Study of Helicobacter pylori genotype status in cows, sheep, goats and human beings

Background Helicobacter pylori is one of the most controversial bacteria in the world causing diverse gastrointestinal diseases. The transmission way of this bacterium still remains unknown. The possibility of zoonotic transmission of H. pylori has been suggested, but is not proven in nonprimate reservoirs. In the current survey, we investigate the presence of H. pylori in cow, sheep and goat stomach, determine the bacterium virulence factors and finally compare the human H. pylori virulence factors and animals in order to examine whether H. pylori might be transmitted from these animals to human beings. Methods This cross- sectional study was performed on 800 gastric biopsy specimens of cows, sheep, goats and human beings. The PCR assays was performed to detection of H. pylori, vacA and cagA genes. The PCR products of Ruminant’s samples with positive H. pylori were subjected to DNA sequencing analysis. Statistical tests were applied for data analysis. Results Overall 6 (3%) cows, 32 (16%) sheep and 164 (82%) human beings specimens were confirmed to be H. pylori positive; however we were not able to detect this bacterium in all 200 goat samples. The vacA s1a/m1a was the predominant H. pylori genotype in all three kinds of studied population. There was 3.4–8.4% variability and 92.9-98.5% homology between sheep and human samples. Conclusions Considering the high sequence homology among DNA of H. pylori isolated from sheep and human, our data suggest that sheep may act as a reservoir for H. pylori and in the some extent share the ancestral host for the bacteria with human.


Background
Helicobacter pylori is a gram negative, spiral shaped bacterium which its main reservoir is humans, particularly the human stomach. It colonizes most of the population, making it one of the most controversial bacteria in the world that cause gastritis, peptic ulcer, duodenal ulcer, lymphoma and gastric cancer [1].
According to the reports the main routs of infection has not been clarified yet [2]. However it is likely that H. pylori infection occurs during childhood or adolescence both in developing and developed countries [3] and its transmission occurs by person to person, either by fecaloral or oral-oral routes [4].The possibility of zoonotic transmission of H. pylori has been suggested, but is not proven in nonprimate reservoirs [4]. Some reports indicated that there is a high prevalence of antibody against this bacterium in veterinarians, butchers and slaughters rather than other people, so it suggests that H. pylori might be transmitted from animals to human [5,6]. Recently some researchers have been isolated H. pylori from cow, sheep, camel, pigs and dogs milk [7,8]. Therefore, it seems that these animals can be a reservoir of this bacterium. The severity of clinical manifestations varies depends on several factors such as host genetic, immune system, bacterial load and virulence factors [9]. This bacterium has several virulence factor genes which are generally classified into three categories: I) strain-specific genes, such as cag pathogenesity island (PAI) and genes located in plasticity island region (e.g. jhp0947 and dupA genes), which are present in only some H. pylori strains. II) phase-variable genes which change during different growth conditions. Based on comparison of three completed genomes of H. pylori, six genes encoding outermembrane proteins (babB, oipA, hopZ sabA, sabB and babC) are supposed to have phase variation. III) The genes with polymorphisms, for instance, specific vacA genotypes have been associated with different clinical outcomes [10]. The cag pathogenicity island (PAI) which belongs to the first category, encodes a type IV secretion system [2,3]. The cagA gene is located in the end of the cag PAI and has been proposed as a marker for the cagPAI. Different type of the cagA gene in some region is associated with diverse clinical outcomes; for instance cagA1a in East Asian Strains, is associated with more severe clinical manifestations than the absence of the gene [11]. The other important virulence factor of H. pylori is a vacuolating cytotoxin (VacA), which belongs to the last category and is associated with injury to epithelial cells. The vacA gene is present in virtually all strains of H. pylori but it is polymorphic, comprising variable signal regions (type s1 or s2) and mid-regions (type m1 or m2). Type s1/m1 vacA contribute with more epithelial cell damage rather than type s1/m2, whereas type s2/m2 and the rare s2/m1 are supposed to be non-toxic due to the presence of a short 12-residue hydrophilic extension on the s2 form [12,13]. The s-region is classified into s1 and s2 types and the m-region into m1 and m2 types. The s1 type is further subtyped into s1a, s1b and s1c subtypes, and the m1 into m1a and m1b subtypes. The mosaic combination of s and m-region allelic types determines the particular cytotoxin and, consequently, the pathogenicity of the bacterium [2,14].
In the current survey, we investigated the presence of H. pylori in cow, sheep and goat stomach, as well as bacterium virulence factors distribution among human and other studied population.

