirmed that the matching sequences only exist in H. pylori.
The nested PCR amplification product was electrophoresed in 2% agarose gel (Amresco, USA); Takara 50 bp DNA Ladder was used as a molecular mass marker.
DNA sequencing was completed by Beijing Tianyihuiyuan Life Science & Technology Inc. Alignments were performed between nested PCR production sequences and sequences in GenBank.

Establishment of the 13C-Urea Breath Test Detection
Threshold. Gerbils were challenged with H. pylori by oral gavage at concentrations of 2 × 10 9 CFU/mL, 2 × 10 7 CFU/mL, 2 × 10 5 CFU/mL, 2 × 10 3 CFU/mL, and 2 × 10 1 CFU/mL and sterile saline. Each concentration treatment group contained three animals. Gerbils were placed immediately into a holder tube. The holder tube was connected to a gas propulsion device and a gas collecting device. The gas exhaled by a gerbil for 10 min was collected as the background gas with a ventilation of 0.5 mL/s. The gerbil was removed and was gavaged with 0.8 mg/mL urease solution (Haidewei, Shenzhen, China), and the gerbil was placed back into the holder tube immediately. Sample gas was collected in the same way. Finally, the 13 C tester (Haidewei, Shenzhen, China) was inserted into the gas bag, and the delta-over-baseline (DOB) value was measured.
2.7. Other H. pylori Detection Methods. A rapid urease detection kit (SanQiang, China) was performed on gerbil gastric tissue.
Immunohistochemistry and Warthin-Starry silver staining were also performed with gastric tissue to detect H. pylori. The gastric tissue was embedded in paraffin and cut into 4 μm-thick sections. Pyloric gastric sections were immunostained for H. pylori with primary antibodies (1 : 30 dilution, Batch B-0471, DAKO, Glostrup, Denmark). The Warthin-Starry silver staining kit was purchased from Solarbio (Beijing, China).

Statistical
Analysis. ROC (receiver-operating characteristic) curve analysis was used to determine the cut-off value of the detection threshold of the 13 C-urea breath test. The sensitivity, accuracy, and specificity of the 13 C-urea breath test methods and nested PCR were compared with several conventional detection methods. All analyses were performed using Graph Pad Prism 8.

Sensitivity and Specificity
Assay of Nested PCR. The results showed that target fragments could be amplified from H. pylori at a concentration of 2 × 10 2~1 0 8 CFU/mL (Figure 1(a)). The lowest concentration of H. pylori that can be detected is 2 × 10 2 CFU/mL and the total PCR reaction volume is 20 μL, containing 4 CFU of bacteria. The expected band can only be observed in H. pylori but not in other bacteria (Figures 1(b) and 1(c)). In order to exclude the influence of other bacteria including Helicobacter species, we compared the target fragments amplified by nested PCR. The GenBank database was used to perform BLAST analysis on the sequencing results to confirm that the nested PCR amplified fragments were specific fragments of H. pylori. Taken together, nested PCR detection method for H. pylori has high sensitivity and specificity.
3.2. Detection of H. pylori Infection by Nested PCR. After 10 weeks of infection with H. pylori, the gastric fluid of 10 pathogen-free gerbils was extracted. Gastric tissue, duodenal content, and faecal samples were collected after euthanizing the gerbils. DNA extraction, PCR amplification, and gel electrophoresis were performed on all the samples.

Results from the Mongolian Gerbil Screening Groups.
In order to investigate the natural infection of H. pylori in Chinese laboratory gerbils, we performed nested PCR detection of H. pylori in gastric fluid and gastric tissue of the main Chinese laboratory gerbil populations. There were three conventional gerbils infected with H. pylori at CMU, while the positive rate was 9% (2/35); two conventional gerbils were infected with H. pylori at ZJCLA with a 6% (1/31) positive rate. However, the clean gerbils at CMU and ZJCLA and the conventional gerbils at DMU were not infected with H. pylori as determined by evaluation of gastric fluid samples with the nested PCR detection method (Table 1). These observations suggested the presence of natural infection of H. pylori among laboratory gerbils in China, although the positive rate was low.

