Experimental Helicobacter pylori infection of Rhesus macaques (Macaca mulatta)

IJMM IJ

Int. J. Med. Microbiol. 291, 33-43 (2001) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/ijmm

Experimental Helicobacter pylori infection of Rhesus macaques (Macaca mulatta) Kerstin Mätz-Rensing1, Emanuela Kunz1*, Christian Kraft2, 3*, Dirk Lorenzen2, Sebastian Suerbaum3, Franz-Joseph Kaup1 1 2 3

Department of Veterinary Medicine and Primate Husbandry, German Primate Centre, D-37077 Göttingen, Germany Department of Virology and Immunology, German Primate Centre, D-37077 Göttingen, Germany Institute of Hygiene and Microbiology, University of Würzburg, D-97080 Würzburg, Germany

Received February 12, 2001 · Accepted February 13, 2001

Abstract The aim of the present study was to establish an animal model for Helicobacter pylori (H. pylori) infection at the German Primate Centre in rhesus monkeys (Macaca mulatta). During the experiments the susceptibility of three animals to different H. pylori strains of human origin was tested. In a follow-up study gastric biopsies from three different sites were investigated in regular intervals using microbiological, histological, electron microscopical and molecular biological methods to evaluate the presence of bacterial colonization and the occurrence of gastritis. It was possible to establish a persistent experimental infection. The rather long follow-up period of 18 months offered the possibility to demonstrate a permanent H. pylori infection in the gastric mucosa of the test animals. The three animals have now been successfully colonized with H. pylori for 18 months and presented a chronic active gastritis confirmed by microbiological and histological methods. By molecular typing, the identity of the isolates recovered from the animals was shown. It was possible to demonstrate that one infection strain outcompeted the second one. Taken together, prerequisites exist for making use of an attractive and useful animal model in rhesus monkeys especially for long term observations. Key words: animal model – experimental infection – H. pylori – rhesus monkey

Introduction The presence of spiral bacteria in human gastric mucosa was first recognized more than one hundred years ago. In 1982 these bacteria were isolated for the first time in cultures of endoscopic specimens from patients with gastritis and peptic ulceration (Marshall and Warren, 1984). Since the discovery of H. pylori and its causative role in human gastric disease, several animal

models of Helicobacter-induced gastritis have been described, including gnotobiotic piglets (Krakowka et al., 1995), monkeys, mice (Lee et al., 1997), rats and gerbils (Miyata et al., 1999). Other animal models are based on related bacteria like H. felis in mice (Fox et al., 1996) and H. mustelae in ferrets (Fox et al., 1990). With the exception of monkeys and a single cat colony (Handt et al., 1994) none of the animal species used in these models is a natural host of H. pylori. In contrast,

* These authors contributed equally. Corresponding author: Dr. K. Mätz-Rensing, German Primate Centre, Department of Veterinary Medicine and Primate Husbandry, Kellnerweg 4, D-37077 Göttingen, Germany, Phone: +49 551 3851 279, Fax: +49 551 3851 277, E-mail: [email protected] 1438-4221/01/291/1-033 $ 15.00/0

