Protective immunization against “Candidatus Helicobacter suis” with heterologous antigens of H. pylori and H. felis

Vaccine 24 (2006) 2469–2476

Protective immunization against “Candidatus Helicobacter suis” with heterologous antigens of H. pylori and H. felis A. Hellemans a,∗ , A. Decostere a , L. Duchateau b , M. De Bock a , F. Haesebrouck a , R. Ducatelle a a

Laboratory of Pathology, Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium b Departement of Physiology, Biochemistry and Biometry, Faculty of Veterinary Medicine, Ghent University, Belgium Received 4 July 2005; received in revised form 14 December 2005; accepted 14 December 2005 Available online 4 January 2006

Abstract “Helicobacter (H.) heilmannii” type 1 colonizes the human stomach. It has been shown to be identical to “Candidatus H. suis”, a Helicobacter species colonizing the stomach of more than 60% of slaughter pigs. This bacterium is, until now, not isolated in vitro. The effect of vaccination on “Candidatus H. suis” infection was studied in a mouse model. Mice were vaccinated intranasally or subcutaneously with whole bacterial cell lysate of Helicobacter pylori or Helicobacter felis and subsequently challenge infected with “Candidatus H. suis”. Intranasal and subcutaneous immunisation caused a decrease in faecal excretion of “Candidatus H. suis” DNA. Urease tests on stomach tissue samples at 16 weeks after challenge infection were negative in all H. felis intranasally immunized animals and in the majority of the animals of the other immunisation groups. Since PCR on stomach tissue samples at 16 weeks after challenge infection could still detect “Candidatus H. suis DNA” in all immunisation-challenge groups, complete clearance of challenge bacteria was not achieved. © 2005 Elsevier Ltd. All rights reserved. Keywords: Immunisation; Mouse model; Faecal PCR; “Candidatus Helicobacter suis”; Pigs

1. Introduction Helicobacter (H.) pylori infections in humans are a major cause of gastric and duodenal ulceration [1,2] as well as gastric cancer [3]. Triple therapies with proton pump inhibitors and antibiotics, clarithromycin and amoxicillin, are recommended as first line treatment [4]. These standard therapies increasingly face problems with antibiotic resistance [5] and recurrence of infection, especially in areas where H. pylori is endemic [6]. Vaccination may be a useful alternative. Different animal models have been developed in order to test vaccinal approaches [7]. H. pylori proteins expressed in infected mice and hence exposed to the mouse immune system, appear similar to those in human infections, suggesting

∗

Corresponding author. Tel.: +32 9 264 77 45; fax: +32 9 264 77 89. E-mail address: [email protected] (A. Hellemans).

0264-410X/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2005.12.033

that the mouse model is suitable for the preclinical screening of vaccine antigen candidates [8]. H. pylori is not the only bacterial pathogen capable of colonizing the human gastric mucosa. “Helicobacter heilmannii” indeed has been found in approximately 0.96% of gastric biopsies [9]. Recently it has been shown that H. heilmannii does not represent a single species, but a group of different bacterial species with a similar spiral morphology, most of which are probably zoonotic in origin [10–13]. On the basis of 16S rRNA gene sequences, “H. heilmannii” has been classified into two types [14]. ‘H. heilmannii’ type 2 organisms are closely related, if not identical, to the canine and feline Helicobacter spp., namely Helicobacter felis, Helicobacter bizzozeronii and Helicobacter salomonis. More than 50% of the “H. heilmannii” infections in humans, however, are due to “H. heilmannii” type 1 [13]. It is now accepted that “H. heilmannii” type 1 is identical to “Candidatus H. suis” [15,16], a spirally shaped bacterium that colonizes the stomach of more

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than 60% of slaughter pigs [17–19]. The role of “Candidatus H. suis” in gastric disease in the pig is still unclear, although it has been suggested that this bacterium is associated with gastric ulceration of the pars oesophagea [20] and chronic pyloric gastritis in pigs [18]. In vitro cultivation of “Candidatus H. suis” currently is not possible, but mouse inoculation can be used to grow and maintain this bacterium viable for more than two years starting from infected pig stomach mucosa [21,22]. Little information is available regarding treatment and prevention of “Candidatus H. suis” infection in both humans and pigs. The purpose of the present study was to determine whether prophylactic vaccination against “Candidatus H. suis” with antigens derived from related species, H. pylori or H. felis, is feasible, using a mouse model.