Establishment of the Mongolian Gerbil 13 C-Urea Breath
Test and Detection Threshold. H. pylori can produce abundant urease enzymes that hydrolyse urea to release CO 2 and NH 3 . The hydrolysed urea will form 13 CO 2 after taking 13 C-urea capsules orally and enter the lungs with blood and be discharged by gas. Based on this principle, we designed a 13 C-urea breath test gas collection device (Figure 3(a)) for gerbils that consists of a gerbil holder tube, a gas propulsion device and a gas collecting device. We can determine whether the gerbils are infected with H. pylori by detecting the 13 C in the exhaled gas before and after taking the 13 Curea solution. We then studied the relationship between the DOB value and the dose of 13 C-urea, H. pylori concentration and time. The DOB value increased with increasing doses of 13 C-urea (Figure 3(b)). With the increase in detection time after gavage, the DOB value reached a peak and then gradually decreased (Figure 3(c)). The DOB value increased with increasing H. pylori concentration (Figure 3(d)). By detecting the change in DOB with different amounts of H. pylori (Figure 3(e)), we determined that by collecting the exhaled gas of gerbils within 10 min after a gavage of 0.5 mg/mL 13  animals that were considered positive by the 13 C-urea breath test also tested positive by nested PCR. The detection rate of nested PCR in gastric tissue was higher than that of the 13 C detection method.
3.6. Other H. pylori Detection Methods. To verify the accuracy of the nested PCR method, immunohistochemistry and rapid urease test were used for comparison in the present study. The rapid urease test showed that the detection rate of 10 animals infected with H. pylori was 100% (Table 3). We observed H. pylori in gerbils that were H. pylori-positive by nested PCR and the 13 C-urea breath test at the same time through immunohistochemistry ( Figure 4). H. pylori was cultured and identified from the gastric mucosa of 5 gerbils that were determined to be H. pylori-positive by nested PCR performed on gastric fluid and gastric tissue ( Figure 5). Bacterial culture proves the presence of H. pylori.

