Gastritis in dogs can be caused by several factors. The main triggers are systemic disease, diet, environmental influences, stress, and behavioral factors.1,2 Similar to the situation for humans, it has been proposed that Helicobacter spp infections may play a role in gastritis and cancer in dogs.1,3 However, no clear association between Helicobacter spp infections and gastric pathological conditions has been established.1,4–10 Most of the data implicating non–Helicobacter pylori as a cause of gastric pathological conditions in domestic animals has been obtained from experimentally infected mice or Mongolian gerbils.11–13 In contrast, investigations focused on natural infections have been unable to establish a positive correlation between Helicobacter spp colonization and disease.1,2,4–6,9,10,14,15
It has been suggested16 that a high density of Helicobacter-like organisms could trigger clinical signs in dogs. Difficulty in evaluating the association between Helicobacter spp and gastric conditions relates to the high prevalence of Helicobacter spp in healthy dogs.14,17,18 Additionally, the pathogenic potential of various Helicobacter spp in dogs has not been clearly determined.5,19 Therefore, the objective of the study reported here was to investigate the relationship between the density or numbers of native gastric Helicobacter spp in dogs and the presence of gastric lesions.
Materials and Methods
Animals
A random sample of 80 client-owned dogs was selected by use of an epidemiological computer program.20,a This number was based on the fact that the total canine population of Costa Rica in 2011 was 870,000 dogs, as determined on the basis of data from the World Society for the Protection of Animals,21 and that the estimated prevalence of Helicobacter spp in the canine population was 95% (assumed error, 0.5%). Dogs included in the study were randomly selected from those brought to the Small Animal Hospital of the School of Veterinary Medicine for neutering. They included 49 females and 31 males, and they ranged from 4 months to 12 years of age. Eight had a history of gastrointestinal disorders. All the dogs were consuming a dry pellet diet and were up-to-date on vaccinations and dewormings. Dogs that had received any drugs during the 15 days preceding the study were excluded.
Procedures for gastroscopy and collection of biopsy specimens were explained to the owners. Informed written consent was obtained for each dog included in the study. Animal handling and other procedures were performed in accordance with guidelines established by the Animal Welfare Committee and Bioethics of the School of Veterinary Medicine, National University, Heredia, Costa Rica, and in agreement with the corresponding law (Law No. 7451 on Animal Welfare).
Gastroscopic evaluation and collection of biopsy specimens
Before gastroscopic examination was performed, each dog underwent routine hematologic and clinical biochemical analysis. Results were used to anticipate complications during gastroscopy and anesthesia.
Gastroscopic evaluation was performed with a flexible endoscope coupled to a light source and camera. Dogs were anesthetized, and gastroscopy was performed in accordance with the World Small Animal Veterinary Association standards.22,23 At least 2 biopsy specimens were collected from each of 3 gastric zones (fundus, antrum, and body). Additional samples were collected when macroscopic lesions were observed during gastroscopy. Specimens used for bacteriologic culture and fast urease tests were processed immediately after collection. Biopsy specimens for PCR assay were frozen at −80°C until further processing. Samples for histologic examination were placed in neutral-buffered 10% formalin until further processing.