2. Materials and methods 2.1. Animals Five-week-old male SPF BALB/c mice (free from Helicobacter spp.) were purchased from an authorized breeder (HARLAN, Horst, The Netherlands). The animals were housed individually in autoclaved filter top cages and provided with a commercial diet (TEKLAD, HARLAN) and water ad libitum. After an adaptation period of 1 week, the animals were used in the experiments. All experiments involving animals were approved by the Animal Care and Ethics Committee of the Faculty of Veterinary Medicine, Ghent University. 2.2. Antigens for vaccination H. pylori SS1 and H. felis CS1 (ATCC 49179) were grown on brain heart infusion agar (BHI, Oxoid, Basingstoke, England) containing 10% horse blood, 5 mg amphotericin B/l, Campylobacter Selective Supplement (Skirrow (Oxoid) containing 10 mg vancomycin/l, 5 mg trimethoprim lactate/l and 2500 units polymyxin B/l) and Vitox supplement (Oxoid). Plates were incubated at 37 ◦ C in microaerobic conditions. The antigens used for immunization were prepared by harvesting 3-day-old cultures from H. pylori SS1 or H. felis CS1 in sterile phosphate buffered saline. The bacterial suspension was sonicated (eight times 30 s, 50% capacity; MISONIX, Farmingdale, USA). After centrifugation (5000 × g, 5 min, 4 ◦ C) the supernatant was filtered through a 0.22-␮m pore filter (Schleicher and Schuell, Gent, Belgium) and stored at −70 ◦ C. Afterwards, protein concentration was determined by the Lowry assay [23]. 2.3. “Candidatus Helicobacter suis” for challenge Since in vitro isolation of “Candidatus H. suis” is not possible with current methods, in vivo isolation was performed by mouse inoculation.

Thirty pig stomachs were obtained from the slaughterhouse and transported to the lab. The stomachs were opened and the remaining food was rinsed off with autoclaved tap water (37 ◦ C). A small mucosal tissue sample from the antrum (1 cm from the torus pyloricus) was taken to screen for the presence of “Candidatus H. suis”. Half of this fragment was used for rapid urease test (CUT, Temmler Pharma, Marburg, Germany): one tablet was dissolved in 500 ␮l of distilled water, and the mucosal tissue sample was incubated in the solution at 37 ◦ C. The test was regarded positive when the solution turned red within 1 h. The other half of the mucosal tissue sample was frozen (−20 ◦ C) and used for specific detection of “Candidatus H. suis” by PCR as previously described [24]. From one stomach that yielded a positive urease test, the upper cell layers and mucus from the antrum were scraped off. Scrapings were homogenized in lyophilisation medium (LYM) consisting of two volumes of horse serum, one volume of Brain Heart Infusion broth (Oxoid, Basingstoke, England) and 10% glucose. The homogenate was then centrifuged (5000 × g; 5 min) to remove large particles. Supernatant was diluted 1/10 in LYM and intragastrically inoculated in three BALB/c mice of 6 weeks old (Harlan, Horst, the Netherlands). After this first passage in mice, two extra mouse passages were performed. Each mouse passage was performed, 2 weeks after inoculation, by homogenizing whole urease positive mouse stomachs in LYM (5 ml LYM/stomach). Mouse stomach homogenate was then used as inoculum without previous centrifugation step. For each mouse passage, except for the last one, three new BALB/c mice were inoculated. The last mouse passage was performed in 15 BALB/c mice. From these 15 mice the urease positive stomachs were pooled and homogenized. The homogenate was frozen at −70 ◦ C. During mouse passages, each mouse stomach was screened for the presence of “Candidatus H. suis” with a urease test, performed as described above, on a mucosal tissue sample of the glandular region. A second stomach mucosal tissue sample of the glandular region was used in a PCR, specific for “Candidatus H. suis”, as previously described [24]. 2.4. Intranasal immunisation experiment Twenty-one mice were divided into three groups of six animals (groups 1–3) and one group of three animals (group 4). All animals from group 1 were immunized intranasally with H. felis CS1 sonicate and those of group 2 with H. pylori SS1 sonicate, twice with 3 weeks time interval. Intranasal immunisation was done by applying 100 ␮g of sonicate mixed with 5 ␮g cholera toxin (List, Campbell, CA, USA), in a total maximum volume of 30 ␮l, on the external nares of unanaesthetized mice. Mice from group 4 were not immunized. Four weeks after the final immunization all animals from groups 1, 2 and 3 were challenged with “Candidatus H. suis”. Therefore, the frozen stock from “Candidatus H. suis”, prepared as described above, was placed at 37 ◦ C for 15 min, and each

