Introduction

During the last decade, the number of species in the genus Helicobacter has quickly developed, and at least 38 formally named Helicobacters have been recognized. Helicobacter pylori (H. pylori) is the most important species globally in human disease; however, Helicobacter heilmannii and Helicobacter felis are also associated with gastric disease in humans and are worthy of discussion (Harbour and Sutton 2008; Heilmann and Borchard 1991; Lee et al. 1988).

H. heilmannii has the largest number of known mammalian hosts among the known gastric Helicobacter spp. (Trebesius et al. 2001). These bacteria accompany with H. felis have been microscopically observed in the stomach of dogs, wild rats, cheetahs, cats, swine, various species of nonhuman primates, and in a small percentage of humans with gastritis. But H. felis was occasionally observed in human gastric biopsies (Eaton et al. 1993; Lavelle et al. 1994; Queiroz et al. 1990; Stolte et al. 1994).

The prevalence of gastric Helicobacter spp. in dogs and cats is high, but exact mode of transmission and the role of Helicobacter spp. in gastrointestinal diseases are not clear. It suggested that oral–oral and fecal–oral are probable routes of transmission (Brown 2000). Detection of Helicobacter spp. in saliva and dental plaque supported this hypothesis that the oral cavity could be a reservoir and source of Helicobacter spp. infection in human and cats (Recordati et al. 2007; Shojaee Tabrizi et al. 2010). To the best of the authors’ knowledge, there is no documented report investigating prevalence and association of different species of Helicobacter spp., including H. pylori, H. felis, and H. heilmannii in the oral cavity and stomach of stray dogs.

Consequently, the principal aim of the present study was to determine the prevalence of Helicobacter spp. in the oral cavity of stray dogs, as a possible route of transmission, and to identify the association between the oral and gastric Helicobacters.

Materials and methods

Animals and sampling procedure

This study has been approved by the Iranian laboratory animal ethics framework under the supervision of the Iranian Society for the Prevention of Cruelty to Animals. Forty-eight stray dogs (29 males and 19 females (mean age = 5.2 years)) were captured from different suburban locations of Mashhad, Iran. In Iran, government policy dictates that overpopulating stray dogs should be euthanized to preserve the wild life in suburban areas. Health status of dogs was not ascertained in our study. Sampling performed immediately after euthanasia with acepromazine (Neurotranq 1, 0.2 mg/kg, IM) and Thiopental Sodium (Nesdonal 1, 25–30 mg/kg, IV).

Sterile cotton swabs were used to collect oral secretions, and dental plaque was removed from the first upper premolar with a sterile curette. Then, samples were immediately transferred to the 500 ml phosphate buffer saline. At necropsy, stomachs were removed and opened along the greater curvature, and samples were collected from gastric fundus, gastric body, and gastric juice. All gastric and oral specimens were frozen immediately at −20 °C until further analysis.

DNA extraction and PCR assays

DNA was extracted from the saliva, dental plaque, and gastric samples using QIA amp DNA Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Subsequently, presence of Helicobacter genus was investigated using 16S rRNA gene and, then, by using specific primers. Positive samples were evaluated for different species included H. pylori, H. heilmannii, and H. felis. PCR amplification was performed in a final volume of 25 μl containing 3 μl of extracted DNA, 2.5 μl of 10× PCR buffer (Fermentas, Lithuania), 1.5 mM MgCl2, 0.2 mM of each deoxynucleotide triphosphate, 1.6 × 10−7 mM of each primer and 0.04 U μl−1 Super Taq DNA Polymerase (Gen Fanavaran Co., Iran). Primer sequences and PCR conditions are presented in Table 1. The resulting PCR products underwent gel electrophoresis [1.2 % (w/v) agarose gel with 0.3 % ethidium bromide in 10 % Tris–Borate–EDTA buffer] and visualized under UV transilluminator. PCR results were interpreted as follows: appearance of (1) the 764 bp band only, Helicobacter spp. positive (H. heilmannii and H. felis negative); (2) the 764 and 580 bp bands, H. heilmannii positive and H. felis negative; (3) the 764 and 241 bp bands, H. felis positive and H. heilmannii negative; (4) the 764 and 296 bp bands, H. pylori positive (H. heilmannii and H. felis negative); and (5) all four bands: H. felis, H. heilmannii, and H. pylori-positive.

Table 1 Oligonucleotide primers and PCR conditions
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Sequencing

Positive samples for 16S rRNA specific primers that did not meet the specified species of Helicobacter genus were randomly subjected to sequencing. The sequences were analyzed by nucleotide data bank, and their similarities were determined.