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several reports have documented natural spontaneous gastric infections with H. pylori in nonhuman primates (Baskerville and Newell, 1988; Newell and Harwood, 1989). H. pylori appears to be enzootic in at least some breeds of rhesus monkeys (Drazek et al., 1994; Dubois et al., 1991, 1995; Handt et al., 1997). More than half of these animals are reported to be colonized by the age of 2 years, although some remain uninfected for many years (Dubois et al., 1996). H. pylori strains from macaques were indistinguishable from human isolates in biochemical and phenogenotypical analysis (Dubois et al., 1996). Similar to the human infection, the infection of macaques is persistent and associated with acute as well as chronic gastritis (Baskerville and Newell, 1988). Furthermore, gastric ulcer and cancer have been reported in this species although the incidence is very low (Eaton, 1999). While many experimental questions can be validly addressed with small animal models, primate models bear promise to mimick human H. pylori infection more closely than any other model due to the evolutionary relationship between humans and primates. Chimpanzees (Hazell et al., 1992) and several macaque species (Euler et al., 1990, Fujioka et al., 1994; Reindel et al., 1999) have been used to study the effects of experimental colonization by H. pylori. Data from the literature show that Macaca mulatta and Macaca fuscata are susceptible for experimental infection (Shuto et al., 1993; Dubois et al., 1999). In both species a persistent chronic infection can be established. Problems exist in the availability of H. pylori-free animals prior to experimental infection (Solnick et al., 1999) and in the fact that most animals harbour a mild chronic gastritis due to spontaneous infection with large gastrointestinal spirals or H. pylori or both (Mätz-Rensing et al., 1999). Here we report about our experience with a model of experimental H. pylori infection in three rhesus monkeys at the German Primate Centre.

Materials and methods Animals Three male rhesus monkeys, (Macaca mulatta) from Indian origin, aged 3 to 4 years were used in this study. During quarantine the animals tested negative for tuberculosis, HTLV (Human T Cell Leukaemia Virus), SIV (simian immunodeficiency virus) and Foamy virus. The animals were housed in individual cages with free access to tap water at the German Primate Centre according to the Guide for the Care and Use of Laboratory Animals, published by the National Research Council and the Institute for Laboratory Animal Resources. The diet consisted of primate pellets (sniff, special diet GmbH, Soest) and fresh fruit. During quarantine all animals were tested by endoscopic, bacterio-

logic and microbiologic methods for the presence of natural Helicobacter infection. Although the animals appeared to be negative for H. pylori they were naturally colonized with socalled large gastrointestinal spirals (LGIS). Histological and microbiological examination gave evidence that some of the large gastrointestinal spirals belong to uncultivated Helicobacter species like H. heilmannii. To eliminate these large gastrointestinal spirals all monkeys were treated with oral application of amoxicillin sirup (30 mg/kg) daily for three weeks followed by a dual therapy in form of a combination of amoxicillin and metronidazol (30 mg/kg each) given daily orally for 1 week. Inocula (bacterial strains) A first attempt to colonize the monkeys with laboratory strains of H. pylori (NCTC11 637, N6) that had been frequently passaged for years was unsuccessful. Therefore, two clinical isolates that had been passaged less than five times were used for a second series of experiments. The bacterial strains used for experimental infection were the two isolates BO417 and BO418. Both strains were isolated from patients with severe acute gastritis receiving medical treatment at the Ruhr University, Bochum, Germany. The bacteria presented vacA, cagA and flaB. The strains were stored at –80 °C in H. pylori storage medium containing brain heart glucose broth (Oxoid, CM225), yeast extract (Oxoid, L 21), horse serum (Gibco, 16 050–098) and glycerol (Riedel-de Haen, 33 224). They were thawed at room temperature for ten minutes, brought into brucella broth and then cultured on horse blood agar plates under microaerobic conditions (Campypac Plus, 90 % N2, 5 % CO2, 5 % O2 at 37 °C for 3 to 4 days). Bacterial colonies of both isolates were resuspended in 1 ml brucella broth and the concentration was determined as 106 –108 colony forming units (CFU)/ml. The inoculum was prepared by mixing a suspension of the two bacterial strains diluted in 1 ml brucella broth. Inoculation protocol and follow-up study For the experimental infection an H. pylori cocktail of the strains BO417 and BO418, first generation, was given intragastrally in a concentration of 2.56  106 CFU/ml, for the superinfection three days later strains BO417 and BO418, 2nd generation in a concentration of 3.32  106 CFU/ml. Three weeks after the end of antibiotic therapy to reduce colonization with large gastric spiral bacteria, experimental infection was begun. After an overnight fast the animals were anesthetized with GM II (“Göttinger Mischung”) (Rensing, 1999) in doses of 0.1 ml/kg weight. The gastroscopic examination was carried out using a fiberscope (FG-100FP) from Fujinon with an outer diameter of 10 mm. Firstly, the macroscopic appearance of corpus and antral mucosa was assessed qualitatively. Secondly, 5 ml of natriumbicarbonate were applied to neutralize the gastric juice. Afterwards, 1 ml of the prepared bacterial suspension was sprayed endoscopically on the corpus, antrum and fundus mucosa. The endoscope canal was cleaned with water to remove the remaining bacterial material. After infection and after each endoscopy the endoscope was rinsed with water and desinfected by soaking for 15 minutes in GigaseptFF. The equipment was