Bacteriologic analysis
Biopsy specimens were placed in sterile 10% urea solution (pH, 6.8) for fast urease testing, as described elsewhere.24 Samples were homogenized in brain-heart infusion broth containing 7% horse serum, and preparations were spread onto plates containing fresh Müeller-Hinton agar and Columbia agar (both of which were supplemented with 7% sheep blood) as well as Skirrow Campylobacter-selective agar. Plates were incubated in a microaerophilic atmosphereb at 35°C for 10 days, as described elsewhere.24,25
Biopsy specimens for PCR assay were used for DNA extraction and PCR amplification of 16S rRNA, as described elsewhere.26–29 Obtained amplicons were prepared for sequencingc by the use of a commercial kit.d Sequences were visually examined, edited, and analyzed by use of bioinformatics tools.30,31,e,f
Histologic examination
Formalin-fixed biopsy specimens were sectioned and stained with H&E stain in accordance with standard procedures.32 Selected tissue sections also were stained with Warthin-Starry modified staing and used for microscopic detection of Helicobacter-like organisms. Gastric samples were evaluated by 2 separate investigators (MSE and AAA). In accordance with the World Small Animal Veterinary Association standards,22 samples were classified on the basis of a gastritis severity score on a scale of 0 to 3 (0 = microscopically normal [not observed], 1 = mild lesions, 2 = moderate lesions, and 3 = severe lesions). The mean value was calculated and expressed as the gastritis severity score. Additionally, score values obtained for each of 3 stomach zones (fundus, antrum, and body) of each dog were added to obtain the total gastritis severity score. Lesions were documented by use of a camera adapted to a microscope, and photographs were editedh when necessary.
Estimation of the density of Helicobacter-like organisms
The density of Helicobacter-like organisms was calculated by counting the number of organisms in 10 hpfs (400X) for each of the 3 stomach zones on biopsy specimens stained with Warthin-Starry modified stain. Density was classified into 4 categories on a scale of 0 to 3 as described elsewhere33: 0 = not observed, 1 = low (1 to 5 Helicobacter-like organisms), 2 = moderate (6 to 15 Helicobacter-like organisms), and 3 = high (> 16 Helicobacter-like organisms). The mean value was calculated and expressed as the Helicobacter-like organism density score. Additionally, scores obtained for each of the 3 stomach zones of each dog were added to obtain the total Helicobacter-like organism density score.
Criteria for positive results with Helicobacter spp
Dogs were assessed for Helicobacter spp. Dogs were considered positive for Helicobacter spp when there were ≥ 2 positive results for bacteriologic culture, microscopic observation, the fast urease test, or 16S rRNA PCR assay.
Statistical analysis
Each histologic finding and Helicobacter-like organism density score per gastric region (fundus, antrum, and body) were analyzed by use of Kruskal-Wallis tests. To detect a preferential zone for gastritis development or density of Helicobacter-like organisms, significance of the gastritis severity score or Helicobacter-like organism density score for each gastric zone was also analyzed by use of Kruskal-Wallis tests. Correlation between density of Helicobacter-like organisms and gastritis severity score was analyzed by the use of the Spearman correlation analysis.i,j Values of P < 0.05 were considered significant.
Results
Endoscopic and histologic examinations of gastric mucosa
The most important gastroscopic findings for the 80 dogs were hyperemia (23 dogs) and mild erosion and ulceration (15 dogs; Table 1). On the basis of the World Small Animal Veterinary Association standards, 55 dogs had some degree of gastritis. Gastritis was classified as mild (n = 31), moderate (16), or severe (8). The main histologic findings for the 80 dogs differed in severity and were edema (n = 72), hyperemia (69), lymphocytic-plasmacytic infiltrate (55), fibrosis and glandular atrophy (30), neutrophil infiltration (22), lymphoid aggregates (17), and eosinophil infiltration (16; Table 2). Only 1 dog had injury of the epithelial surface. These findings were similar to those reported for healthy dogs without gastritis.7,33,34
Findings and results during gastroscopic examination of 80 client-owned dogs (55 of which had gastritis*) in Costa Rica.
| Gastroscopic finding | Mild | Moderate | Severe | Total |
|---|---|---|---|---|
| Contents (mucus, bile, or fluid) | 7 | 10 | 16 | 33 |
| Hyperemia or vascularity | 8 | 9 | 6 | 23 |
| Erosion or ulcer | 15 | 0 | 0 | 15 |
| Irritation of gastroesophageal sphincter | 5 | 7 | 2 | 14 |
| Scope passed through pylorus | 3 | 5 | 4 | 12 |
| Edema | 1 | 15 | 2 | 8 |
| Hemorrhage | 3 | 4 | 1 | 8 |
| Friable appearance of body | 3 | 1 | 0 | 4 |
| Uninflatable lumen | 2 | 0 | 0 | 2 |
| Discoloration of body | 1 | 0 | 0 | 1 |
Gastritis severity was scored as mild lesions, moderate lesions, and severe lesions for each of 3 zones of the stomach (fundus, antrum, and body).