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animal was intragastrically inoculated with 0.3 ml. Animals from group 4 were not challenged. At 1, 2, 4, 5, 6, 7, 9, 11, 13 and 15 weeks after challenge, faecal material was collected in each week for 4 consecutive days, from each individual mouse to screen for the presence of bacterial DNA. DNA extraction and PCR was performed on 200 mg of faecal samples collected at a specific day using a protocol described below. Sixteen weeks after challenge, all animals were euthanized by cervical dislocation following isoflurane anaesthesia (IsoFLo, Abbot, IL, USA). The non-glandular part of the stomach was discarded, and then the stomach was divided longitudinally into two halfs from the esophageal opening to the pylorus. One half of the stomach from each of the animals was used for a semi-quantitative urease assay as described below. From the other half, 3 mm2 mucosal tissue samples were frozen (−20 ◦ C) and used for PCR specific for “Candidatus H. suis” as described below.

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Table 1 Helicobacter strains used to test the specificity of the PCR-SK developed for detection of “Candidatus H. suis” DNA in mouse faecal samples Strain

Species

CCUG 38995 CCUG 29260 CCUG 32350 NCTC 11961 LMG 6444 LMG 16318 LMG 18044 LMG 16316 LMG 18086 LMG 11759 LMG 14378 LMG 12678 LMG 12684 R 1051 R 1053 R 3647 LMG 7543

H. bilis H. pametensis H. nemestrinae H. pylori C. jejuni H. pullorum H. mustelae H. hepaticus H. canis H. fenelliae H. nemestrinae H. pametensis H. acinonychis H. bizzozeronii H. salomonis H. felis H. cinaedi

2.5. Subcutaneous immunisation experiment Twenty-one mice were divided into three groups of six animals (groups 1–3) and one group of three animals (group 4). Animals from groups 1 and 2 were immunized subcutaneously with H. felis or H. pylori sonicates, respectively, three times with 3 weeks time intervals. For this purpose 100 ␮g of the sonicate in solution was mixed in equal volume amounts with a proprietary saponin formulation (Pfizer Company, USA) and injected subcutaneously at the lower back of the animals. Four weeks after the final immunisation, animals from groups 1, 2 and 3 were challenged with “Candidatus H. suis” as described in experiment 1. Animals from group 4 were not immunized and not challenged. Sampling of faecal material and sampling of stomach material, from all animals, was done as described for Section 2.4. 2.6. PCR for detection of “Candidatus Helicobacter suis” in mouse faecal samples (PCR-SK) At the moment of the study a PCR for specific detection of “Candidatus H. suis” in stomach samples was already described [24]. This PCR amplifies a 433 bp fragment of the 16S rRNA gene of “Candidatus H. suis” but could not detect the bacterial DNA in the faeces of infected mice. In faeces, fragmentation of DNA has been shown [25], therefore, other primers HS 586 (5 -GGGAGGACAAGTCAGGTGTGAA3 ) and HS 641 (5 -TCTCCCACACTCCAGAAGGATAG3 ), amplifying a 79 bp fragment of the 16S rRNA gene (Genbank accession no. AF1027028), were selected. DNA from 200 mg faecal samples was extracted using QIAamp® DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to manufactures’s instructions. PCR reaction mixtures (50 ␮l) contained 50 pM of each primer (Invitrogen Life Technologies, Merelbeke, Belgium), 200 ␮M of each deoxynucleoside triphosphate (Amersham Pharmacia Biotech, Puurs, Belgium), 0.03 U/␮l Taq platinum, 1.5 mM