Experimental H. pylori infection in Rhesus macaques

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then rinsed with sterile demineralised water and air dried. The superinfection was carried out in the same way. Gastric biopsy sampling Three days after infection the first endoscopic investigation was performed. This endoscopic examination was repeated at two weeks, three weeks, five weeks, 3, 5, 10, 12, 18, and 24 months. At each investigation point 5–6 pinch biopsies of gastric mucosa of the corpus, fundus and antrum, respectively, were obtained endoscopically, brought either into urea medium, or into brucella broth for bacteriological investigations, fixed in formalin or glutaraldehyde for morphologic investigations or stored natively at –80 °C for PCR techniques. The urea medium consisted of Urea Broth Base and 40 % urea solution with phenol red as indicator. From each location brush cytology was carried out on Helicobacterspecific horse blood agar plates and blood agar plates. In addition, peripheral blood samples were collected for immunological studies. For experimantal design see Figure 1. Histological examination Light microscopy was performed on biopsies of gastric tissue fixed in 10 % neutral buffered formalin and embedded in paraffin. Five-micrometer sections were stained with haematoxylin and eosin and viewed under  63 to 100 magnification. Sections were scored for chronic gastric inflammation using a scale of 0 to 3 as modified from the Sydney system: 0  intact mucosal lining and essentially no inflammatory cell infiltration of the lamina propria; 1  mild increase of mononuclear infiltration, localised in the upper part of the mucosa (superficial gastritis); 2  mononuclear infiltration extending from the epithelial surface deeper into the lamina propria (severe gastritis); 3  marked inflammatory cell infiltration with disruption of gastric glands and infiltration with polymorphonuclear leucocytes in glands and surface erosions (diffuse severe gastritis). The occurrence of lymphoid follicles in the lamina propria was scored as present (, , ) or absent (–). The gastric lesions were evaluated on the basis of haematoxylin- and eosin-stained sections. The presence of gastric bacteria was assessed in Warthin-Starry-stained sections using a Steiner modification of the Warthin-Starry silver stain (Steiner and Steiner, 1944). For transmisson electron microscopy (TEM), biopsies were placed in freshly prepared fixative (glutaraldehyde, 3 %). After postfixation in 1 % osmium tetroxide, specimens were dehydrated in a graded ethanol series and embedded in epoxy resin. Semithin sections were cut on an ultramicrotome Reichert ultracut S and stained with methylene blue. Selected areas were trimmed and thin sectioned, contrasted with uranyl acetate and lead citrate and viewed in a Zeiss transmission electron microscope EM 10C. Microbiology Unfixed samples taken from the mucosa were streaked on horse blood agar plates containing 5.0 % horse blood (Oxoid, Columbia agar) supplemented with vancomycin, trimethoprim, cefsulodin, amphotericin B (H. pylori selective supplement, Oxoid, SR 147). The plates were incubated at

Fig. 1. Experimental design.

