• View in gallery
    Figure 1—

    Phylogenetic consensus tree depicting the genetic relationship among 16S rRNA gene sequences amplified with the C97 and C05 primer set in organisms cultured from gastric biopsy specimens, dental plaque, and saliva of 8 dogs. Sequences from vomiting dogs are indicated (asterisks). The numbers at the nodes are the bootstrap percentages (1,000 replications; 65% cutoff). Vertical distance has no meaning. Reference isolates were obtained from GenBank; accession numbers for each reference isolate are indicated in parentheses. Scale in the lower left corner represents nucleotide substitutions per site.

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    Figure 2—

    Photograph of an agarose gel depicting PCR products for Wolinella-specific primers performed on DNA extracted from the saliva samples of 10 pet dogs (lanes 1 through 10), Helicobacter heilmannii (lane 11), Helicobacter felis (lane 12), Helicobacter cinaedi (lane 13), Helicobacter bilis (lane 14), Helicobacter bizzozeronii (lane 15), Helicobacter hepaticus (lane 16), H pylori (lane 17), Helicobacter canis (lane 18), Wollinella succinogenes (lane 19), and negative control samples (lanes 20 and 21). Lane 22 is a 110-bp ladder.

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    Figure 3—

    Fluorescence in situ hybridization image of a saliva smear obtained by use of probes for Helicobacter spp (HEL274 and HEL818 probes conjugated to the fluorescent dye Cy3) and eubacteria (EUB338 probe conjugated to the fluorescent dye 6FAM). The characteristic lazy s–shape of W succinogenes is visible (red-orange structure) and is surrounded by non-Helicobacter bacteria with a spiral morphology (green structures).

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Evaluation of the Helicobacteraceae in the oral cavity of dogs

Melanie CravenDepartment of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853

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Camilla RecordatiDipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Sezione di Anatomia Patologica Veterinaria e Patologia Aviare, Facoltà di Medicina Veterinaria, Università degli Studi di Milano, 20133 Milano, Italy

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Valentina GualdiIZSLER Sezione di Lodi, via A. Einstein, Località Cascina Codazza, 26900 Lodi, Italy

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Graziano PengoClinica Veterinaria Oriolo, SS 415, Km 41.5, 26012 Castelleone, CR, Italy.

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Mario LuiniIZSLER Sezione di Lodi, via A. Einstein, Località Cascina Codazza, 26900 Lodi, Italy

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Eugenio ScanzianiDipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Sezione di Anatomia Patologica Veterinaria e Patologia Aviare, Facoltà di Medicina Veterinaria, Università degli Studi di Milano, 20133 Milano, Italy

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Kenneth W. SimpsonDepartment of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853

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Abstract

Objective—To determine the Helicobacter spp present in the oral cavity of dogs and the relationship of those organisms with gastric Helicobacter spp to better define the potential for dog-human and dog-dog transmission.

Sample—Saliva and dental plaque from 28 dogs and gastric biopsy specimens from a subset of 8 dogs.

Procedures—PCR-based screening for Helicobacter spp was conducted on samples obtained from the oral cavity of 28 dogs. Comparative analysis was conducted on Helicobacteraceae 16S rDNA clone libraries from the oral cavity and stomach of a subset of 8 dogs (5 vomiting and 3 healthy) that had positive PCR results for Helicobacter spp.

Results—Helicobacteraceae DNA was identified in the oral cavity of 24 of 28 dogs. Analysis of cloned 16S rDNA amplicons from 8 dogs revealed that Wolinella spp was the most common (8/8 dogs) and abundant (52/57 [91%] clones) member of the Helicobacteraceae family in the oral cavity. Only 2 of 8 dogs harbored Helicobacter spp in the oral cavity, and 1 of those was coinfected with Helicobacter heilmannii and Helicobacter felis in samples obtained from the stomach and saliva. Evaluation of oral cavity DNA with Wolinella-specific PCR primers yielded positive results for 16 of 20 other dogs (24/28 samples were positive for Wolinella spp).

Conclusions and Clinical RelevanceWolinella spp rather than Helicobacter spp were the predominant Helicobacteraceae in the oral cavity of dogs. The oral cavity of dogs was apparently not a zoonotically important reservoir of Helicobacter spp that were non–Helicobacter pylori organisms.