MgCl2 and 1 × PCR buffer (Invitrogen Life Technologies). Then, 2 ␮l of template DNA was added to the vials. The PCR conditions were as follows: initial denaturation at 95 ◦ C for 3 min; followed by 40 cycles of 30 s at 94 ◦ C, 30 s at 62 ◦ C and 30 s at 72 ◦ C. Final extension was performed for 5 min at 72 ◦ C. PCR products were run on 1.5% agarose gels containing 50 ng/ml ethidium bromide. After 1 h at 160 V the products were visualized with an UV transilluminator. Specificity of the primers was tested on DNA extracts from 17 different Helicobacter species (Table 1). No amplification products were seen when Helicobacter species other than “Candidatus H. suis” were used as template. To test the sensitivity of the PCR-SK, 200 mg faecal samples from SPF mice were spiked with respectively 1010 , 109 , 108 , 107 , 106 , 105 , 104 , 103 , 102 and 10 copies of the 16S rRNA gene sequence from “Candidatus H. suis” present as plasmid DNA. Therefore, the whole 16S rRNA gene sequence (1.4 kbp) of “Candidatus H. suis” was amplified by PCR as previously described [15]. This was followed by cloning into the pCR2.1-TOPO vector using the TOPO TA Cloning kit (Invitrogen, Life Technologies) according to the manufacturer’s instructions for chemical transformation. DNA sequencing further confirmed that the expected 16S rRNA gene sequence had been cloned into the pCR2.1-TOPO vector. Plasmid DNA was extracted with the S.N.A.P.TM Miniprep kit (Invitrogen, Life Technologies). The circular plasmid was eluted in sterile water and linearized with EcoRV (Invitrogen, Life Technologies). The absorbance of the DNA solution was measured three times at 260 nm on a UV–vis spectrophotometer, and the mean value was taken as the actual absorbance. The copy number of the rRNA gene sequence was calculated as “copy number of 16S rRNA gene/␮l = (concentration of linearized plasmid (g/␮l)/molecular weight of pCR2.1-TOPO (g/mol)) × 6.023 × 1023 (copies/mol)”. Serial dilutions of the purified linear plasmid were made and 10 ␮l of each dilu-

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tion (ranging from 109 copies/␮l to 1 copy/␮l) was used to spike a 200 mg faecal sample. DNA from spiked faecal samples was extracted using QIAamp® DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. PCR-SK performed on faecal samples spiked with different amounts of 16S rRNA gene sequence copies (ranging from 1010 to 101 copies), showed that PCR-SK on faecal samples could detect at least 105 copies of 16S rRNA gene sequence of “Candidatus H. suis”. Two copies of 16S rRNA gene are present in the H. pylori genome [26]. Assuming that two copies of the 16S-rRNA gene are also present in the genome of “Candidatus H. suis”, this detection limit represents 5 × 104 bacteria. 2.7. Urease assay for assessment of colonization of Helicobacter bacteria in mouse gastric tissue samples Urease activity in the stomach of mice was assessed using the method of Corth´esy-Theulaz et al. [27] with some modifications. One half of the stomach was immersed in 500 ␮l of CUTest (Temmler Pharma) and incubated at 37 ◦ C for 3 h. After centrifugation (5 min, 100 × g) the supernatant was used for spectrophotometric quantification at an OD of 550 nm. The cut-off value was calculated in each experiment and corresponded to the mean + 5S.D. of the absorbance values obtained with gastric samples of non-immunized, nonchallenged mice. 2.8. PCR for detection of Helicobacter in mouse gastric tissue samples DNA from mucosal tissue samples was extracted by using the Dneasy Tissue kit (Qiagen, Hilden, Germany) according to the instructions of the manufacturer. PCR for specific detection of “Candidatus H. suis” was performed as described previously [24].