37 °C in microaerobic conditions in vented jars containing 90 % N2, 5 % O2 and 5 % CO2. H. pylori isolates were identified as forming pinhead-sized colonies that grew within 3 to 5 days and had urease, catalase and oxidase activities, and by microscopy as gram-negative and s-shaped rods. DNA extraction A DNA extraction kit (QIAmp DNA Mini Kit, Qiagen, Hilden, Germany) was used for total DNA isolation from H. pylori cultures. Briefly, pure cultures from single colony picks were harvested from agar plates and treated with lysozyme, proteinase K, RNase A in lysis buffer. DNA was purified following the manufacturer’s instructions and quantified spectrophotometrically at 260/280 nm using a GeneQuant apparatus (Amersham Pharmacia Biotech, Uppsala, Sweden). Strain typing The reisolated H. pylori strains were compared with the inoculum strains BO417 and BO418 by two different methods. Random amplified polymorphic DNA fingerprinting (RAPD) was performed as described by Akopyanz et al. (1992) using the primers AP 1254 and AP1281. These gave characteristic band patterns for the two inoculum strains. Strains could also be differentiated by partial sequence analysis of the 16S rDNA genes as described below. 16S rDNA-PCR amplification The 16S rDNA genes were amplified using the broad-range primers pBr5se 5-GAAGAGTTTGATCATGGCTCAG-3 and p13B 5-GTACAAGACCCGGGAACGTATTC-3 (Monstein et al., 2000). Approximately 200 ng bacterial DNA was amplified by PCR in a total volume of 50 µl reaction puffer (100 mM Tris-HCl, 500 mM KCl, 2 mM MgCl2, pH 9.0) containing 20 pmol of each primer, 0.2 µM of each dNTP and 1.5 U Taq polymerase (Life Technologies, USA). PCR was performed in reaction tubes in a minicycler (UNO Thermoblock, Biometra, Göttingen, Germany) under the following conditions: denaturation at 94 °C for 40 s, annealing at 56 °C for 40 s, elongation at 72 °C for 60 s (35 cycles) and a final exten-

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Table 1. Summary of findings in animal no. 7743. Time after H. pylori inoculation

Gastric side of biopsy pinch

Endoscopical Results

Prae-inoculation

Cardia Fundus Antrum

normal mucosa

2 wpi

Cardia Fundus Antrum

3 wpi

Gastritis score

Occurrence of lymphoid follicle

Urease test

1

–

– – –

normal mucosa

1–2

–

Cardia Fundus Antrum

mucosa with hyperemia; Fundus: diffuse white macula Ø 2 mm

2

5 wpi

Cardia Fundus Antrum

normal mucosa

5 mpi

Cardia Fundus Antrum

10 mpi

Colonization

Results of RAPD-PCR

16S rDNA sequence

– – –

– – –

– – –

+ + –

+ – –

BO417 – –

n. d. – –

–

– + –

– + –

– n. d. –

– n. d. –

1

–

– + –

+ – +

n. d. – n. d.

n.d. – n.d.

normal mucosa

2

–

– – –

– – –

– – –

– – –

Cardia Fundus Antrum

normal mucosa

1–2

+

+ + +

+ – +

BO417 – BO417

BO417 – BO417

12 mpi

Cardia Fundus Antrum

mucosa with hyperemia; Fundus: diffuse white macula Ø 3 mm

2

–

– – –

+ – –

n. d. – –

n.d. – –

18 mpi

Cardia Fundus Antrum

mucosa with hyperemia; Fundus: diffuse white macula Ø 3 mm

2

+

– – –

+ + +

n. d. BO417 BO417

n.d. n. d. n. d.

wpi: weeks post infection; mpi: months post infection; n. d.: not investigated; +: positive; –: negative.