Abstract

Objective—To determine the Helicobacter spp present in the oral cavity of dogs and the relationship of those organisms with gastric Helicobacter spp to better define the potential for dog-human and dog-dog transmission.

Sample—Saliva and dental plaque from 28 dogs and gastric biopsy specimens from a subset of 8 dogs.

Procedures—PCR-based screening for Helicobacter spp was conducted on samples obtained from the oral cavity of 28 dogs. Comparative analysis was conducted on Helicobacteraceae 16S rDNA clone libraries from the oral cavity and stomach of a subset of 8 dogs (5 vomiting and 3 healthy) that had positive PCR results for Helicobacter spp.

Results—Helicobacteraceae DNA was identified in the oral cavity of 24 of 28 dogs. Analysis of cloned 16S rDNA amplicons from 8 dogs revealed that Wolinella spp was the most common (8/8 dogs) and abundant (52/57 [91%] clones) member of the Helicobacteraceae family in the oral cavity. Only 2 of 8 dogs harbored Helicobacter spp in the oral cavity, and 1 of those was coinfected with Helicobacter heilmannii and Helicobacter felis in samples obtained from the stomach and saliva. Evaluation of oral cavity DNA with Wolinella-specific PCR primers yielded positive results for 16 of 20 other dogs (24/28 samples were positive for Wolinella spp).

Conclusions and Clinical RelevanceWolinella spp rather than Helicobacter spp were the predominant Helicobacteraceae in the oral cavity of dogs. The oral cavity of dogs was apparently not a zoonotically important reservoir of Helicobacter spp that were non–Helicobacter pylori organisms.

Spiral bacteria of the family Helicobacteraceae, principally Helicobacter pylori, colonize the stomach of approximately 50% of the world's humans1 and are causally associated with chronic superficial gastritis, peptic ulcers, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma.2–4 Gastric colonization by Helicobacter spp is also common in dogs (61% to 100%),5,6 but the infecting species are a diverse group of non–H pylori Helicobacter spp, which include Helicobacter bizzozeronii, Helicobacter heilmannii, Helicobacter felis, Helicobacter salomonis, Helicobacter bilis, Helicobacter cyanogastricus, and Flexispira rappini.5,7–10 The consequences of Helicobacter infection in dogs are less defined, with infection linked to lymphoid hyper-plasia and gastritis, rather than to cancer and pepticulcers.11 Infection with non–H pylori Helicobacter spp, typically categorized as H heilmannii, is reported in 0.25% to 6.0% of humans and is also associated with gastritis and mucosa-associated lymphoid tissue lymphoma.3,12,13 However, information reported in 1 study13 highlights the presence of Helicobacter spp typically associated with the stomachs of dogs (eg, H bizzozeronii or H salomonis rather than H Heilmannii) in up to 48% of humans with non–H pylori Helicobacter infection.

The high prevalence of non–H pylori Helicobacter infection in dogs5,6 raises the possibility that dogs are a potential reservoir for infections of humans. The potential for zoonotic spread is supported by results of a study14 on the increased risk of H heilmannii infection in humans in contact with dogs, cats, and pigs. A questionnaire-based study15 of 125 human patients with gastritis attributable to H heilmannii revealed that 70.3% had contact with 1 or more animals, compared with 37% who had animal contact but did not have gastritis. More direct evidence is provided by results of studies16,17 on similar strains of Helicobacter spp being isolated from humans and their pets. However, the potential zoonotic risk posed by dogs for H heilmannii infection has been challenged on the basis of the relatively low prevalence of H heilmannii in dogs and the fact that dogs have gastric colonization with H heilmannii types 2 and 4, rather than colonization with H heilmannii type I, which is the dominant subtype in humans.18,19

The precise mode of transmission for Helicobacter spp is unresolved in all species, with fecal-oral, oral-oral, and gastric-oral routes hypothesized as possibilities.2,7,20 The recognition of the oral cavity as an entry point for gastric Helicobacter spp in humans was first determined by Marshall et al,1 who found that ingestion of H pylori could subsequently lead to the development of gastritis. Helicobacter pylori DNA has since been isolated from the saliva, supragingival and subgingival plaque, oral mucosa, and dorsal aspect of the tongue of human patients, although detection rates range from 0% to 90%.21 Dogs can acquire Helicobacter infection at an early age through close contact with their dams,22 and in 1 study,23 investigators detected Helicobacter DNA in the saliva and dental plaque in 19 (50.0%) and 17 (44.7%) of 38 dogs examined, with 27 of 38 (71.1%) dogs having positive results for Helicobacter DNA in plaque or saliva. Because contact with oral secretions is the most plausible route of zoonotic infection transmitted from dogs, the purpose of the study reported here was to more critically examine the potential zoonotic risk posed by dogs by defining the Helicobacter spp present in the oral cavity of dogs and the relationship of those organisms with gastric Helicobacter spp.