isonwise significance level of 1.6% (adjusted by Bonferroni’s technique with three comparisons).

3. Results During the study three animals died from a cause unrelated to the treatment. For the intranasal immunisation experiment these animals comprised one animal from group 2 and one animal from group 3. For the subcutaneous immunisation experiment this comprised one animal from group 1. 3.1. Faecal excretion of “Candidatus Helicobacter suis” DNA in faeces of nonimmunized and immunized mice detected by PCR-SK All faecal samples from non-challenged control animals were negative in PCR-SK. Faecal excretion of “Candidatus H. suis” DNA assessed in individual mice is presented in Figs. 1 and 2. There was a significant difference (P < 0.01) in overall percentage PCR positivity between nonimmunized and intranasally immunized animals, both for the H. pylori and for the H. felis sonicate vaccines (Fig. 1). There was no significant difference in percentage PCR positivity between the two intranasally immunized groups, challenged with “Candidatus H. suis”. A significant difference in overall percentage PCR positivity was found also between nonimmunized mice and mice immunized subcutaneously (Fig. 2) with H. pylori sonicate (P < 0.01). The difference in overall percentage PCR positivity between nonimmunized mice and mice immunized subcutaneously with H. felis sonicate was not significant. There was no significant difference in percentage PCR positivity between the two subcutaneously immunised groups, challenged with “Candidatus H. suis”.

2.9. Statistical analysis The analysis of PCR positivity of faecal samples was based on the percentage PCR positivity of faecal samples of individual animals over the whole study period. This overall PCR positivity was compared between the nonimmunized and immunized groups using the Kruskal–Wallis test. A more detailed analysis was done based on the percentage PCR positivity using a logistic regression model with animal as random effect (to model the repeated measures structure) and time, treatment group and their interaction as categorical fixed effects. The semi-quantitative urease assay results were compared by a fixed effects model with OD value as response variable and treatment group as fixed effect. Pairwise comparisons were performed between the unimmunized group and the H. pylori and H. felis immunized groups at a global significance level of 5%, and a compar-

Fig. 1. Intranasal immunisation experiment: overall PCR positivity (expressed as percentage of positive faecal samples over the entire sampling period), specific for “Candidatus H. suis”, in faecal samples, per mouse (each
represents one animal) according to treatment group (bar represents mean) (Cs: challenge with “Candidatus H. suis”; INp: intranasal immunisation with H. pylori sonicate; INf: intranasal immunisation with H. felis sonicate). Treatment groups with * are significantly different (P < 0.01) from the nonimmunized group.

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Fig. 2. Subcutaneous immunisation experiment: overall PCR positivity (expressed as percentage of positive faecal samples over the entire sampling period), specific for “Candidatus H. suis”, in faecal samples, per mouse (each
represents one animal) according to treatment group (bar represents mean) (Cs: challenge with “Candidatus H. suis”; SCp: subcutaneous immunisation with H. pylori sonicate; SCf: subcutaneous immunisation with H. felis sonicate). Treatment groups with * are significantly different (P < 0.01) from the nonimmunized group.