Histologic findings associated with lymphocytic-plasmacytic gastritis* in 80 client-owned dogs (55 of which had gastritis).
| Histologic finding | Mild | Moderate | Severe | Total |
|---|---|---|---|---|
| Edema | 62 | 7 | 3 | 72 |
| Hyperemia | 58 | 2 | 9 | 69 |
| Lymphocytic-plasmacytic infiltrate | 31 | 16 | 8 | 55 |
| Fibrosis and gland atrophy | 8 | 19 | 3 | 30 |
| Neutrophilic infiltrate | 22 | 0 | 0 | 22 |
| Lymphoid aggregate formation | 10 | 7 | 0 | 17 |
| Eosinophilic infiltrate | 16 | 0 | 0 | 16 |
See Table 1 for key.
Lymphocytic-plasmacytic inflammatory infiltrate was consistently detected in all dogs with moderate and severe gastritis; it was mainly detected in the lamina propria. Additionally, mild eosinophil and neutrophil infiltrates and lymphoid aggregates in the lamina propria were also observed in dogs with the severest gastritis (Figure 1). Both inflammatory infiltrates and lymphoid aggregates resulted in separation of gastric glands and disruption of the normal architecture.
Photomicrographs of gastric tissues from representative dogs with various degrees of gastritis. A—Severe lymphocytic-plasmacytic infiltrate in the body of the stomach. B—Moderate fibrosis and glandular atrophy in the body of the stomach. C—Moderate lymphocytic-plasmacytic infiltrate in the body of the stomach. D—Moderate lymphocytic-plasmacytic infiltrate with moderate lymphoid aggregate formation (asterisk) in the fundus of the stomach. H&E stain; bar = 100 μm.
Citation: American Journal of Veterinary Research 78, 12; 10.2460/ajvr.78.12.1414
These observations were consistent with descriptions of dogs with mild to severe gastritis or with lymphocytic-plasmacytic infiltrates in other studies.4–6,17,33–35 Lymphocytic-plasmacytic gastritis was most frequently detected in the body (n = 42), which was followed by the fundus (35) and then by the antrum (25; Figure 2). Inflammatory infiltrate was distributed in all stomach areas (n = 15), body and fundus (13), fundus and antrum (3), body and antrum (1), body only (13), fundus only (4), and antrum only (6). This suggested a preferential pattern for development of gastritis in the body and fundus.
Venn diagram of the distribution of lymphocytic-plasmacytic infiltrate among 55 dogs with gastritis for each of 3 gastric zones (fundus, antrum, and body).
Citation: American Journal of Veterinary Research 78, 12; 10.2460/ajvr.78.12.1414
Testing for Helicobacter spp
Of all 80 dogs, positive results for Helicobacter spp were detected for 75 (94%) on the basis of observation of Helicobacter-like organisms, 72 (90%) by use of the Helicobacter spp PCR assay, 58 (72.5%) by use of the fast urease test, and 4 (5%) by use of bacteriologic culture. On the basis of the criteria used to establish a positive result, 76 of 80 (95%) dogs were considered to have positive results when tested to detect Helicobacter spp.