Materials and Methods

Samples—Samples of saliva and dental plaque were obtained from 28 client-owned dogs; the dogs had various clinical signalments. Saliva samples were obtained from 20 dogs randomly selected from the general practice and medicine caseload at the Cornell University Hospital for Animals and included healthy dogs as well as those undergoing clinical investigation because of a wide variety of clinical signs. Samples were collected and placed in sterile PBS solution on ice. A subset of 8 dogs (4 males and 4 females; 5 vomiting and 3 healthy) that ranged from 6 months to 14 years of age (mean, 6.8 years) underwent gastric biopsy at the University of Milan. Biopsy samples were collected into sterile microcentrifuge tubesa and placed on ice. Endoscopy and collection of biopsy samples were performed as part of the clinical investigation. Results for these 8 dogs have been reported elsewhere.23 Owner consent was obtained for all dogs and procedures. The study was approved by the Ethical Committee of the Veterinary School of the University of Milan.

PCR assay for Helicobacter spp—The DNA was extracted from oral and gastric samples as described elsewhere23,24 and stored at −80°C until analyzed. Aliquots of DNA were amplified by use of a PCR assay with Helicobacter-specific primers C97 (5′-GCTATGACGGGTATCC-3′) and C05 (5′-ACTTCACCCCAGTCGCTG-3′), which resulted in the generation of 1,200-bp 16S rDNA gene amplicons.6,25 Briefly, samples were thawed on ice, and 2 μL of sample was added to a 50-μL volume that contained 25 pmol of each primer and 25 μL of Taq polymerase.b Samples were heated in a thermocyclerc to 94°C for 10 minutes; then subjected to 35 cycles of denaturation for 1 minute at 94°C, primer annealing for 1.5 minutes at 58°C, and extension at 72°C for 2 minutes; and then subjected to a final extension at 72°C for 15 minutes. Negative control samples in which the DNA extract was omitted were included with each reaction. The PCR products were developed via agarose gel (1%) electrophoresis with ethidium bromide in Trisacetate EDTA buffer. Size of the expected fragments was compared with a 100-bp reference marker.d

Construction and analysis of 16S rDNA clone libraries—The DNA from the oral cavity and stomach of 8 dogs (5 vomiting and 3 healthy) with positive results for Helicobacter spp when tested by use of the PCR assay was amplified with the primers C97 and C05 as described previously. A PCR purification kite cloned into a thymine-adenine cloning vectorf was used to purify 1,200-bp 16S rDNA products and introduce them into Escherichia coli DH5α. Plasmid DNA from up to 10 clones of each sample was purified by use of a DNA-purification kit,g and clones were sequenced at the Cornell University Bio-Resource Center with M13 primers and DNA polymeraseh by use of DNA-sequencing kits.i,j The DNA sequences obtained with forward and reverse primers were extracted from flanking vector sequences by use of computer software,k and the same software was used to align contiguous sequences. The identity of partial 16S rDNA sequences was determined by comparison with data from an online databasel and a bioinformatic search tool.m Phylogenetic analyses were conducted by use of the neighbor-joining method in a software program.26,n

PCR assay for Wolinella spp—On the basis of the results of 16S rDNA sequence analysis, we designed primers against Wolinella spp (WOL1, forward: 5′-AAAGAGCACGTAGGCGGC-3′ [position 316,294 through 316,272]; WOL2, reverse: 3′-CAGGATTCTATCAATGTCAAGCCC-5′ [position 315,857 through 315,874]) that resulted in generation of a 440-bp amplicon. Specificity of the Wolinella spp primers was tested on DNA extracts from H pylori (ATCC 43504), H felis (ATCC 49179), H bizzozeronii (ATCC 700030), H heilmannii (DNA from the stomach of an infected cat), H bilis (ATCC 51630), Helicobacter cinaedi (ATCC 35683), Helicobacter hepaticus (ATCC 51450), Helicobacter canis (ATCC 51401), and Wolinella succinogenes. The DNA extract (2 μL) was heated to 95°C for 15 minutes; then subjected to 35 cycles of 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 45 seconds; and then subjected to a final extension step at 72°C for 7 minutes. The DNA extracted from saliva samples of 20 dogs that were not examined by use of 16S rDNA sequencing was analyzed via PCR assay for the presence of Wolinella spp DNA.