Discussion
Though the transmission route of H. pylori infection remains unclear, it is generally believed that the transmission of H. pylori person-to-person mainly occurs via the oral-oral or faecal-oral route [20]. Mongolian gerbils are a good animal model for studying H. pylori infection and transmission because of the tumor progression associated with H. pylori in gerbils is similar to that of humans [9,21]. Besides, gerbils are often kept as pets for children who are susceptible to H. pylori [22]. In Charles River Laboratories International, Inc. (Wilmington, USA)'s latest Gerbil VAF Report, 100.0% (72/72) of gerbils were positive for Helicobacter species through PCR detection. Our general survey of the major Mongolian gerbil population in China also showed the 5 BioMed Research International natural infection of H. pylori through the gastric fluid nested PCR detection method. The positive rate was highest (9%) in the conventional gerbils at the Capital Medical University. In rodents, direct transmission of H. pylori occurs from challenged mice to unchallenged mice via saliva and faeces in a single cage [23]. Studies have found that H. pylori infection in Mongolian gerbil pups are transmitted by the faecal-oral route from an infected mother [21]. Mongolian gerbils are susceptible to H. pylori as previously reported [6]. We also found that the rate of colonization in the Mongolian gerbils infected with H. pylori was 100% after 10 weeks. Furthermore, Mongolian gerbils have been kept as pets for children, and the transmission of H. pylori between humans and pets has been reported [24]. It is necessary to determine the prevalence of H. pylori in pet gerbils through noninvasive detection methods. In addition to being used in the investigation 6 BioMed Research International of H. pylori, Mongolian gerbils are also considered to be a good animal model for studying hearing loss and brain ischemia. In these studies, unexpected results may be produced since H. pylori infection leads to increased expression of IL12 and IFNγ in Mongolian gerbils, which seriously affects the accuracy of experimental results [25][26][27]. Besides, H. pylori infection may induce extragastric diseases, making it difficult to determine the health status of experimental gerbils [28]. Though H. pylori detection before replicating the H. pyloriinfected model in gerbils is necessary, we found few studies that used H. pylori-free gerbils or detected the natural infection before challenge with H. pylori [10,11]. However, we cannot confirm whether the animal had been infected after modelling for a long time unless the animal was sacrificed, which affected the efficiency of modelling and did not conform to the "3R principle" (reduce, refine, and replace). Therefore, there is an urgent need for the detection of H. pylori infection before modelling through a more accurate and noninvasive detection method and for long-term detection [29]. Hence, we developed a method of gastric fluid nested PCR that only needs to extract the gastric fluid of live gerbils through gavage needle without harming the animals [12]. The positive rate of detection from gastric fluid with nested PCR was consistent with the results of the rapid urease detection method, indicating that the gastric fluid nested PCR method had convincing accuracy.
Chronic H. pylori infection can reduce the secretion of gastric acid and allow the growth of the gastric bacterial community [30]. The extracted gastric fluid may contain other bacteria besides H. pylori. We amplified several common bacterial DNAs including H. bilis with nested PCR primers, and no target fragments were detected [31]. After using GenBank database to match primer 1 and primer 2 of nested PCR to Helicobacter species (taxid:209), we found that the matching sequence of both primer 1 and primer 2 only existed in H. pylori. Thus, we confirmed that the nested PCR method is specific to H. pylori. The gastric fluid nested PCR method has high specificity. Considering the primers are a target to bacteria themselves, nested PCR could theoretically be applied in rodents which needs further investigation.
Nested PCR amplification was then performed with genomes from different concentrations of H. pylori. Target fragments could be amplified successfully from H. pylori at a concentration of 2 × 10 2~2 × 10 8 CFU/mL, that is, the copies of the bacterial genome are 10 6~1 , indicating that the nested PCR method has a high sensitivity. In Figure 1(a), agarose gel electrophoresis was performed for the second PCR product of nested PCR. Two bands of PCR products were shown, the lower band was the target band of the second PCR, with a size of 100 bp, and the upper band was the unreacted DNA template, with a size of 499 bp from the first PCR.
The positive rate of H. pylori infection determined by nested PCR was different in different parts of the alimentary canal. During the gavage process, H. pylori entered the junction of the duodenum and stomach with water or food, which caused a high positive rate of detection in the duodenal contents. The positive rate of H. pylori in faeces was low which may be due to the PCR inhibitors contained in the faecal material. Bacterial culture was considered the most specific method for the detection of H. pylori; therefore, we performed bacterial culture from gastric mucosa of 5 gerbils. Nested PCR of gastric tissue and gastric fluid has determined that all the gerbils are H. pylori positive. Gram staining, oxidase, catalase, and urease reactions confirmed the presence of H. pylori. These results demonstrate the reliability of the nested PCR method. 13 C-urea breath test has been applied in clinic and in many experimental animals except for gerbils, such as H. pylori infected mice model and barrier born pigs [32,33]. Although the detection standards are different due to the differences in size of animals and in volume of expired gas, the 13 C-urea breath test is harmless and reliable in these animals. Hence, we designed a gas collection equipment suitable for gerbils and defined the detection method and threshold of gerbil 13 C-urea breath test.
In our research, when gerbils were examined every 5 weeks after infection for 85 weeks, the 13 C-urea breath test showed that 50% of gerbils were infected with H. pylori, while the positive rate of gastric mucosa by nested PCR was 95.2%. The accuracy of the 13 C-urea breath test is unsatisfactory because the urease test basically depends on bacterial density [34]. It is difficult to detect the presence of H. pylori by 13 C-urea breath test at a very low density. Meanwhile, in our study, the air in the holder tube of gerbil gas collection equipment diluted the 13 CO 2 concentration. As there is no special 13 C-urea breath equipment for rodents, we have no choice but to select 13 CO 2 detector for humans.

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We aim to improve the detection method and establish a better detection threshold in the future.

Conclusions
In conclusion, there is a strong need to detect H. pylori with a noninvasive method in gerbils before replicating the model of H. pylori infection because of the considerable natural infection rate and acute effects in animal colonies and individuals after infection. It can improve the validity, reliability, and consistency of subsequent experiments and lay the foundation for research on the pathogenic mechanism and a therapeutic strategy for gastric disease caused by H. pylori.

Data Availability
All data, models, and code generated or used during the study appear in the submitted article.