Establishment of Helicobacter pylori infection model in Mongolian gerbils

Abstract

AIM: To establish a stable and reliable model of Helicobacter pylori infection model in Mongolian gerbils and to observe pathological changes in gastric mucosa in infected animals.

METHODS: Mongolian gerbils were randomly divided into 18 groups; 6 groups were infected with H pylori clinical strain Y06 (n = 6, groups Y), 6 groups were infected with H pylori strain NCTC11637 (n = 6, groups N), and 6 uninfected groups as negative controls (n = 4, groups C). H pylori suspensions at the concentrations of 2 × 10⁸ and 2 × 10⁹ CFU/mL of strain NCTC11637 and strain Y06 were prepared. The animals in three groups N and in three groups Y were orally challenged once with 0.5 mL of the low concentration of the bacterial suspension. The animals in another three groups N and in another three groups Y were orally challenged with 0.5 mL of the high concentration of the bacterial suspension for 3 times at the intervals of 24 h, respectively. For the negative controls, the animals in six groups C were orally given with the same volume of Brucella broth at the corresponding inoculating time. The animals were killed after 2nd, 4^th and 6^th week after the last challenge and the gastric mucosal specimens were taken for urease test, bacterial isolation, pathological and immunohistochemical examinations.

RESULTS: Positive isolation rates of H pylori in the animals of groups Y at the 2nd, 4^th and 6^th week after one challenge were 0%, 16.7% and 66.7%, while in the animals of groups N were 0%, 0% and 16.7%, respectively. Positive isolation rates of H pylori in the animals of groups Y at the 2nd, 4^th and 6^th week after three challenges were 66.7%, 100% and 100%, while in the animals of groups N were 66.7%, 66.7% and 100%, respectively. In animals with positive isolation of H pylori, the bacterium was found to colonized on the surface of gastric mucosal cells and in the gastric pits, and the gastric mucosal lamina propria was infiltrated with inflammatory cells.

CONCLUSION: By using H pylori suspension at high concentration of 2 × 10⁹ CFU/mL for multiple times, the orally challenged Mongolian gerbils can be used as a stable and reliable H pylori infection model. The 2 strains of H pylori can colonize in gastric mucosa of the infected animals and cause mild inflammation reaction.

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INTRODUCTION

Helicobacter pylori, a microaerophilic, spiral and Gram-negative bacillus, is recognized as an important pathogen causing human gastritis and peptic ulcer and a high risk factor for gastric carcinoma[1,2]. In China, chronic gastritis and peptic ulcer are two of the most common digestive diseases, and gastric cancer is one of the malignant tumors with high morbidities[3-34]. An ideal public measure to prevent and control these H pylori infection-associated diseases may be a vaccine that could induce strong humoral and cellular immune responses. However, no commercial H pylori vaccine is available so far, and the development of H pylori vaccine by using genetic engineering techniques is being active[35-39]. A stable and reliable H pylori infection animal model would be necessary for evaluating vaccine efficacy and helpful for understanding the pathological mechanism of the organism. Therapeutic drugs for H pylori eradication differ from those for many other bacteria such as using metronidazole[40,41]. Therefore, H pylori infection animal models would contribute to screen new drugs against H pylori. In recent published data, Mongolian gerbils have been considered as ideal animals to establish infection model by using internationally collected H pylori strains[42-44]. In this study, we used a clinical H pylori isolate named Y06 to establish a stable and reliable infection model in Mongolian gerbils. The colonization sites of H pylori and pathological changes in gastric mucosa of the animals were also observed.

MATERIALS AND METHODS

H pylori strains

A clinical strain of H pylori named Y06 was isolated from a patient with gastric ulcer. This strain was identified based on their characteristic morphology by Gram staining under microscope, and positive for urease and oxidase activities, further confirmed by slide agglutination test using commercial rabbit antiserum against whole cell of H pylori (DAKO). A reference H pylori strain, NCTC11637, was used as an infection control. The two strains were subcultured in Columbia agar (bioMérieux) containing 80 mL/L sheep blood under microaerobic conditions containing 100 mL/L CO₂, 50 mL/L O₂ and 850 mL/L N₂.

Animals

Eight-week-old specific pathogen-free male Mongolian gerbils with body mass of 75±5 g were provided by Experimental Animal Center, Zhejiang Academy of Medical Sciences. These gerbils were randomly divided into eighteen groups: six groups infected with the clinical strain Y06 (groups Y, n = 6 in each group), six groups infected with the reference H pylori strain NCTC11637 (groups N, n = 6 in each group) and six groups as negative controls (groups C, n = 4 in each group).

Dosages and pathway of inoculation

Bacterial cells grown on the 2 strains on Columbia agar for 3-5 d were collected and diluted to the final concentrations of 2 × 10⁸ CFU/mL and 2 × 10⁹ CFU/mL, respectively, by using Brucella broth (bioMérieux). Each Mongolian gerbils in 3 groups Y and 3 groups N were orally challenged with 0.5 mL of 2 × 10⁸ CFU/mL H pylori suspension, while the animals in another 3 groups Y and 3 groups N were attacked with 0.5 mL of 2 × 10⁹ CFU/mL H pylori suspension through the same pathway. For the negative controls, the animals in 6 groups C were orally given with 0.5 mL of Brucella broth. Each animal in the groups was given with the different concentrations of H pylori suspensions or Brucella broth, respectively, as the described above for 3 times at an interval of 24 h. The animals were deprived of food but offered with water for 12 h before the challenge, and supplied with food and water after 4 h of H pylori inoculation.