Distribution of Helicobacter spp
In previous reports,3,6,35–37 Helicobacter spp have been found mainly in the body and fundus of dogs. However, most dogs in the study reported here had a homogeneous distribution and low bacterial density in all 3 gastric zones (Figure 3). Histologically, Helicobacter-like organisms were observed predominantly in the superficial mucus of the gastric mucosa and the lumens of gastric glands. This was consistent with areas in which Helicobacter spp have previously been described in dogs.16,36
Distribution and density of Helicobacter-like organisms on canine gastric mucosa for each of 3 gastric zones (fundus, antrum, and body) for 75 dogs (A) and photomicrographs depicting the distribution (mild density [top], moderate density [middle], and high density [bottom]) of Helicobacter-like organisms on the mucus and epithelial surface of a representative dog (B). Warthin-Starry modified stain; bar = 50 μm. In panel A, the density for Helicobacter-like organisms was calculated by counting the number of organisms in 10 hpfs (400X) and was classified as follows: low = 1 to 5 Helicobacter-like organisms (white bars), moderate = 6 to 15 Helicobacter-like organisms (gray bars), and high = > 16 Helicobacter-like organisms (black bars).
Citation: American Journal of Veterinary Research 78, 12; 10.2460/ajvr.78.12.1414
Correlation between gastritis severity and the density of Helicobacter spp
To estimate the correlation between gastritis severity and density of Helicobacter-like organisms in dogs, the distribution of Helicobacter-like organisms in various areas of the stomach was determined. There were no significant differences among gastric zones for density of Helicobacter-like organisms and gastritis severity (Figure 4). When gastritis severity and Helicobacter-like organism density (total for all 3 gastric zones of each dog) were analyzed, a significant correlation was not detected. Similarly, no correlation was found between other histologic findings and Helicobacter-like organism density (Table 3) or between sex or age (data not shown). These findings were in agreement with findings of other reports1,4–10,33,38 that indicated the presence of Helicobacter spp was not necessarily associated with gastritis in dogs.
Median and range values for gastritis severity score (A) and Helicobacter-like organism (HLO) density score (B) for each of 3 gastric zones for 55 dogs with gastritis and the correlation between total gastritis severity score and total HLO density score (C). Gastritis severity was scored on a scale of 0 to 3 (0 = microscopically normal [not observed], 1 = mild lesions, 2 = moderate lesions, and 3 = severe lesions). The HLO density was calculated by counting the number of organisms in 10 hpfs (400X) and was scored on a scale of 0 to 3 (0 = not observed, 1 = low [1 to 5 HLOs], 2 = moderate [6 to 15 HLOs], and 3 = high [> 16 HLOs]. For panel C, the total gastritis severity score and total HLO density score (total for all 3 stomach zones for each dog) were plotted (each circle represents results for 1 dog), and the correlation was estimated by use of Spearman correlation analysis; there was not a significant correlation (ρ = 0.0014; P = 0.17). The dotted line corresponds to the trend line of the data.
Citation: American Journal of Veterinary Research 78, 12; 10.2460/ajvr.78.12.1414
Histologic findings and density of Helicobacter-like organisms for each of 3 gastric zones (fundus, antrum, and body) in 55 dogs with gastritis.
| Density of Helicobacter-like organisms | |||||
|---|---|---|---|---|---|
| Gastric zone | Histologic finding | Not observed | 1–5 | 6–15 | > 16 |
| Body | Hyperemia | 6 | 30 | 15 | 7 |
| Edema | 6 | 28 | 15 | 7 | |
| Lymphocytic-plasmacytic infiltrate | 4 | 22 | 9 | 6 | |
| Fibrosis and gland atrophy | 1 | 7 | 0 | 4 | |
| Neutrophilic infiltrate | 0 | 5 | 1 | 1 | |
| Lymphoid aggregate formation | 0 | 3 | 1 | 2 | |
| Eosinophilic infiltrate | 2 | 1 | 2 | 1 | |
| Fundus | Hyperemia | 4 | 37 | 10 | 5 |
| Edema | 3 | 39 | 10 | 4 | |
| Lymphocytic-plasmacytic infiltrate | 0 | 26 | 4 | 5 | |
| Fibrosis and gland atrophy | 1 | 11 | 6 | 4 | |
| Neutrophilic infiltrate | 0 | 7 | 1 | 1 | |
| Lymphoid aggregate formation | 0 | 5 | 1 | 1 | |
| Eosinophilic infiltrate | 0 | 3 | 2 | 0 | |
| Antrum | Hyperemia | 2 | 16 | 2 | 3 |
| Edema | 2 | 14 | 2 | 6 | |
| Lymphocytic-plasmacytic infiltrate | 1 | 19 | 2 | 2 | |
| Fibrosis and gland atrophy | 2 | 14 | 1 | 3 | |
| Neutrophilic infiltrate | 0 | 9 | 0 | 1 | |
| Lymphoid aggregate formation | 1 | 4 | 0 | 0 | |
| Eosinophilic infiltrate | 0 | 9 | 0 | 0 | |
Density of Helicobacter-like organisms was calculated by counting the number of organisms in 10 hpfs (400X) for each gastric zone.