FISH for Helicobacteraceae in saliva—Smears of saliva samples were also analyzed by use of FISH for Helicobacteraceae (HEL274 and HEL717 probes conjugated to Cy3 fluorescent dye) and eubacteria (EUB338 probe conjugated to 6FAM dye), as described elsewhere.18,27

Data analysis—Differences in the proportion of Wolinella and Helicobacter spp sequences in 16S rDNA libraries constructed from the stomach and oral cavity were evaluated by use of χ2 tests. Signifcance was set at P < 0.05.

Results

Helicobacter PCR assay—The PCR assay with primers C97 and C05 yielded positive results for Helicobacter spp in the oral cavity DNA samples of 14 of 28 (50%) dogs and the gastric DNA samples of 8 of 8 dogs.

Analysis of 16S rDNA clone libraries—The 16S rDNA clone libraries were constructed from DNA obtained from the oral cavity and stomach of each of 8 dogs (5 vomiting and 3 healthy) that had positive results for Helicobacter spp when tested by use of PCR assays.

Oral cavity

A total of 57 clones from the oral cavity of the 8 dogs with positive results for Helicobacter spp determined by use of PCR assay on samples from the oral cavity and stomach were sequenced. Sequences with highest homology to a Wolinella sp (GenBank M88159) were the most prevalent (8/8 dogs) and abundant (52/57 [91.2%] clones) in samples obtained from the oral cavity. Wolinella spp were the only members of the Helicobacteraceae family amplified from the dental plaque of 6 of 8 dogs. Sequences for Helicobacter spp were detected in 2 dogs: analysis of the dental plaque from a healthy dog yielded organisms that had sequences with the highest homology to H heilmannii (1/7 clones; GenBank AF506786), and the saliva from a vomiting dog yielded organisms that had sequences with the highest homology to H felis (3/6 clones; GenBank AY366428) and H heilmannii (1/6 clones; GenBank AF506786).

Gastric mucosa

A total of 63 clones from the stomach of the 8 dogs with positive PCR results for Helicobacter spp were sequenced. All of these sequences had highest homology to Helicobacter spp known to be present in the canine stomach, including H felis (6 dogs; 32/63 [50.8%] clones; GenBank M57398, U51870, AY686607, and AY366428), H heilmannii (5 dogs; 22/63 [34.9%] clones; GenBank AF506786 and AF506775), and H bizzozeronii (2 dogs; 9/63 [14.3%] clones; GenBank AF302107). The proportion of Wolinella and Helicobacter spp sequences in 16S rDNA libraries differed significantly (P < 0.001) between the oral cavity and stomach.

Of the 5 vomiting dogs, organisms that had sequences consistent with H heilmannii were detected in 4 (20/40 [50%] clones) and H felis were detected in 3 (20/40 [50%] clones), with 2 dogs having organisms with a mixture of sequences. In the 3 healthy dogs, organisms that had sequences with the highest homology to H felis were most prevalent (12/23 [52.2%] clones), followed by homology to H bizzozeronii (2 healthy dogs; 9/23 [39.1%] clones) and H heilmannii (1 healthy dog; 2/23 [8.7%] clones). Organisms with a mixture of sequences with the highest homology to various Helicobacter spp were detected in the 3 healthy dogs (H bizzozeronii and H felis in 2 dogs and H felis and H heilmannii in 1 dog).

In the 2 dogs with Helicobacter spp sequences in their oral cavity and stomach, only 1 dog had Helicobacter spp with highly similar sequences in both sites: a vomiting dog (dog 5) had sequences with highest homology to H felis and H heilmannii in both the saliva and gastric mucosa, whereas a healthy dog (dog 3) had oral cavity sequences with highest homology to H heilmannii and gastric sequences with highest homology to H felis and H bizzozeronii.