Population and sampling
Over all 800 samples; 200 from human and 600 from ruminant were included in the current study. In the ruminants group, over all 600 healthy domestic animals; 200 cows, 200 sheep and 200 goats referring for Zarrinshahr slaughterhouse in Isfahan, center of Iran, during February to August of 2012 were selected randomly. Considering sterile conditions, the sample from the rumen in size of 2 mm to 3 mm was obtained immediately after slaughtering. Samples were placed in 0.1 ml of sterile saline solution and were transported rapidly to the laboratory. The histological examinations were performed by the specialized veterinarians of Shahrekord Azad University. For analysis of H. pylori DNA from human origin, regardless to career of patients, two hundred patients with dyspepsia symptoms referring to gastroenterology department of Hajar Hospital Shahrekord, Iran, from December 2011 to April 2012 were selected and gastric biopsies from antrum were obtained during endoscopy by endoscopist. All patients provided written informed consent prior to endoscopy. All the specimens were placed in 0.1 ml of sterile saline solution and were transported to the laboratory immediately and were stored at -70°C until further investigation.

DNA analysis
From all of biopsy specimens, DNA was extracted by using Genomic DNA purification kit (DNP™, CinnaGen, Iran) considering sterile condition according to manufacture recommendations. The H. pylori presence in studied samples was detected by PCR method using housekeeping gene; glmM gene as a target gene. Due to low sensitivity and difficulty of H. pylori culture particularly from animal sources, the samples were not cultured. The primers sequences for glmM gene amplification were as follows: GlmM-F (5′-GCTTACTTTCTAACACT AACGCGC-3′) and GlmM-R (5′-GGATAAGCTTTTA GGGGTGTTAGGGG-3′) [2]. Primers were used for PCR assays of vacA allels and cagA genes has been described before [15,16]. DNA samples H. pylori (D0008, Genekam, Germany) were used as positive control of cagA and vacA genes, and sterile distilled water was used as negative control. PCR was done in 20 μL (for glmM) or 25 μL (for vacA and cagA) of total reaction volume containing 1.5 mM MgCl 2 (2.0 mM for cagA), 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 200 μM dNTPs each (Fermentas), 0.4 μM primers, 0.3 U of Taq DNA polymerase (Fermentas), and 2 μL (40-260 ng/μL) of DNA. PCR was performed in a DNA Thermal Cycler (Eppendrof Mastercycler 5330, Eppendorf-Nethel-Hinz GmbH, Hamburg, Germany), with 40 cycles for GlmM primer and 35 cycles for vacA and cagA primers. Each cycle consisted of denaturation at 95°C/45 seconds; annealing at 59°C/30 seconds for glmM, 52°C/45 seconds for vacA, and 58°C/45 seconds for cagA; and extension at 72°C/45 seconds [16]. There was another longer extension of 6 minute at 72°C. PCR products were visualized by electrophoresis in 1% agarose gel, were stained with ethidium bromide, and were examined under ultraviolet illumination.

DNA sequencing analysis
DNA sequencing analysis was performed on 6 H. pylori positive sample; 3 samples from cows and 3 samples from sheep which were selected randomly. Due to limitations we were not able to do sequence on all positive samples for glmM gene. For this purpose the DNA extraction was done by the same method as mentioned before for PCR. The amplified 296-bp PCR products (glmM gene) from 6 positive samples were purified with High pure PCR product purification kit (Roche Applied Science), according to manufacturer's recommendations. Single DNA strands were sequenced with ABI 3730 XL device and Sanger sequencing method (Macrogen, Korea).
After the sequence of 6 isolates were trimmed by using Edit View v.1.0.1 (Applied Bioscience, Australia), the sequences of 8 isolate with human source, which has been stored in GenBank with accession numbers: FN598874, CP003476, DQ462665, M60398, NC017361, GU445163, DQ141576, AB664954 were aligned separately against obtained animal isolate sequences using the Clustal W v1.81 in order to obtain a consensus sequence for the gene, glmM (H. pylori ureC). BioEdit Pakage V.7.0.4.1 was used to edit all sequence alignments. The nucleotide sequences of the Iranian ruminant H. pylori glmM (ureC) gene was compared with the correspond sequences reported from other regions via NCBI. By using Njplot software and 1000 bootstrap replicate, an unrooted dendrogramme was constructed.

Ethical considerations
The present study was accepted by the ethical committee of the Hajar Hospital of Shahrekord, Iran and Microbiology and Infectious Diseases Center of the Islamic Azad University of Shahrekord Branch, Iran. Written informed consent was obtained from all of the study patients or their parents.

Results
Totally 600 ruminants and 200 human gastric samples were collected in the current investigation. According to clinical and histopathological examinations, 10 cows and 2 sheep had moderate gastric inflammations while all goats were healthy, however none of the animals showed clinical manifestations. Based on gastroendoscopic and histopathologic finding, out of 200 human biopsy specimens, sixteen patients (11.8%) had gastric ulcers, 22 (16.2%) had duodenal ulcers, 194 (97.5%) had chronic gastritis and 3 (2.2%) had gastric cancer. Among 200 cow samples and 200 sheep samples, 6 (3%) and 32 (16%) were confirmed to be H. pylori positive; however, we were not able to find any H. pylori in goat samples. Out of 200 humans samples, 164 (82%) were infected with this bacterium.