Discussion
Gastritis in dogs can be triggered by various factors,1,2 one of which is purportedly the presence of Helicobacter spp organisms in the stomach.1,39 However, results for the study reported here did not support this supposition. In agreement with results of other reports,3,5,35,40 we detected no correlation between the presence of Helicobacter spp in the stomach of dogs and gastritis. Inconsistent results have been reported, probably because of the multifactorial nature of gastritis and the high prevalence of Helicobacter spp organisms in healthy dogs.1,8 For example, a substantial proportion of the dogs in the present study had microscopic gastric alterations that were not associated with Helicobacter spp or clinical signs. Absence of clinical signs in dogs with endoscopic and histologic lesions has been reported for patients with chronic gastritis41,42 and inflammatory bowel disease.43 The incongruity between clinical signs in dogs and histologic findings has been attributed to errors of assessment by owners or caretakers,33,43 procedural limitations that prevent evaluation of the entire stomach,33,43,44 or injury patterns that did not induce clinical signs.45,46
In the present study, the most frequent histopathologic finding in dogs with any degree of gastritis was plasmacytic-lymphocytic infiltrate with lymphoid aggregates. This inflammatory pattern has been described in dogs with chronic gastritis (including those without clinical signs) attributable to various causes.16,47
Prevalence of Helicobacter spp in the dogs of the present study (76/80 [95%]) was higher than that detected for dogs in Portugal (87%),38 Japan (86%),10 Germany (82%),7 Korea (78.4%),16 Denmark (76.7 %),35 and the United States (67%) but similar to that reported for dogs in Brazil (90%),36 Switzerland (91%),48 and Venezuela (95%)49 and in laboratory dogs in Ohio (100%). Thus, it is evident that dogs are highly colonized by Helicobacter spp organisms, independent of geographic region.
In the present study, we did not detect a correlation between density of Helicobacter-like organisms and gastritis in dogs. However, it is possible that virulence and pathogenicity still could be linked to particular Helicobacter spp and strains.13 To test this possibility, further research in taxonomic characterization and potential pathogenicity of Helicobacter spp is required because apparent variation in virulence has been described even within a single Helicobacter sp.13
The high prevalence of infection, absence of clinical signs in most colonized dogs, and lack of positive correlation between bacterial density and gastritis severity supported the notion that there is not an apparent correlation between Helicobacter spp density or numbers and gastritis in dogs.1,4,6–10 Findings for the present study should be relevant for veterinary clinicians who could mistakenly associate a large Helicobacter spp load in the gastric mucosa as a cause of gastritis without considering the multiple factors associated with this pathological condition.
Acknowledgments
Supported in part by grants from the FIDA-2009-UNA and NeTropica 02-N-10.
The authors thank Laura Alvarado, José Pablo Solano, and Reynaldo Pereira for technical assistance; Bernardo Vargas and Juan José Romero for statistical assistance; Edgardo Moreno for technical consultation; and Kate S. Baker for assistance with the manuscript.
Footnotes
WIN EPISCOPE, version 2.0, Ignacio de Blas, Department of Animal Pathology, Faculty of Veterinary Medicine, University of Zaragoza, Zaragoza, Spain.
