Introduction

Bacteria belonging to the Helicobacter genus are gram-negative, urease positive and spiral shaped. They have been detected in the gastrointestinal tracts of humans and a wide variety of other mammalian species (Farthing 1998, Jalava et al. 1998, Jenkins & Bassett 1997, Jonkers et al., 1997, Lehours 2003, Neiger & Simpson 2000; Simpson et al. 1999, 2000a, b). Helicobacter pylori infects human beings and is associated to several gastroduodenal diseases, including peptic ulcer (Crawford 2000, Tokunaga et al. 1998, Veldhuyzen van Zanten & Lee 1999, Warren & Marshall 1984). This organism also seems to have a strong relation to swine gastric ulceration (Szeredi et al 2005). Helicobacter infection in dogs includes a wider variety of species than does Helicobacter infection in human beings (Camargo 2002). It has been shown that 61% of sick dogs (Yamasaki et al., 1998) and up to 100% of healthy dogs may have Helicobacter infection (Happonen et al. 1998). In Brazil, it was found that around 95% of healthy dogs have Helicobacter spp in their gastric mucosa (Camargo 2002, Mota 2003, Moutinho et al 2007). Helicobacter spp. infection has been diagnosed in man through invasive and non-invasive tests. The first ones include fast urease test, cytology, histopathology, culture and polymerase chain reaction and need endoscopy in order to obtain gastric samples (Buckley & O'Morain 1998, Tokunaga et al., 1998). Because data regarding Helicobacter spp. infection pathogenic potential and diagnosis is scarce and controversial, research on that matter is essential (Camargo 2002).The purpose of this work was to evaluate the presence of Helicobacter spp. in endoscopic samples from healthy dogs and from dogs with signs of upper gastrointestinal disease through fast urease test, cytological and histopathological tests.

Materials and Methods

Endoscopic samples from the stomachs of 18 dogs were analyzed. Specimens collected from dogs with signs of upper gastrointestinal disease (10 dogs) were considered as group 1 and specimens from healthy dogs (8 dogs) were considered as group 2. Two samples of the gastric antrum and two samples of the gastric body were collected from each dog. One sample of each stomach region was first rolled over a clean microscopic glass slide which was fixed in absolute ethanol and stained with phenicated fuchsin 1% for cytological evaluation. Then, the sample was put into a tube with 1ml of urea 10% solution with phenol red as pH indicator in room temperature for the fast urease test. This test is considered positive when there is a color changing from yellow to red in 24 hours. The other sample was fixed in 10% buffered formalin solution and routinely processed for paraffin embedding. Sections obtained were stained with Giemsa and Warthin-Starry technique for histopathological evaluation. This research was in accordance with the principles of COBEA (Colégio Brasileiro de Experimentação Animal).

Results

A total of 36 specimens were evaluated through all tests and 63, 9% (23/36) were positive for the fast urease test. Fifty percent (5/10) of antrum specimens from dogs with signs of upper gastrointestinal disease and 75% (6/8) of antrum specimens from healthy dogs were positive for the fast urease test. When analyzing the body specimens, the fast urease test revealed 60% (6/10) positivity for samples from dogs with signs of upper gastrointestinal disease and 75% (6/8) positivity for samples from healthy dogs. Both antrum and body specimens were negative for the fast urease test in three animals (30%-3/10) from group 1 and one animal (12, 5%-1/8) from group 2. The cytological evaluation revealed elongated coiled spiral bacteria, compatible to Helicobacter genus in 23 specimens (63, 9%-23/36), 12 (52, 2%-12/23) of which from the gastric antrum and 11 (47, 8%-11/23) from the gastric body. That means that 10 animals were positives for Helicobacter spp in both antrum and body specimens. Two animals had positivity only in body samples and one had only in the antrum sample. Histopathological evaluation of Warthin-Starry stained sections showed that long spiral bacteria compatible with Helicobacter spp. genus were present in 63,9% (23/36) of the samples. These bacteria were found frequently colonizing the superficial mucus of the gastric mucosa, the lumen of gastric glands and less often the parietal cells. Within the 23 positive specimens, 13 (56, 5%-13/23) were from the gastric body and 10 (43, 5%-10/23) from the gastric antrum. Moreover, the test showed that 50% (5/10) of antrum specimens of group 1 dogs (dogs with signs of upper gastrointestinal disease) and 62, 5% (5/8) of antrum specimens of group 2 (healthy dogs) were positive. Also, 50% (5/10) of the body specimens of group 1 and 100% (8/8) of the body specimens of group 2 showed the spiral bacteria. Thirteen animals (72, 2%-13/18) were positive for Helicobacter spp in at least one gastric region. The Giemsa stain was unable to detect spiral bacteria in 2 (5, 6%-2/36) specimens, one from the gastric antrum and the other from the gastric body of two different animals.

Discussion and Conclusions

The invasive methods for detection of Helicobacter spp. infection require endoscopy of the upper gastrointestinal system, in order to directly observe the mucosa and to biopsy it (Buckley & O'Morain 1998, Tokunaga 1998). Those methods were chosen here because they are the most used for the diagnosis of Helicobacter spp. infection in man and dogs (Jenkins & Basset, 1997). The advantage of endoscopic biopsies is that they allow sampling the alimentary tract after visual inspection of internal aspects of the organs, guiding correctly the sampling procedure. Nevertheless, biopsies are small size and the pathologist will have much less tissue for evaluation (Else 1996). The present research showed that 63, 9% of the samples were positive for Helicobacter spp. but it might be possible that the size of the biopsy interferes on finding the bacteria as suggested by Leite (2005). On the other hand, three different diagnostic methods were used and two different stomach regions were analyzed which minimizes that problem. Helicobacter infection seems to be as disseminated in dogs as in man since 72,2% of the studied animals had Helicobacter spp. infecting least one gastric region, if the Warthin Starry stain is considered the gold standard. When studying separately the samples from dogs with signs of upper gastrointestinal diseases, this research shows that 50% are positive. Yamasaki et al. (1998) described that 61% of sick dogs are infected with Helicobacter spp. When evaluating samples from healthy dogs, the present study revealed that 100% are positive the same percentage found by Happonen et al (1998). Brazilian healthy dogs presented 93, 2%, (Mota 2003), 95% (Camargo 2002) and 96% (Moutinho 2007) of positivity. Factors like previous antibiotic administration, sampling method and place of sampling must be considered for the purpose of understanding why the sick dogs have a lower percentage of positive samples. Also, the Helicobacter spp. distribution in the gastric mucosa is scattered and irregular, with some areas with high colonization and areas with no microorganism (Crawford, 2000; Simpson et al., 2000a). When associating the two gastric regions, antrum and body with the presence of Helicobacter spp., it seems that both are able to give the animal Helicobacter spp. infection status. However, if only one biopsy in only one region using only one diagnostic method is used, the chances of diagnosis of Helicobacter spp. on the individual is lower. As shown in the present research, there was positive body specimens and negative antrum specimens from the same individual and a small number of samples had disagreement between the different diagnostic methods employed. The detailed study of the specimens from dogs with signs of gastrointestinal disease and healthy dogs makes it possible to conclude that (a) endoscopic biopsies must be taken from two or more biopsy sites, (b) two or more specimens of each gastric region seems to be adequate, (c) fast urease test, cytology and histopathology, mainly, Warthin-Starry stain should be chosen for a more precise diagnostic of Helicobacter spp. in the dog gastric mucosa.

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