Genotyping pattern of the vacuolating cytotoxin A and cytotoxin associated gene A of the Helicobacter pylori strains detected in fecal samples of household dogs

In despite of the high clinical impact of Helicobacter pylori, its exact sources and routes of transmission are unknown. Dogs may play an imperative role in the transmission of H. pylori to humans. The current investigation was done to study the status of vacA and cagA genotypes in the H. pylori strains of dogs. One-hundred and fifty fecal samples were collected from healthy and complicated household dogs. Genomic DNA was extracted from fecal samples and presence of 16S rRNA gene was studied using the PCR amplification. Distribution of vacA and cagA genotypes were studied by the multiplex PCR. Thirteen out of 150 fecal samples (8.66%) were positive for H. pylori 16S rRNA gene. Prevalence of H. pylori in healthy and complicated dogs were 5.55% and 8.57%, respectively. Male had the higher prevalence of H. pylori (P=0.038). The most commonly detected genotypes among the H. pylori strains were vacAs1A (61.53%), cagA (38.46%), vacAm1a (38.46%), vacAs2 (30.76%) and vacAm2 (30.76%). The most commonly detected combined genotypes were s1aCagA (30.76%), s1am1a (23.07%), s2m1a (23.07%) and s2CagA (23.07%). Iranian household dogs harbor H. pylori in their fecal samples similar in genotypes of the vacA and cagA alleles which suggest that complicated and even healthy dogs may be the latent host of the H. pylori and its genotypes. However, supplementary studies are required to found the exact role of dogs as a definitive host of the H. pylor


Introduction
Dogs are one of the most useful and popular companion animal.Dogs chiefly can affluence loneliness, decrease allergies, depression, anxiety, stress, and variety of human immunological diseases and indorse social interaction, encourage exercise and playfulness, and deliver unqualified love.Dogs are also skilled guard, decent hunters, real narcotic detector and sharp probes.Company of dogs with children can increase their immune levels against many kinds of zoonotic diseases. 1elicobacter pylori (H.pylori) is a gram negative, spiral shaped bacterium of the human stomach.It is considered as one of the most contentious bacteria in the world.H. pylori is responsible for peptic ulcer, gastritis, lymphoma, duodenal ulcer, and gastric cancer. 2,35][6][7] Documented data revealed a high homology in the DNA sequence of H. pylori strains isolated from household dogs and also those which cause severe gastroenteritis and other types of gastrointestinal diseases in their owners. 8][6][7][8] Study the distribution of putative genotypes is essential to assess the microbiological and epidemiological prospective of H. pylori in clinical cases.The most imperative virulence genes accompanied with severe cases of H. pylori infections are vacuolating cytotoxin A (vacA) and cytotoxin associated gene A (cagA). 9,10The vacA belongs to the group of genes with mutable structures.This gene is polymorphic, comprising mutable signal regions (type s1 or s2) and mid-regions (type m1 or m2).The s1 type is supplementary subtyped into s1a, s1b and s1c and the m1 into m1a and m1b subtypes.The mosaic combination of s and m-regions regulates the specific cytotoxin and therefore, the pathogenicity of the H. pylori. 9,100][11] Genotyping using these virulence markers is measured as one of the best methods for assess associations between H. pylori isolates from diverse clinical samples.
From clinical, epidemiological, microbiological and hygienic prospective, it is important to assess the distribution of putative genotypes of the H. pylori in companion animals like dogs.Considering the unclear epidemiological aspects of H. pylori in fecal samples of dogs, the present investigation was done in order to study the precise status of vacA and cagA genotypes of H. pylori strains detected in the fecal samples of healthy and complicated household dogs.

Ethical issues
All samples were taken from volunteer dogs and written signed consent was obtained from the owners of examined dogs.Sampling procedure was non-invasive and all stages of sampling were done in a way that the lowest levels of pain and damages faced to the examined dogs.

Samples
From March 2015 to May 2016, overall 150 fecal samples were collected from the household dogs of different sexes (male and female, age 8 months to 12 years) who were referred for routine checks to the clinics of the Small Animal Internal Medicine, Tehran, Iran.Samples were collected randomly from the healthy dogs (n=90) and those that had history of gastric disorders (n=60).The dogs of the treatment group had a history of gastric ulcer, gastritis, chronic

DNA extraction and Helicobacter pylori 16S rRNA gene amplification
Detection of H. pylori strains was done based on the 16S rRNA-based PCR amplification.Genomic DNA was extracted from the fecal samples using a DNA extraction kit for cells and tissues (Fermentas, Germany) according to the manufacturer's instructions and its density was assessed by optic densitometry.

Genotyping of vacA and cagA genes of Helicobacter pylori
Presence of the cagA and the genotypes of vacA (s1a, s1b, s1c, m1a, m1b and m2) alleles were determined using PCR technique.List of primers is shown in Table 1. 13 The PCR were performed in a total volume of 50 μl containing 1 μM of each primer, 1 μL of genomic DNA (approximately 200 ng), 1 mM of dNTPs mix (invitrogen), 2 mM of Mgcl2, and 0.05 U/μL Taq DNA polymerase (invitrogen).PCR amplifications were performed in an automated thermal cycler (Biometra Co., Germany).The following cycle conditions were used for PCR amplification: for vacA: 32 cycles of 45s at 95°C, 50s at 64°C, and 70s at 72°C; for cagA: 1 min at 94°C, 1 min at 56°C, and 1 min at 72°C.All runs included one negative DNA control consisting of PCR grade water and two or more positive controls (26695, J99, SS1, Tx30, 88-23 and 84-183).