CampyGen gas-generating system, Oxoid Ltd, Basingstoke, Hampshire, England.
3130 genetic analyzer, Applied Biosystems, Foster City, Calif.
Big Dye terminator, version 3.1 cycle sequencing kit, Applied Biosystems, Foster City, Calif.
MEGA, Molecular Evolutionary Genetics Analysis, version 6.0, Pennsylvania State University, State College, Pa. Available at: www.megasoftware.net/home. Accessed Mar 14, 2016.
NCBI Blast, National Center for Biotechnology Information, Bethesda, Md. Available at: www.ncbi.nlm.nih.gov/BLAST. Accessed Mar 14, 2016.
Steiner silver kit, Thermo Scientific, Cambridge, Mass.
Adobe Photoshop C5, version 8.0, Adobe Systems Inc, Mountain View, Calif.
IBM SPSS Statistics for Windows, version 19.0, IBM Corp, Armonk, NY.
InfoStat, Departments of Statistics and Experimental Design, Faculty of Agricultural Science, Universidad Nacional de Córdoba, Córdoba, Argentina. Available at: www.infostat.com.ar. Accessed Jul 25, 2016.
References
1. Jergens AE, Pressel M, Crandell J, et al. Fluorescence in situ hybridization confirms clearance of visible Helicobacter spp. associated with gastritis in dogs and cats. J Vet Intern Med 2009; 23:16–23.
2. Simpson K, Neiger K, DeNovo R, et al. The relationship of Helicobacter spp. infection to gastric disease in dogs. J Vet Intern Med 2000; 14:223–227.
3. Lanzoni A, Faustinelli I, Cristofori P, et al. Localization of Helicobacter spp. in the fundic mucosa of laboratory Beagle dogs: an ultrastructural study. Vet Res 2011; 42:42.
4. Eaton KA, Dewhirst FE, Paster BJ, et al. Prevalence and varieties of Helicobacter species in dogs from random sources and pet dogs: animal and public health implications. J Clin Microbiol 1996; 34:3165–3170.
5. Ekman E, Fredriksson M, Trowald-Wigh G. Helicobacter spp. in the saliva, stomach, duodenum and faeces of colony dogs. Vet J 2013; 195:127–129.
6. Happonen I, Saari S, Castren L, et al. Occurrence and topographical mapping of gastric Helicobacter-like organisms and their association with histological changes in apparently healthy dogs and cats. Zentralbl Veterinarmed A 1996; 43:305–315.
7. Hermanns W, Kregel K, Breuer W, et al. Helicobacter-like organisms: histopathological examination of gastric biopsies from dogs and cats. J Comp Pathol 1995; 112:307–318.
8. Neiger R, Simpson KW. Helicobacter infection in dogs and cats: facts and fiction. J Vet Intern Med 2000; 14:125–133.
9. Straubinger RK, Greiter A, McDonough SP, et al. Quantitative evaluation of inflammatory and immune responses in the early stages of chronic Helicobacter pylori infection. Infect Immun 2003; 71:2693–2703.
10. Yamasaki K, Suematsu H, Takahashi T. Comparison of gastric lesions in dogs and cats with and without gastric spiral organisms. J Am Vet Med Assoc 1998; 212:529–533.
11. De Bock M, Decostere A, Hellemans A, et al. Helicobacter felis and Helicobacter bizzozeronii induce gastric parietal cell loss in Mongolian gerbils. Microbes Infect 2006; 8:503–510.
12. Court M, Robinson PA, Dixon MF, et al. Gastric Helicobacter species infection in murine and gerbil models: comparative analysis of effects of H. pylori and H. felis on gastric epithelial cell proliferation. J Infect Dis 2002; 186:1348–1352.
13. Joosten M, Blaecher C, Flahou B, et al. Diversity in bacteriumhost interactions within the species Helicobacter heilmannii sensu stricto. Vet Res 2013; 44:65.