The relationship of Helicobacter sequences in 16S rDNA clone libraries constructed from the oral cavity and gastric mucosa with each other and with reference sequences for a variety of Helicobacteraceae in GenBank was evaluated further by use of phylogenetic analysis (Figure 1). Helicobacteracea sequences from the dental plaque of 8 of 8 dogs clustered most closely with Wolinella spp (specifically W succinogenes, as opposed to Candidatus Wolinella africanus) and were clearly distinct from Helicobacter spp (bootstrap values, 100%; nucleotide similarity scores, 93.4% to 98.5%). The sequences of organisms cultured from the gastric mucosa clustered with reference sequences for H felis, H heilmannii type 2, H bizzozeronii, and H Salomonis, with similarity scores of 95.2% to 99.8%. Sequences of organisms cultured from the gastric mucosa and oral cavity were clearly distinct (bootstrap value, 97%) from the sequences for H heilmannii type 1 (L10079; the predominant subtype reported in infections in humans), H pylori, and enteric Helicobacter spp (Helicobacter fenelliae, H cinaedi, and H canis).

Figure 1—
Figure 1—

Phylogenetic consensus tree depicting the genetic relationship among 16S rRNA gene sequences amplified with the C97 and C05 primer set in organisms cultured from gastric biopsy specimens, dental plaque, and saliva of 8 dogs. Sequences from vomiting dogs are indicated (asterisks). The numbers at the nodes are the bootstrap percentages (1,000 replications; 65% cutoff). Vertical distance has no meaning. Reference isolates were obtained from GenBank; accession numbers for each reference isolate are indicated in parentheses. Scale in the lower left corner represents nucleotide substitutions per site.

Citation: American Journal of Veterinary Research 72, 11; 10.2460/ajvr.72.11.1476

In the 2 dogs with Helicobacter sequences in their oral 16S rDNA library, sequences from the saliva and gastric mucosa of a vomiting dog (dog 5) clustered on a branch with highest homology to H felis and H heilmannii, which suggested colonization with similar strains at each site. In contrast, Helicobacter spp sequences from the oral cavity and gastric mucosa of a healthy dog (dog 3) had sequences that segregated in 5 clusters, which suggested colonization with different strains.

Examination for Wolinella spp

Results of 16S rDNA sequence analysis were unexpected because we had anticipated that the C97 and C05 primer set would be specific for the Helicobacter genus, as reported in other studies.25,28 To investigate the prevalence of Wolinella spp, we designed PCR primers for Wolinella spp and checked their specificity by use of a PCR assay against a panel of Helicobacter spp (H pylori, H heilmannii, H felis, H cinaedi, H bilis, H bizzozeronii, H hepaticus, H pylori, H canis, and H cinaedi); we used W succinogenes DNA as the positive control sample. We then performed a PCR assay for Wolinella spp on DNA extracted from the saliva of the 20 dogs that had not been examined via 16S rDNA sequencing. Sixteen of 20 dogs had positive results for Wolinella spp when tested by use of the PCR assay (Figure 2). Thus, 24 of 28 (85.7%) dogs had Wolinella spp DNA present in their oral cavity.

Figure 2—
Figure 2—

Photograph of an agarose gel depicting PCR products for Wolinella-specific primers performed on DNA extracted from the saliva samples of 10 pet dogs (lanes 1 through 10), Helicobacter heilmannii (lane 11), Helicobacter felis (lane 12), Helicobacter cinaedi (lane 13), Helicobacter bilis (lane 14), Helicobacter bizzozeronii (lane 15), Helicobacter hepaticus (lane 16), H pylori (lane 17), Helicobacter canis (lane 18), Wollinella succinogenes (lane 19), and negative control samples (lanes 20 and 21). Lane 22 is a 110-bp ladder.

Citation: American Journal of Veterinary Research 72, 11; 10.2460/ajvr.72.11.1476

The presence of Wolinella spp in the oral cavity was further examined by use of FISH for Helicobacteraceae on saliva smears (Figure 3). Although the probes were not specific for Wolinella spp, the characteristic lazy s–shape of the organism,29,30 rather than the spiral morphology of Helicobacter spp, was readily visible.

Figure 3—
Figure 3—

Fluorescence in situ hybridization image of a saliva smear obtained by use of probes for Helicobacter spp (HEL274 and HEL818 probes conjugated to the fluorescent dye Cy3) and eubacteria (EUB338 probe conjugated to the fluorescent dye 6FAM). The characteristic lazy s–shape of W succinogenes is visible (red-orange structure) and is surrounded by non-Helicobacter bacteria with a spiral morphology (green structures).