No significant interaction between time and treatment group was detected in the more detailed analysis, so that it can be assumed that the differences between immunisation groups over time are constant and reflected by the differences found in the Kruskal–Wallis analysis of the overall percentage PCR positivity (Figs. 3 and 4). 3.2. Urease assay on gastric tissue The results of the semi-quantitative urease assay for the intranasal immunisation experiment are presented in Fig. 5. Immunization of animals with H. pylori or H. felis sonicate before “Candidatus H. suis” challenge resulted in a significantly lower (P < 0.05) urease activity compared to the nonimmunized challenged group. This was reflected in a negative (below the cut-off value) urease test in 80% (4/5) of the H.

Fig. 3. Intranasal immunisation experiment: to illustrate the evolution in time for the PCR (specific for “Candidatus H. suis”) positivity of faecal samples in challenge infected groups, weekly percentages were calculated, according to treatment group (, intranasal immunisation with H. pylori sonicate; ♦, intranasal immunisation with H. felis sonicate;
, no immunisation).

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Fig. 4. Subcutaneous immunisation: to illustrate the evolution in time for the PCR (specific for “Candidatus H. suis”) positivity of faecal samples in challenge infected groups, weekly percentages were calculated, according to treatment group (, intranasal immunisation with H. pylori sonicate; ♦, intranasal immunisation with H. felis sonicate;
, no immunisation).

pylori immunized animals and in all of the H. felis immunized animals. There was no significant difference in urease activity between the two intranasally immunised groups, challenged with “Candidatus H. suis”. The results of the semi-quantitative urease assay for the subcutaneous immunisation experiment are presented in Fig. 6. A significantly lower (P < 0.05) urease activity was found for both H. pylori and H. felis immunized groups, challenged with “Candidatus H. suis”, when compared to the non-immunized challenged group. Eighty percent (4/5) of the H. pylori immunized and 66% (4/6) of the H. felis immunized animals had a negative urease test. There was no significant difference in urease activity between the two subcutaneously immunised groups, challenged with “Candidatus H. suis”. 3.3. PCR analysis of gastric tissue All non-challenged animals were negative in PCR.

Fig. 5. Intranasal immunisation experiment: semi-quantitative urease activity of gastric stomach tissue represented as OD value (550 nm) per mouse (each
represents one animal) according to treatment group (bar represents geometric mean) (Cs: challenge with “Candidatus H. suis”; INp: intranasal immunisation with H. pylori sonicate; INf: intranasal immunisation with H. felis sonicate). Treatment groups with * are significantly different (P < 0.05) from the relevant non-immunized group. The dashed line represents the cutoff value (mean + 5S.D.) determined from negative mice stomachs.

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Fig. 6. Subcutaneous immunisation experiment: semi-quantitative urease activity of gastric stomach tissue represented as OD value (550 nm) per mouse (each
represents one animal) according to treatment group (bar represents geometric mean) (Cs: challenge with “Candidatus H. suis”; SCp: subcutaneous immunisation with H. pylori antigens; SCf: subcutaneous immunisation with H. felis antigens). Treatment groups with * are significantly different (P < 0.05) from the relevant non-immunized group. The dashed line represents the cut-off value (mean + 5S.D.) determined from negative mice stomachs.

Intranasal or subcutaneous immunisation of mice with H. pylori or H. felis antigens, and challenge with “Candidatus H. suis” resulted in all stomach samples being positive in PCR.

4. Discussion No information is available in the literature on the potential of vaccine induced protection against “Candidatus H. suis”. Since this bacterium is still unculturable we were interested to investigate whether immunisation with antigens from closely related Helicobacter species would lead to protection. Some studies revealed sufficient antigenic cross-reactivity between H. felis and H. pylori to generate protection to H. felis challenge following immunization with H. pylori sonicates [28,29]. Given the high degree of amino-acid conservation between H. pylori and H. felis urease enzyme, this protein most probably plays a role in induction of cross-protection [30,31]. There is one study showing that H. heilmannii infection can be prevented by vaccination with H. heilmannii UreB and H. pylori UreAB, confirming that protective immunity against Helicobacter infections can be elicited by homologous as well as heterologous Helicobacter urease [32]. Phylogenetically, H. felis is more closely related to “Candidatus H. suis” than H. pylori [15], therefore, one could expect to obtain better results with heterologous H. felis immunization. However, no significant differences between these two groups were found. The protective effect of H. felis or H. pylori immunization, found in this study, probably is due to antigens conserved between different Helicobacter species with urease and heat shock proteins as possible candidates [33,34]. An accurate diagnosis and quantification of Helicobacter infection is extremely important for evaluating immunization