14. Harbour S, Sutton P. Immunogenicity and pathogenicity of Helicobacter infections of veterinary animals. Vet Immunol Immunopathol 2008; 122:191–203.
15. Van den Bulck K, Baele M, Hermans K, et al. First report on the occurrence of “Helicobacter heilmannii” in the stomach of rabbits. Vet Res Commun 2005; 29:271–279.
16. Hwang CY, Han HR, Youn HY. Prevalence and clinical characterization of gastric Helicobacter species infection of dogs and cats in Korea. J Vet Sci 2002; 3:123–133.
17. Prachasilpchai W, Nuanualsuwan S, Chatsuwan T, et al. Diagnosis of Helicobacter spp. infection in canine stomach. J Vet Sci 2007; 8:139–145.
18. Simpson J. Helicobacter infection in dogs and cats: to treat or not to treat? In Pract 2005; 207:204–207.
19. Sailasuta A, Prachasilpchai W. The role of Helicobacter spp. infection in domestic animals. In: Mozsik G, ed. Current topics in gastritis. Rijeka, Croatia: InTech, 2013;23–36.
20. Thrusfield M, Ortega C, de Blas I, et al. WIN EPISCOPE 2.0: improved epidemiological software for veterinary medicine. Vet Rec 2001; 148:567–572.
21. Sociedad Mundial para la Protección Animal. Situación de la población canina en los hogares de la Gran Área Metropolitana. Available at: www.colegioveterinarios.or.cr/files/doc/Situacion-de-la-poblacion-canina-en-los-hogares-de-la-Gran-Area-Metropolitana-Costa-Rica-WSPA.pdf. Accessed Oct 24, 2017.
22. Day MJ, Bilzer T, Mansell J, et al. Histopathological standards for the diagnosis of gastrointestinal inflammation in endoscopic biopsy samples from the dog and cat: a report from the World Small Animal Veterinary Association Gastrointestinal Standardization Group. J Comp Pathol 2008; 138:S1–S43.
23. Washabau RJ, Day MJ, Willard MD, et al. Endoscopic, biopsy, and histopathologic guidelines for the evaluation of gastrointestinal inflammation in companion animals. J Vet Intern Med 2010; 24:10–26.
24. Alarcón T, Baquero M, Domingo D, et al. Diagnóstico microbiológico de la infeccón por Helicobacter pylori. In: Cercenado E, Cantón R, eds. Procedimientos en microbiología clínica. Madrid, Spain: Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica, 2004:1–25.
25. Hänninen ML, Utriainen M, Tanskanen R. Mycoplasma contamination of canine gastric biopsy samples and cultures of gastric Helicobacter spp. FEMS Immunol Med Microbiol 1998; 22:335–339.
26. Boomkens SY, Kusters JG, Hoffmann G, et al. Detection of Helicobacter pylori in bile of cats. FEMS Immunol Med Microbiol 2004; 42:307–311.
27. Buczolits S, Hirt R, Rosengarten R, et al. PCR-based genetic evidence for occurrence of Helicobacter pylori and novel Helicobacter species in the canine gastric mucosa. Vet Microbiol 2003; 95:259–270.
28. Simmons JH, Riley LK, Besch-Williford CL, et al. Helicobacter mesocricetorum sp. nov., a novel Helicobacter isolated from the feces of Syrian hamsters. J Clin Microbiol 2000; 38:1811–1817.
29. Thoreson AC, Borre MB, Andersen LP, et al. Development of a PCR-based technique for detection of Helicobacter pylori. FEMS Immunol Med Microbiol 1995; 10:325–333.
30. Tamura K, Stecher G, Peterson D, et al. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 2013; 30:2725–2729.
31. Altschul SF, Gish W, Miller W, et al. Basic local alignment search tool. J Mol Biol 1990; 215:403–410.
32. Allen TC. Hematoxylin and eosin. In: Prophet EB, Mills B, Arrington JB, et al, eds. Laboratory methods histotechnology. Washington, DC: Armed Forces Institute of Pathology, American Registry of Pathology, 1992;53–58.