Gel electrophoresis
The PCR amplification products (10 μL) were subjected to electrophoresis in a 1.5% agarose gel in 1X TBE buffer at 80 V for 30 min, stained with ethidium bromide, and images were obtained in a UVIdoc gel documentation systems (UK).The PCR products were identified by 100 bp DNA size marker (Fermentas, Germany).

Statistical analysis
Data were transferred to Microsoft Excel spreadsheet (Microsoft Corp., Redmond, WA, USA) for analysis.Using SPSS 16.0 statistical software (SPSS Inc., Chicago, IL, USA), Chi-square test and Fisher's exact test analysis was performed and differences were considered significant at values of P<0.05.Distribution of H. pylori genotypes detected in fecal samples of dogs were statistically analyzed.

Results
Table 2 represents the prevalence of H. pylori in of fecal samples of healthy and complicated dogs.Results showed that 13 out of 150 fecal samples (8.66%) were positive for H. pylori strains.All of the H. pylori strains harbored the 16S rRNA gene.Figure 1   electrophoresis for amplification of the 16S rRNA gene of H. pylori strains.Prevalence of H. pylori in healthy and complicated dogs were 5.55% and 8.57%, respectively.Statistically significant differences were seen for the prevalence of H. pylori between healthy and complicated dogs (P=0.024) and also between male and female dogs (P=0.038).Table 3 represents the distribution of vacA and cagA genotypes among the H. pylori strains detected in fecal samples of dogs.VacAs1A (61.53%), cagA (38.46%), vacAm1a (38.46%), vacAs2 (30.76%) and vacAm2 (30.76%) were the most commonly detected genotypes among the H. pylori strains of dogs.H. pylori strains of complicated dogs had the higher prevalence and also more diverse studied genotypes.Statistically significant difference was seen for the prevalence of H. pylori genotypes between healthy and complicated dogs (P=0.035).
Table 4 represents the distribution of combined genotypes in the H. pylori strains of fecal samples of dogs.We found hat s1aCagA (30.76%), s1am1a (23.07%), s2m1a (23.07%) and s2CagA (23.07%) were the most commonly detected combined genotypes in the H, pylori strains detected in fecal samples of dogs.Prevalence of cagA positive and cagA negative strains were 46.15% and 53.84%, respectively.

Discussion
Humans are recognized as the major host of H. pylori; though, the histopathological inspection represents high prevalence and occurrence of H. pylori infections in the gastric mucosa of other mammalian which shows their high capacity as latent hosts for H. pylori strains. 14he current research exposed that the dogs and particularly those who had gastric complications like history of gastric ulcer, gastritis, chronic vomiting and weight loss may be the reservoir or original host of the H. pylori.Prevalence of H. pylori in fecal samples of dogs was 8.66% which was considerable high.Low levels of hygiene used for maintenance of dogs, their close contact with stray animals and especially dogs and transmission of pathogenic H. pylori strains from infected owners to dogs are the main factors caused high prevalence of H. pylori in fecal samples of our study.However, the prevalence rate of H. pylori could be more than the reported prevalence and it is due the fact that the fecal samples of our study were taken from the dogs who were kept in the houses.In the other hand, all of the dogs of our study had owners and kept in hygienic conditions.In addition, household dogs of our study were fed with cooked foods which were free from H. pylori strains.This matters will explain the low prevalence rate of H. pylori in fecal samples of dogs in this study.High prevalence of H. pylori in raw foods have been reported previously. 15,16 milar findings have been reported previously from Thailand, 17 Iran, 6 Denmark, 18 Egypt, 5 Italy 4 and Belgium. 19revalence rate of H. pylori in fecal samples of our investigation (8.66%) was lower that of Thailand (17.30%). 20Recent researches reported the high prevalence of Helicobacter-like microorganism in clinical samples taken from canine.They showed that Helicobacter-like organisms are extremely prevalent in dogs, with 100% of random-source dogs, 21 67-100% of clinically healthy dogs [21][22][23][24][25] and 100% of laboratory beagles and shelter dogs infected. 25,26elicobacter spp.have also been established in gastric biopsies of 61-95% of dogs. 26,27Torkan and Sakhaei Shahreza (2016) 28 revealed that 13 out of 240 (5.41%) gastric biopsy samples of Iranian dogs were positive for H. pylori with a higher prevalence of the bacterium in dogs with gastric ulcer (6.36%).As far as we know, the present study is the first prevalence report of the detection of H. pylori in the fecal samples of dogs.

Conclusions
In conclusion, we identified a relatively low prevalence of H. pylori in the fecal samples of healthy and complicated dogs in Iran as well as high presence of vacA and cagA genotypes especially in complicated dogs.Our results indicated that Iranian healthy and complicated household dogs harbor H. pylori in their fecal samples similar in genotype of the vacA and cagA alleles which suggest that complicated and even healthy dogs may be the latent host of the H. pylori and its genotypes.This matter is an important public health threat regarding the close contact of human with household dogs.However, additional studies are required to found the exact role of dogs as a definitive host of the H. pylori.
loss.Samples were collected in a sterile manner, with care taken to avoid cross-contamination between different sites.Fecal samples were transported to the laboratory at 4°C.All samples were kept under refrigeration in plastic bags.

Figure 1 .
Figure 1.Results of the gel electrophoresis for the 16SrRNA gene of the H. pylori strains detected in fecal samples of healthy and complicated dogs.M: 100 bp ladder, 1: Positive control, 2-4: Positive sample for the 16SrRNA gene (110 bp), and 5: Negative control.
found new epidemiological aspects of this bacterium in various clinical samples.

Table 1 . List of primers used for genotyping of Helicobacter pylori strains detected in fecal samples of dogs.
represents the results of the gel

Table 4 . Distribution of combined geno- types amongst the H. pylori strains of fecal samples of dogs.
*From a total of 13 positive strains of H. pylori.