Isolation and identification of H pylori

Six animals in group Y, 6 in group N and 4 animals in group C were killed, respectively, at 2, 4 and 6 wk after the last challenge. Two gastric mucosal specimens at the adjacent position were taken from antrum and corpus, respectively. One of the specimens was used for H pylori isolation, and the others were fixed with 40 g/L formaldehyde solution. The colonies on Columbia plates were identified by microscopy after Gram-staining, assays for urease and oxidase activities and slide agglutination test using the commercial H pylori-specific antiserum. The bacterium was defined to be H pylori if it was Gram-negative with arc shape or “seagull-like”, positive for the two enzymes and immune agglutination.

Pathological and immunohistochemical examinations

The gastric mucosal specimens fixed with formaldehyde were pathologically examined after embedding, section and haematoxylin-esosin (HE) staining. H pylori in gastric mucosal specimens were detected by the immunohistochemical method using a commercial rabbit anti-H pylori antibody and goat anti-rabbit HRP-labeled IgG antibody (Jackson Immunoresearch).

RESULTS

Infection of Mongolian gerbils with H pylori strains

The results of H pylori isolation from gastric mucosal specimens of Mongolian gerbils are shown in Table 1.

Table 1 The detection results of H pylori isolated from gastric mucosa of experimental infected Mongolian gerbils.

Group	Detection time (wk)	Infection rate (%) (positive/total cases)
		1 × 108 CFU (1 challenge)	1 × 109 CFU (3 challenges)
Y	2	0(0/6)	66.7(4/6)
N	2	0(0/6)	66.7(4/6)
C	2	0(0/4)	0(0/4)
Y	4	16.7(1/6)	100(6/6)
N	4	0(0/6)	66.7(4/6)
C	4	0(0/4)	0(0/4)
Y	6	66.7(4/6)	100(6/6)
N	6	16.7(1/6)	100(6/6)
C	6	0(0/4)	0(0/4)

Y, groups infected with strain Y06; N, groups infected with strain NCTC11637; C, negative controls.

Pathological and immunohistochemical findings

In animals with positive H pylori isolation, the organisms were found to colonize the surface of gastric mucosa and the gastric pits (Figure 1). In the presence of H pylori infection, infiltration of chronic inflammatory cells in the lamina propria and erosions on the surface of gastric mucosa were observed (Figure 2).

Figure 1 The H pylori bodies located on the surface of gastric mucosal cells and in gastric pits ( × 1000).

Figure 2 The infiltrated chronic inflammatory cells in the gastric mucosal lamina propria of the specimens with positive H pylori isolation ( × 400).

DISCUSSION

In previous published data, animals for establishment of H pylori infection models included guinea pigs, rats, nude mice, chimpanzee etc.[45-48]. These animal models have many disadvantages such as low infection rates, instability, immunodeficiency and high costs. In 1997, Lee et al[48] successfully established an animal model infected with a H pylori strain named as SS1 in C₅₇BL/6 and BaLb/c mice. This animal model showed a high frequency and stability for H pylori infection. However, strain SS1 is a mutant of H pylori and its virulence is considerably low. Recently, Mongolian gerbils, which has distinct advantages such as high frequency and stability of infection, large colonization of H pylori, longer living suitable for study with long period of time and pathological changes similar to those observed in human with chronic gastritis, have become the predominant animals for preparing H pylori infection model[42-44]. In 1999, Chi et al[49] established a stable model of H pylori infection in Mongolian gerbils, which was orally pretreated with alcohol. Therefore, Mongolian gerbils are regarded as an ideal animal for H pylori infection models.

Previous studies have shown that infection model in Mongolian gerbils by oral challenge once with H pylori suspension at the concentration of 1 × 10⁸ CFU of H pylori is stable[42-44]. In the present study, H pylori was undetectable in the gastric mucosa from Mongolian gerbils at 2 wk after challenge once with 1 × 10⁸ CFU of the bacterium. Furthermore, the infection rates at 6 wk after one challenge by H pylori strain Y06 and strain NCTC11637 were only 66.7% and 16.7%, respectively. On contrast, by using three oral challenges at the dosage of 1 × 10⁹ CFU, H pylori colonization rates in the gastric mucosa at 2 and 6 wk after challenge by H pylori strain Y06 or strain NCTC11637 were both 66.7% and 100%, respectively, which indicates that multiple challenges with a high concentration of H pylori contribute to the increased infection rates.

The infection rates of H pylori strain Y06 and strain NCTC11637 in Mongolian gerbils were 100% and 66.7% at 4 wk for 3 challenges using the low concentration of bacterial suspension, and 16.7% and 0% at 4 wk and 66.7% and 16.7% at 6 wk after one challenge using the high concentration. H pylori strain Y06, a fresh clinical isolate, seems to be more virulent than strain NCTC11637 and more beneficial for establishing H pylori infection model in Mongolian gerbils with a higher infection rate in a shorter period of time.

H pylori was found on the surface of gastric mucosa and in the gastric pits of Mongolian gerbils when infection was established. The observations that there were erosions of mucosal surface and infiltration of chronic inflammatory cells in the lamina propria of gastric mucosa of H pylori infected Mongolian gerbils indicates that the two tested H pylori strains are able to colonize gastric mucosa of Mongolian gerbils and cause chronic inflammation and gastric erosions.