33. Robić M, Artuković B, Beck A, et al. Histopathological changes in stomachs of dogs with naturally acquired Helicobacter infection. Vet Arh 2007; 77:103–111.
34. Rossi G, Fortuna D, Pancotto L, et al. Immunohistochemical study of lymphocyte populations infiltrating the gastric mucosa of Beagle dogs experimentally infected with Helicobacter pylori. Infect Immun 2000; 68:4769–4772.
35. Wiinberg B, Spohr A, Dietz HH, et al. Quantitative analysis of inflammatory and immune responses in dogs with gastritis and their relationship to Helicobacter spp. infection. J Vet Intern Med 2005; 19:4–14.
36. Anacleto TP, Lopes LR, Andreollo NA, et al. Studies of distribution and recurrence of Helicobacter spp. gastric mucosa of dogs after triple therapy. Acta Cir Bras 2011; 26:82–87.
37. Scanziani E, Simpson KW, Monestiroli S, et al. Histological and immunohistochemical detection of different Helicobacter species in the gastric mucosa of cats. J Vet Diagn Invest 2001; 13:3–12.
38. Amorim I, Freitas DP, Magalhães A, et al. A comparison of Helicobacter pylori and non-Helicobacter pylori Helicobacter spp. Binding to canine gastric mucosa with defined gastric glycophenotype. Helicobacter 2014; 19:249–259.
39. Van den Bulck K, Decostere A, Baele M, et al. Identification of non-Helicobacter pylori spiral organisms in gastric samples from humans, dogs, and cats. J Clin Microbiol 2005; 43:2256–2260.
40. Recordati C, Gualdi V, Craven M, et al. Spatial distribution of Helicobacter spp. in the gastrointestinal tract of dogs. Helicobacter 2009; 14:180–191.
41. Carpenter HA, Talley NJ. Gastroscopy is incomplete without biopsy: clinical relevance of distinguishing gastropathy from gastritis. Gastroenterology 1995; 108:917–924.
42. Pregel P, Rota A, Palmerini D, et al. Detection of Helicobacter in gastric washing of cats. J Vet Diagn Invest 2008; 20:780–782.
43. McMahon LA, House AK, Catchpole B, et al. Expression of Toll-like receptor 2 in duodenal biopsies from dogs with inflammatory bowel disease is associated with severity of disease. Vet Immunol Immunopathol 2010; 135:158–163.
44. Guilford WG. Upper gastrointestinal endoscopy. In: McCarthy TC, ed. Veterinary. endoscopy in small animal practice. St Louis: Elsevier Saunders, 2005;279–391.
45. Dooley CP, Cohen H, Fitzgibbons PL, et al. Prevalence of Helicobacter pylori infection and histologic gastritis in asymptomatic persons. N Engl J Med 1989; 321:1562–1566.
46. Kreuning J, Lindeman J, Biemond I, et al. Relation between IgG and IgA antibody titres against Helicobacter pylori in serum and severity of gastritis. J Clin Pathol 1994; 47:227–231.
47. Kleinschmidt S, Meneses F, Nolte I, et al. Retrospective study on the diagnostic value of full-thickness biopsies from the stomach and intestines of dogs with chronic gastrointestinal disease symptoms. Vet Pathol 2006; 43:1000–1003.
48. Neiger R, Dieterich C, Burnens A, et al. Detection and prevalence of Helicobacter infection in pet cats. J Clin Microbiol 1998; 36:634–637.
49. Polanco R, Salazar V, Reyes N, et al. High prevalence of DNA from non-H. pylori helicobacters in the gastric mucosa of Venezuelan pet dogs and its histological alterations. Rev Inst Med Trop Sao Paulo 2011; 53:207–212.