sion at 72 °C for 10 min. The amplified PCR products were analysed by agarose gel electrophoresis (1.5 %), stained with ethidium bromide and visualised under UV light. The fragment size of the amplified PCR product was 1380 bp. To rule out contamination with foreign DNA, negative PCR controls, containing only buffer and PCR primers, were run in parallel. Sequencing of the PCR products Prior to the sequencing of the 16S rDNA PCR-amplified products the DNA was eluted from the agarose gel into 180 µl purification buffer (supplied with the purification kit, see below) and heated to 65 °C for 15 min. The DNA was then further purified using the GFX DNA Gel Band Purification Kit (Amersham Pharmacia Biotech, USA) washed and eluted in double-distilled water. The purified PCR products were sequenced directly with the SequiTherm EXCEL II Long-Read DNA sequencing kit (Epicentre Technologies, Madison, USA). Didesoxynucleotides (ddNTP) for termination and the fluorescence (IRD 800)labelled primer pBR5se and p13B were added to the DNA samples. The PCR protocol was as follows: denaturation at 94 °C for 40 s, annealing at 58 °C for 40 s and elongation at 70 °C for 60 s (35 cycles). The denatured sequencing products

were separated in a sequencing gel (6 %) (Gene-Page Plus, Amresco, Solon, USA) and the DNA sequence analyses were performed automatically using a Li-cor 4000 DNA sequencer (MWG BIOTECH, Germany). The DNA sequences obtained were compared with DNA sequences of the H. pylori inoculum strains and with published DNA sequences through a BLAST database search (EMBL and GenBank databases). The partial 16S rDNA sequences for strains BO417 and BO418 were submitted to GenBank under accession numbers AJ310143 and AJ310144, respectively.

Results Colonization The results indicate that all three animals were successfully colonized by H. pylori. It was possible to reisolate the inoculated H. pylori from biopsies first taken two weeks after inoculation (Tables 1–3). All animals remained infected throughout the 18 months during which they were studied.

Experimental H. pylori infection in Rhesus macaques

Endoscopic investigation Clinically, the monkeys developed a moderate to severe gastritis particularly in the first weeks after infection. Localised to multifocal reddening and mild petechiae were noted endoscopically at different time points in each animal (Fig. 2c, d). Eighteen months after experimental infection all animals developed small whitish foci in their mucosa identified as lymphoid follicles by histological examination. The clinical symptoms varied between the animals with being less pronounced in animal nos. 7743 and 9050 and severe in animal no. 8156 (Tables 1–3). Histological examination The histological changes were compared with the histological picture before experimental infection, which was regarded as basic data for the following examina-

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tions. Until the fifth week all inoculated animals developed a mild to moderate chronic active gastritis up to gastritis score 2 (Fig. 3a–d). The gastritis persisted during the whole observation period in all three animals. Additionally, the mucosa of different stomach sites developed hyperemia and edema (Fig. 3a). After 10 months animal no. 7743 exhibited a marked atrophy of the previously normal antral mucosa, together with activated lymphoid follicles in the lamina propria and the chronic active gastritis still at grade 2 (Table 1) (Fig. 3c). In this animal the lymphoid follicle proliferation continued to expand, corresponding well with the clinical picture 18 months post infection. Together with the histological changes the presence of gastric bacteria was scored. Hematoxylin and eosin stain and Warthin-Starry silver stain each revealed the presence of LGIS in gastric pits and on the mucosa surface of each animal. The bacteria were 7–10 µm in length and tightly coiled with up to 10 spirals. In the

Table 2. Summary of findings in animal no. 8156. Time after H. pylori inoculation

Gastric side of biopsy pinch

Endoscopical Results

Prae-inoculation

Cardia Fundus Antrum

normal mucosa

2 wpi

Cardia Fundus Antrum

3 wpi

Gastritis score

Occurrence of lymphoid follicle

Urease test

0

–

– + –

mucosa with hyperemia; Fundus: petechiae; Antrum: ulcus Ø 5 mm

1–2

–

Cardia Fundus Antrum

normal mucosa

2

5 wpi

Cardia Fundus Antrum

normal mucosa

5 mpi

Cardia Fundus Antrum

10 mpi

Colonization

Results of RAPD-PCR

16S rDNA sequence

– – –

– – –

– – –

– + –

+ + +

BO418 n. d. BO417/ BO418

n. d. n. d. n. d.

–

– + –

– – +

– – n. d.

– – n. d.