Statistical analysis

Frequency of H. felis and H. heilmannii was calculated in dental plaque, saliva, gastric juice, gastric fundus, and body. Oral cavity samples include dental plaque and/or saliva samples, and gastric region samples include gastric juice or gastric fundus and/or gastric body samples. For the assessment of the association between the presence of Helicobacter spp. in the gastric region vs. oral cavity and dental plaque vs. saliva, chi-square test and, if required, Fisher’s exact test were used. p Values less than 0.05 were considered as statistically significant. Kappa coefficient was also calculated to evaluate agreement between existence of H. felis or H. heilmannii in oral cavity and gastric region.

Results

Genus-specific PCR identified Helicobacter spp. DNA in 45/48 (93.7 %) of dental plaque and 43/48 (89.6 %) of saliva samples. All 48 dogs were found to harbor Helicobacter spp. DNA in their oral cavity (dental plaque and/or saliva). Frequency of H. felis and H. heilmannii in dental plaque specimens was 3/45 (6.6 %) and 17/45 (37.7 %). Two samples of dental plaque (4.4 %) had both H. felis and H. heilmannii. PCR in saliva samples detected Helicobacter spp. DNA in 43/48 (89.6 %) of the subjects; 2/43 (4.6 %) were H. felis, and 18/43 (41.8 %), H. heilmannii. No saliva samples had both strains, simultaneously. A total of 5 (10.4 %) and 27 (56.2 %) dogs were found to harbor H. felis and H. heilmannii in oral cavity, respectively. Finally, no association was detected between the presence of H. felis or H. heilmannii in dental plaque and saliva (p = 1 and p = 0.31, respectively).

Helicobacter spp. DNA was identified in gastric juice, fundus, and body of the stomach in 46 (95.8 %), 40 (83.3 %), and 40 (83.3 %) of 48 dogs, respectively. H. felis and H. heilmannii were found in 12/46 (26 %) and 33/46 (71.7 %) of gastric juice, 12/40 (30 %) and 25/40 (62.5 %) of fundus, and 22/40 (55 %) and 16/40 (40 %) of body samples, respectively. Totally, gastric region infection with H. felis and H. heilmannii was 31 (64.6 %) and 43 (89.6 %), respectively. H. pylori was not found in any parts of oral cavity or stomach.

Table 2 presents the frequency of Helicobacter spp., H. felis and H. heilmannii, in oral cavity (saliva and dental plaque) and gastric specimens. There was no statistically difference between the frequency of H. felis in oral cavity and gastric specimens (p = 0.45). Frequency of H. heilmannii was 27 (56.2 %) in oral cavity and 43 (89.6 %) in gastric regions, and no significant differences were identified (p = 0.86). H. felis was simultaneously negative in 16 and positive in 4 dogs in oral cavity and gastric region samples, and there was a little agreement between the presence of H. felis in the oral cavity and gastric region (kappa = 0.05). Two and 24 dogs were simultaneously negative and positive in oral cavity and gastric region for H. heilmannii, respectively. There was less than chance agreement between existence of H. heilmannii in oral cavity vs. gastric region (kappa = 0.02).

Table 2 Frequency (in percents) of Helicobacter spp., H. felis and H. heilmannii, in oral cavity (saliva and dental plaque) and gastric specimens (n = 48)
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Two DNA samples of dental plaques that did not meet used species of Helicobacter by PCR were subjected to sequencing. DNA sequencing of their 16S rRNA revealed that they were related to the Wolinella spp. with homology rates of 99 % (GenBank accession JN869512-JN869513).

Discussion

In this study, all samples of the oral cavity and gastric regions were infected with Helicobacter spp., except two of dental plaques which were related to Wolinella spp. Several studies previously showed that Helicobacter spp. are extremely prevalent in dog’s stomach, so that 67–100 % of healthy pet dogs (Eaton et al. 1996; Happonen et al. 1998; Jalava et al. 1998) and 100 % of random-source dogs (McNulty et al. 1989), laboratory beagles, and shelter dogs have been infected (Eaton et al. 1996; Henry et al. 1987; Strauss-Ayali et al. 1999), which is compatible with our findings.

Besides H. pylori, the most important species of Helicobacter in human, two other species named H. heilmannii and H. felis are also related with gastric disorders in humans (Heilmann and Borchard 1991; Lee et al. 1988). In the current study, H. pylori was not detected in oral cavity and gastric regions of stray dogs. In contrast with H. pylori, the prevalence of H. heilmannii was about 56.3 and 89.6 % in dog’s oral cavity and gastric regions.