Citation: American Journal of Veterinary Research 72, 11; 10.2460/ajvr.72.11.1476

Discussion

The canine oral cavity frequently has positive results when tested for Helicobacter DNA (> 70% of dogs).23 This raises the possibility that dogs may be an important zoonotic reservoir for increasingly recognized gastritis attributable to non–H pylori Helicobacter spp in humans.15,31 To more clearly define the risk posed by contact with secretions and materials from the oral cavity of dogs, we sought to determine the specific Helicobacter spp present in dental plaque and saliva. The initial PCR testing with the widely used C97 and C05 primer set indicated that Helicobacter DNA was present in the oral cavity of 14 of 28 (50%) dogs. However, analysis of 16S rDNA libraries from the oral cavity revealed that sequences with the highest homology to Wolinella spp, rather than to Helicobacter spp, represented 91% of the Helicobacteraceae present. In marked contrast, clone libraries of the gastric mucosa contained predominantly H felis and H heilmannii, but there were no detectable Wolinella organisms. These results were unexpected because we anticipated that the C97 and C05 primer set would be specific for the Helicobacter genus, as has been reported in other studies.25,28 Alignment of the C97 and C05 primer set with the W succinogenes genome sequenceo reveals a 100% match at positions 1,382,756 and 1,381,565, respectively, and yields an approximately 1,200-bp DNA fragment that is indistinguishable in size from that of Helicobacter spp. Another commonly used Helicobacter-specific primer pair, C97 and C98 (which yields a 400-bp 16S rRNA amplicon), also closely matches the W succinogenes genome, with just 1 mismatched base at the 5′ end (position 1,382,375). Thus, the C97 and C98 primer set may also generate similarly sized amplicons of W succinogenes and Helicobacter spp, and this may have yielded false-positive results for the presence of Helicobacter spp DNA in the oral cavity in another study.23 Therefore, we concluded that the 16S rRNA primers C97, C98, and C05 are specific for Helicobacteraceae and not for the Helicobacter genus, as has been previously assumed.25,28 The Wolinella-specific PCR primers designed for the study reported here should be helpful for detecting positive results attributable to W succinogenes rather than those attributable to Helicobacter spp.

Wolinella organisms were originally isolated from the rumen of cattle,32 and the Wolinella genus belongs to the epsilon subclass of the proteobacteria.33,34 Wolinella succinogenes has been isolated from the canine oral cavity35 and was considered to be nonpathogenic until sequence analysis of the W succinogenes genome29,o revealed homologous genes for virulence factors (eg, the type IV secretory pathway, adhesins, invasins, and cytotoxins) in H pylori and Campylobacter jejuni. This has led researchers to question the nonpathogenic status of W succinogenes.29 In fact, a novel uncultured Wolinella organism (Candidatus Wolinella africanus) was identified in human patients with squamous cell carcinoma of the esophagus29; in a composite sample of the stomach, esophagus, and oral cavity of asymptomatic Venezuelans36; and in the stomach of a horse with gastric ulcers.37 A new putative Wolinella sp has also been identified in the stomach of a sea lion with gastritis.38 However, the Wolinella spp identified in the dogs of the study reported here clustered more closely with W succinogenes than with Candidatus W africanus.

Based on the adaptation of Helicobacter spp to different ecological niches of the gastrointestinal tract, it has been suggested30 that Wolinella spp may be more adapted to colonization of the squamous epithelium because the rumen and esophagus are both lined by this type of epithelium. The canine oral cavity, which is also lined by squamous epithelium, may represent another colonization site for W succinogenes. Findings of the present study support the presumption that W succinogenes and Helicobacter spp have distinct preferential colonization sites in dogs. The reason for the relative oral abundance of W succinogenes, compared with that of Helicobacter spp, is unclear but may relate to subtleties in the oral microenvironment, such as pH, reduction-oxidation potential, and nutrient availability. The growth of specific species may also be dependent on the presence or absence of other organisms in the microenvironment. For example, bacteria, such as Streptococcus mutans and Prevotella intermedia, in bio-film can inhibit the growth of H pylori strains.39,40 In one of those studies,40 investigators also found that the pathogenic periodontal bacteria Porphyromonas spp and Fusobacterium nucleatum can adhere to and trap H pylori, and the incidence of H pylori in the oral cavity of humans may therefore vary according to oral health status. Whether the incidence of Helicobacter spp in the oral cavity of dogs is interlinked with oral health status remains to be determined.