1

–

+ + –

– – –

– – –

– – –

normal mucosa

2

–

– – –

– – +

– – BO417

– – BO417

Cardia Fundus Antrum

antrum: petechiae

2

–

+ + –

+ – +

BO417 – BO417

BO417 – BO417

12 mpi

Cardia Fundus Antrum

mucosa with hyperemia

2

–

– + –

+ – –

n. d. – –

n. d. – –

18 mpi

Cardia Fundus Antrum

mucosa with hyperemia

2

–

+ + +

– + +

– n. d. BO417

– n. d. BO417

wpi: weeks post infection; mpi: months post infection; n. d.: not investigated; +: positive; –: negative.

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Table 3. Summary of findings in animal no. 9050. Time after H. pylori inoculation

Gastric side of biopsy pinch

Endoscopical Results

Prae-inoculation

Cardia Fundus Antrum

normal mucosa

2 wpi

Cardia Fundus Antrum

3 wpi

Gastritis score

Occurrence of lymphoid follicle

Urease test

1

–

– – +

normal mucosa

1

–

Cardia Fundus Antrum

normal mucosa

2

5 wpi

Cardia Fundus Antrum

mucosa with hyperemia; fundus: petechiae

5 mpi

Cardia Fundus Antrum

10 mpi

Colonization

Results of RAPD-PCR

16S rDNA sequence

– – –

– – –

– – –

– + –

+ + –

BO417 BO417 –

n. d. n. d. –

+

– + +

– – –

– – –

– – –

1

–

– + –

+ + +

n. d. n. d. n. d.

n. d. n. d. n. d.

normal mucosa

1–2

–

– – –

– – –

– – –

– – –

Cardia Fundus Antrum

normal mucosa

2

–

+ + –

– – +

– – BO417

– – BO417

12 mpi

Cardia Fundus Antrum

mucosa with hyperemia

2

–

+ + –

– – –

– – –

– – –

18 mpi

Cardia Fundus Antrum

mucosa with hyperemia; diffuse white maculae

2

–

+ + +

– + –

– BO417 –

– n. d. –

wpi: weeks post infection; mpi: months post infection; n. d.: not investigated; +: positive; –: negative.

gastric pits some fragmented parts of Helicobacter-like bacteria were distinguishable. It was not possible to confidently identify organisms compatible with H. pylori in paraffin-embedded tissue sections. Electron microscopic examination showed different spiral gastric bacteria. All animals harboured helical, tightly coiled bacteria with up to 10 spirals either in the gastric pits or attached to the epithelium. The bacteria had unipolar, in some cases bipolar tufts of 4 to 6 sheathed flagella. Where the bacteria were attached to the epithelium, the epithelial cells formed cell fingers fusing with the bacteria. Apart from these long LGIS, short, only twice curved bacteria were identified within the gastric pits and interpreted as H. pylori because of their different morphology. Microbiological examination Culture and urease testing of biopsy specimens obtained at each endoscopy performed subsequent to in-

oculation showed persistent colonization with H. pylori in each animal. Microbiological results revealed the presence of H. pylori in all gastric compartments investigated with different intensity (Tables 1–3). On selective horse blood agar plates the H. pylori isolates were identified as forming small colorless pinhead-sized colonies that grew slowly within 3 to 5 days. Using negative staining techniques it was possible to demonstrate the typical H. pylori morphology of the re-isolated bacteria showing tightly curved rods with 3–6 sheathed flagella at one pole (Fig. 4). Typing of reisolated H. pylori strains by 16SrDNA sequence analysis and RAPD PCR H. pylori could be cultured from gastric biopsy specimens taken after 2 weeks, 5, 10, 12, and 18 months. H. pylori colonies could be preferentially isolated from the cardia and antral biopsies (Tables 1–3). Two animals (animal no. 8156, animal no.7743) were positive