H. heilmannii is an important Helicobacter that can cause gastritis, peptic ulcer, and even gastric low-grade lymphoma in humans (Morgner et al. 2000; Regimbeau et al. 1998). Unlike many species of Helicobacter spp., such as H. pylori and H. felis, it has been isolated from different types of mammals (Trebesius et al. 2001). Several studies showed that the prevalence of H. heilmannii was less than 0.5 % among human patients who underwent upper gastrointestinal endoscopy for dyspeptic symptoms (Flejou et al. 1990; Heilmann and Borchard 1991; McNulty et al. 1989; Morris et al. 1990); however, it has been as high as 6 % in Thailand and China (Yali et al. 1998; Yang et al. 1998). This controversy could be related to different parameters in various geographic regions, but, as our study revealed a high prevalence of H. heilmannii in stray dogs, human contact with infected animals may be an important risk factor for acquiring this infection.

This study showed low prevalence of H. felis in oral cavity (10.4 %) and high prevalence of H. felis in gastric regions (64.6 %). In other studies, 8.4 and 62.7 % of dogs were affected with H. felis in gastric regions (Jalava et al. 1998; Van den Bulck et al. 2005).

Even though the exact route of transmission is unclear, direct contact, fecal–oral, oral–oral, and gastro–oral are likely ways (Axon 1995). With respect to some reports of non-pylori Helicobacter gastritis in humans and high prevalence of H. felis and H. heilmannii in dogs, zoonotic features of Helicobacter infections should be considered (Meining et al. 1998; Stolte et al. 1994).

There are a few reports about the prevalence of Helicobacter spp. in oral cavity and its association with occurrence of Helicobacter spp. in gastric regions (Solnick and Schauer 2001). In a study by Recordati et al. (2007), nested PCR showed Helicobacter spp. DNA in 36 (94.7 %) gastric biopsies, 17 (44.7 %) dental plaque, and 19 (50 %) saliva samples. In this study, no statistical relationship (p = 0.053) between the degree of gastric colonization in histology and the presence of Helicobacter spp. DNA in the oral cavity was shown (Recordati et al. 2007).

In accordance with previous studies, in the current study, the association between H. felis and H. heilmannii in oral cavity and gastric region was not shown. Shojaee Tabrizi et al. (2010) also found no correlations between Helicobacter colonization at oral cavity and gastric regions of stray cats (Shojaee Tabrizi et al. 2010). In contrast, a number of authors reported statistically significant correlation between the existence of H. pylori in the gastric region and the oral cavity (Morales-Espinosa et al. 2009; Rasmussen et al. 2010). Although we did not find any relationship between presence of H. felis or H. heilmannii in oral cavity and gastric region, high prevalence of H. heilmannii in oral cavity favors the oral–oral spread and proposes that the oral cavity may be a reservoir for Helicobacter spp., though may not inevitably indicate the colonization status of the gastric mucosa.

Our study showed relatively low prevalence of H. felis (6.6 and 4.6 %) and high prevalence of H. heilmannii (37.7 and 41.8 %) in dental plaque and saliva samples. There was no association between presence of H. felis or H. heilmannii in dental plaque and saliva. In another study on humans, H. pylori DNA was detected in 42.3 and 47.4 % of saliva and dental plaque samples, respectively (Rasmussen et al. 2010), although no statistically significant difference was observed between strains in the saliva and dental plaque (Rasmussen et al. 2010).

Direct sequencing of two 16S rRNA gene-specific PCR products of dental plaque was related to the Wolinella spp. with homology rates of 99 %. Wolinella spp. is related to Helicobacteraceae family with ribosomal DNA similar with Helicobacter spp. members. Frequency of this bacterium in the oral cavity of dogs is believed to be high (Craven et al. 2011). Results of this study, in accordance with Craven et al. (2011), revealed that Wolinella spp. may be found in the oral cavity of dogs. However, further investigation is required to detect the real prevalence of Wolinella in the oral cavity of dogs and its role in pathogenesis of gastrointestinal disorders.

In conclusion, this study showed low prevalence of H. felis and high prevalence of H. heilmannii in oral cavity of stray dogs. Meanwhile, it revealed that the contamination of dog’s oral cavity may not neccessarily equal to the gastric Helicobacter infection, but it may possibly operate as a reservoir for gastric infectivity. Furthermore, the oral–oral route should be considered as an important route of transmission for non-pylori Helicobacter spp. in stray dogs.