Results of sequence-based analysis of gastric Helicobacter spp in the present study were broadly consistent with results reported in other studies, with sequences that had the highest homology to H heilmannii, H felis, and H bizzozeronii commonly identified. However, Helicobacter spp with highest homology to H bizzozeronii were less predominant than those reported in other studies9,30,41,42 and were detected only in the gastric mucosa of the healthy dogs. In contrast, H heilmannii was detected almost exclusively in the vomiting dogs, which perhaps implies a more pathogenic role of H heilmannii in dogs and warrants further investigation in a larger population. The gastric mucosa of most (5/8 [63%]) of the dogs appeared to be dual colonized with 2 Helicobacter spp, which is a higher rate of coinfection than reported in some studies (13.3%9 and 16.6%42) but is similar to the rate (55%) reported in another study.31 Helicobacter felis and H heilmannii sequences in the organisms cultured from the gastric mucosa and oral cavity of a vomiting dog clustered on the same phylogenetic branch, and this may have been related to recent vomiting (10 hours before sample collection). Despite concordance in the vomiting dog, sequences of DNA for Helicobacter spp cultured from the oral cavity did not reliably correlate with species cultured from the gastric mucosa, as indicated by results for a healthy dog, which had organisms cultured from the gastric mucosa that had sequences with highest homology to H felis and H bizzozeronii and organisms cultured from the oral cavity that had sequences homologous to H heilmannii. Moreover, the presence of microbial DNA determined solely via PCR assay is not indicative of colonization by viable bacteria.

For the study reported here, we concluded that the predominant member of the Helicobacteraceae family inhabiting the canine oral cavity was Wolinella spp, which may preferentially colonize the oral squamous epithelium. Because only 5 of 57 (8.8%) clones cultured from the oral cavity were Helicobacter spp, the oral route may not be the predominant mode for transmission of gastric Helicobacter spp between dogs. The low frequency of Helicobacter spp clones, coupled with the absence of clustering with H heilmannii type 1, also implied that contact with the oral cavity of dogs likely poses little zoonotic risk for non–H pylori Helicobacter infection in humans. Larger studies encompassing a range of sites for collection of samples in the oral cavity and various oral health statuses in dogs are required to confirm these initial findings and to investigate whether Wolinella spp have a potential pathogenic role in oral disorders in dogs, as has been suggested in studies on humans and other mammals.

ABBREVIATIONS

ATCC

American Type Culture Collection

FISH

Fluorescence in situ hybridization

a.

Eppendorf GA, Hamburg, Germany.

b.

Taq PCR Master Mix, QIAGEN, Valencia, Calif.

c.

Mastercycler gradient, Eppendorf GA, Hamburg, Germany.

d.

Fermentas Inc, Glen Burnie, Md.

e.

Qiaquick, QIAGEN, Valencia, Calif.

f.

pGEM-T Easy, Promega Corp, Madison, Wis.

g.

Qiaprep Spin Miniprep kit, QIAGEN, Valencia, Calif.

h.

AmpliTaq DNA polymerase, Applied Biosystems, Foster City, Calif.

i.

3700 automated DNA sequencer, Applied Biosystems, Foster City, Calif.

j.

PRISM BigDye terminator sequencing kit, Applied Biosystems, Foster City, Calif.

k.

Sequencher, Gene Codes Corp, Ann Arbor, Mich.

l.

Ribosomal Database Project [database online]. Lansing, Mich: Michigan State University, 2010. Available at: rdp.cme.msu.edu/. Accessed May 2010

m.

BLAST, National Center for Biotechnology Information, National Institutes of Health, Bethesda, Md. Available at: blast.ncbi.nlm.nih.gov/. Accessed May 2010.

n.

MEGA, version 3.1, Center for evolutionary software and information, Tempe, Ariz.

o.

Wolinella Genome BLAST, National Center for Biotechnology Information, National Institutes of Health, Bethesda, Md. Available at: www.wolinella.mpg.de/wolinella.html. Accessed May 2010.

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Contributor Notes

The authors thank Francis Davis for technical assistance with the PCR assays, DNA sequencing, and fluorscence in situ hybridization.

Address correspondence to Dr. Simpson (kws5@cornell.edu).