Experimental H. pylori infection in Rhesus macaques

on six of seven examinations, the other one (animal no. 9050) only on the 2-weeks, 5-weeks, 10-months, and 18-months examination by culture. In order to characterize the reisolated bacteria, multiple single colony isolates of H. pylori recovered from biopsies were analysed by RAPD fingerprinting and 16S rDNA sequence analyses. The inoculum strains BO417 and BO418 differed in three nucleotide positions in the V6 region of the 16S rDNA genes, which permitted unequivocal discrimination between these strains. After 2 weeks, strain BO417 could be detected in at least one location in all animals. In one of the animals (no. 8156), both strains could be detected at this time point. At all later time points, only strain BO417 was detectable, indicating that this strain had outcompeted BO418 in all animals (Tables 1–3) (Fig. 5).

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Discussion The aim of the present study was to establish a closeto-human animal model of H. pylori infection. Genetically and immunologically, nonhuman primates are closest to humans, and chronic gastritis caused by H. pylori in these animals strongly resembles the human disease (Dubois, 1998). Furthermore, experimental infection of rhesus macaques with human pathogenic H. pylori allows a long-term investigation of disease development including gastric alterations, microbiological changes and immune responses. In the present study we confirm, that rhesus macaques can be successfully colonized by human pathogenic isolates of H. pylori. Successful colonizations were shown either by histological examination of biop-

Fig. 2. Gastroscopic examination. (a) Technique of brush cytology; (b) biopsy technique; (c) antrum; animal no. 8156; 10 months post infection; multifocal petechiae; (d) antrum, animal no. 8156; 2 weeks post infection, focal ulcera near to the pyloric area.

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K. Mätz-Rensing et al.

Fig. 3. Histological results. Paraplast, haematoxylin and eosin stain, 177x; 10 months post infection. (a) Cardia, animal no. 8156, moderate chronic gastritis with mononuclear infiltration of the gastric mucosa. (b) Antrum, animal no. 9050, moderate chronic gastritis with mononuclear infiltration of the gastric mucosa. (c) Antrum, animal no. 7743, moderate chronic gastritis with lymph follicles in the gastric mucosa. (d) Fundus, animal no. 9050, moderate chronic gastritis with lymph follicles in the gastric mucosa showing activated follicular centers.

Experimental H. pylori infection in Rhesus macaques

sy specimens obtained at each endoscopy after inoculation, or by reisolation of H. pylori from these biopsies. Single bacteria colonies growing on Helicobacterspecific horse blood agar plates could be roughly identified as Helicobacter-like bacteria because of their typical spiral morphology and physiological reaction (gramnegative, urease-positive etc.). However, RAPD-PCR with two different primers and broad-range 16S rDNA PCR combined with the DNA sequence analysis of the PCR products revealed that these colonies are identical with one of the applicated H. pylori strain. This method is useful not only for the specific identification of H. pylori, but also other bacterial species in gastric biopsies (Monstein et al., 1996). The highly sensitive PCR-based molecular methods described allow the identification and assignment of the reisolated bacteria to one of the applied strains which clearly outcompeted the other one. The 16S rDNA sequencing results support the RAPDPCR band patterns with no exception. The failure to colonize the monkeys with highly passaged laboratory strains was probably predictable given the experiences with both primate and non-primate models of H. pylori infection. While we could initially detect a mixed colonization with both strains in one of the three animals, only one strain was able to establish persistent infection in the monkey. It was notable that we were unable to detect colonization with any method after 5 months in two animals. It seems possible that the density of colonization was too low to be detectable due to incomplete adaptation of the strain to the monkey habitat, and that multiple adaptive mutations were necessary to develop the fitness necessary to establish a dense colonization. A more detailed characterization of strain BO417 and possible changes that the strain may have undergone during its adaptation to the monkey stomach is underway. The human type of H. pylori-associated chronic gastritis reveals superficial epithelial degeneration together with mononuclear (lymphocytes, plasma cells) infiltration of the mucosa as well as neutrophilic activity, an almost universal phenomenon in H. pylori gastritis (Dixon et al., 1996). Additionally, the incidence of lymphoid follicles in the basal portion of the mucosa ranges at 70 % (Dixon et al., 1996). Similarly, this and earlier studies with experimentally infected rhesus monkeys as well as with naturally occurring H. pylori infection show the development of active chronic gastritis with alterations close to those observed in humans (Baskerville and Newell, 1988; Dubois et al., 1996). The onset of the disease is characterised by increased neutrophilic activity accompanied by the development of gastritis. In our model the gastritis increased progressively to grade 2 and then persisted, alternating between grade 1 and 2 for the duration of observations. During the course, two animals developed even lymphoid follicles in the lami-