studies. We used an urease assay to assess the effect of vaccination on bacterial colonization. The gastric urease assay has been used before for semi-quantitative detection of H. heilmannii, H. felis and H. pylori infection in mouse immunization studies [35,36]. In a study of Kleanthous et al. [37] it was shown that the urease assay is relatively insensitive in detecting H. pylori in gastric tissue below approximately 103 CFU and thus a negative urease assay does not indicate complete protection (sterilizing immunity). This is probably also true for detection of “Candidatus H. suis” colonization. Since histology has about the same sensitivity as the urease assay [38] a more sensitive quantitative culture approach should be used to detect the Helicobacter colonization. Unfortunately “Candidatus H. suis” is unculturable at this time. Therefore, PCR on stomach tissue samples, which is very sensitive and considered the standard for diagnosis of “Candidatus H. suis” [24], was used in this study to investigate whether immunised mice were actually free of this bacterium as suggested by the urease assay. To our knowledge, PCR on faecal samples is never used before to evaluate success of immunisation. In the immunized groups less faecal samples were positive for “Candidatus H. suis” DNA. It is not clear to what extent excretion of “Candidatus H. suis” DNA in the faeces reflects colonization level in the stomach. Nevertheless, performing this PCR on faecal samples was very useful to demonstrate differences between immunized and unimmunized mice. Helicobacter vaccination studies have mainly employed mucosal vaccination, although it has been demonstrated that parenteral immunization with appropriate schedules and formulations constitutes a valuable approach to cure Helicobacter infections [39–41]. We demonstrated that a protective response against “Candidatus H. suis” can also be induced by parenteral immunization with heterologous antigens. The immunization in the present study was prophylactic. In the case of therapeutic immunization, when the organisms have already oriented the host immune response to their benefit, parenteral immunization may be more successful than mucosal immunization. Parenteral immunization may mobilize cells from systemic origin that have not been already primed in one given direction by a Helicobacter infection [40]. Infection of mice with “Candidatus H. suis”-positive mucus was previously performed by Moura et al. [42] and this resulted in an inflammatory response in both antral and oxyntic mucosa. The pattern of histological lesions was similar to that observed in pigs infected with “Candidatus H. suis”. Moreover, the infection with “Candidatus H. suis, in both animal species, is not transient since the animals remain infected for a long period [40]. These results strenghten the hypothesis that “Candidatus H. suis” is a true pathogen. This study was aimed at showing the employability of the mouse model to study protection against “Candidatus Helicobacter suis” by immunisation. It was not our intention to study the development of post immunisation gastritis, although we realize that this is an important issue, especially when

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using sonicates or cell extracts for prophylactic immunisation [29]. In conclusion, we report for the first time that (1) both mucosal and systemic immunization with heterologous H. felis or H. pylori antigens hold promise for inducing protective immunity against “Candidatus H. suis”; and (2) this mouse model may be a useful tool in testing “Candidatus H. suis” vaccine formulations for eventual use in pigs and in humans.

Acknowledgements This work was supported by the Federal Government service of Public Health, Safety of the Food Chain and Environment grant S-6137. We thank Dr. R. Ferrero, Monash University, Clayton, Australia for providing the H. pylori SS1 strain. We are grateful to Nathalie Van Rysselberghe and Karolien Hermy for their technical assistance.

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