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Fig. 4. Fundus, animal no. 8156, 2 weeks post infection; negative staining of colony material with typical H. pylori morphology of the isolated bacteria showing tightly coiled rods with 4 unipolar flagella; TEM, 40 000 .

na propria and one animal showed the beginning of glandular atrophy in the antrum. In addition, we found a weak cellular immune response against H. pylori antigens (data not shown). The antigen-specific proliferation of peripheral blood lymphocytes (PBL), however, was associated with the experimental infection, because the T-cell reactivity against H. pylori antigens could be only measured in the acute phase 2–5 weeks after inoculation. A proliferative response to H. pylori antigens was neither found before infection or in PBL cultures of an uninfected, H. pylori-free animal, suggesting that the cellular immune response corresponds specifically to the H. pylori infection. In line with our findings Mattapallil et al. (2000) recently showed that the frequency of specific and IFN-γ producing T cells in gastric lamina propia and peripheral blood was increased during early acute H. pylori infection of rhesus macaques. We assume that this T cell-driven cell-mediated immune response is probably H. pylori-specific and the proliferation of T cells in our studies might be due to the primed T cells as described by others. The colonization of the monkeys with LGIS, which was not successfully eradicated in this study, did not seem to interfere with the H. pylori infection. While the existence of this coinfection may make the interpretation of some experiments more difficult, and was hence undesired, the mixed infections also open up some additional experimental avenues. It has been suggested that coinfections between H. pylori and other Helicobacter species may be a source of genetic diversity for H. pylori due to interspecific recombination (Suerbaum et al., 1998). The long-term effects of such coinfections on the genetic stability of a resident H. pylori population can be assessed in our model. The analyses

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Fig. 5. Typing of inoculum strains BO417, BO418 and bacteria reisolated from the rhesus monkeys by RAPD-PCR with primers AP1281 (left) and AP1254 (right). The time between inoculation of the monkeys and the reisolation of the strains is indicated above the lanes. The numbers 7743, 8156 and 9050 are the code numbers of the three monkeys experimentally infected with H. pylori in this study. wpi: weeks post infection; mpi: months post infection. Note the mixed infection (BO417+BO418) in animal 8156 at two wpi. In later samples, BO418 has been outcompeted by BO417.

reported here have not yielded evidence of genetic changes within BO417 in the first 18 months of infection, but a longer follow-up and more detailed examination should permit an assessment of the frequency of interspecies recombination events. Our results are consistent with previous reports indicating that the rhesus monkey is of special value concerning the establishment of these infections as a model for human disease (Dubois et al., 1996, 1999; Solnick et al., 1999). In addition, the similarities to the human disease render the rhesus monkey useful not only for infection studies but even more for follow-up studies of longer duration, given the long lifespan of Macaca mulatta. The great similarities between primates and humans make this animal model attractive for investigating the onset and progression of H. pyloriassociated gastrointestinal diseases and evaluating potential vaccine candidates in preclinical studies. Acknowledgements. The authors thank Helga Gilhaus, Hafiza Zuri and Karin Kaiser-Jarry for their excellent technical assistance. Work in the lab of S. Suerbaum was supported by grant SU 133/3-2 from the Deutsche Forschungsgemeinschaft.

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