Discussion
Human is known as the main host of H. pylori; however the histopathology of this bacterium is in contrasts with many other gastric Helicobacter spp. In which their natural host response against bacteria involve mild or even no inflammatory response [4], so it is possible that H. pylori has originated from another mammalian host in the distant past [17]. In the current survey we evaluated whether cow, sheep and goat can be the original host of this bacterium. In order to achieve this goal we collected 600 gastric specimens from healthy cows, sheep, goats and 200 gastric biopsy from human with dyspepsia in west of Iran where the prevalence of H. pylori is higher than 70% [18]. In our investigation, the prevalence of H. pylori in cows and sheep's population was 3% and 16% respectively, we were not able to detect it in goat's gastric tissue samples whereas the Italian survey on 400 milk samples by nested PCR assay, indicated that the prevalence of this bacterium in cow, sheep and goat populations were 50%, 33% and 25.6% respectively [8]. Scandinavian researchers found H. pylori in 60% of 38 sheep gastric tissue [4]. Also Rahimi and Kheirabadi declared H. pylori existed in 12.2% of sheep, 8.7% of goat and 14.1% of cow milk by PCR method [19]. In the other study which was conducted in Japan, H. pylori was found in 72.2% of cow raw milk specimens [20]. The diversity of H. pylori frequency in various hosts and regions may relate to animal, microbe and environmental factors. The prevalence of this bacterium in Iranian patients was 82% which is similar to previous reports from Iran and also Japan, South America, Turkey and Pakistan where more than 80% of dyspepsia patients were H. pylori positive, however in Scandinavia and England the prevalence ranges between 20% to 40% [21,22]. According to our results, screening of goats' stomach for H. pylori was negative which is in accordance with Gueneau et al. study in 2003 in which they failed to detect H.pylori in studied goats [23]. This finding may support by two possible reasons: Ones that goats are an exception among ruminants in having particular natural mechanisms of resistance to this bacterium. Another hypothesis is that some other microorganisms like Candidatus H. bovis may colonize the goat's stomach and establish the extent of the resistance of goats to the super infection with H. pylori [23]. H. pylori strains with the cagA gene is supposed to be more virulent rather than cagA-negative strains [12] however this is not constant [2]. The prevalence of cagA-positive H. pylori varies from one geographic region to another, e.g., 97% in Korea, 94% in Malaysia, 90% in China, 78% in Turkey, 53% in Kuwait, 85% North America and 65% in Slovenia [14,[24][25][26][27]. In the current study cagA gene was found in 92% of Iranian populations which is in accordance with previous local report [18]. Since the most of H. pylori isolated from human samples regardless to clinical outcomes harbor the cagA gene (P > 0.05), thereby as it was declared previously, our finding did not support the role of the cagA as predictive marker for increased virulence feature of H. pylori in Iranian dyspepsia patients [1]. The cagA gene was found in 66%, 75% of cow and sheep populations respectively, which was not studied on animals' samples yet. There was no statically meaningful difference in status of the cagA in human and animal samples, which may reflect that all H. pylori recovered from human and animals have same ancestors. According to our results for the vacA, all of our samples with positive PCR for H. pylori, irrespective to source of strains was positive for vacA. Although Dore et al. [4] in 2001 detected vacA gene in 60.3% and 7.9% of H. pylori strains isolated from sheep tissue and sheep milk samples respectively; now it is supposed that all H. pylori strains should possess the vacA gene, as it was supported by many studies around the world [12,26,27]. The vacA s1a/m2 were predominant vacA alles among all three studied population including human, cow and sheep.
Based on statistical analyses, there was a significant correlation between s1a/m2 genotype of H. pylori in sheep and human beings. Also s1a allele was significantly prevalent among cow and human. To our knowledge, this is the first comparison study of H pylori DNA sequence among specimens from cow, sheep and human in Middle East. As it was shown in Table 3 there was a high DNA sequence homology between H. pylori strains of sheep and human however this homology was low between cows    [29]. Some other reports also support zoonotic transmission of H. pylori via close contact with domestic animals [4,[30][31][32][33]. These studies, along with those have been shown that H. pylori can survive in sheep milk [4,19,[34][35][36] are supportive for reservoir role of sheep for human infection. Due to lack of any recorded sequence for H. pylori with animal source in Gene bank, we compared our isolates sequences with recorded human isolates sequences. Despite the little diversity in studied sequences, we were able to justify the genetic diversity of the bacterium based on its diverse hosts. As the origin of many Iranian noble cows and sheep refer to the America and Germany, so the perceived genetic similarities among sequences of H. pylori FN598874-Germany and CP003476-USA with those of Iranian cow and sheep isolates in this research can justify this claim.
On the other hand, transportation of livestock between Far-East Countries (Japan and China) and South Africa and Iran basically does not have historical background [37]. Thus, placing of Japanese, Chinese and South Africans strains in other branches of phylogenetic tree is indicating more differences in the sequence of H. pylori between Iran and mentioned countries.

Conclusion
In conclusion cows and sheep in Iran harbor H. pylori in their gastric tissue similar in genotype of the cagA and vacA allels with isolates recovered from human. Also since there was a high homology sequence of H. pylori DNA among sheep and human, suggest that sheep may are the natural reservoir of the bacteria and can transmit H. pylori to human community.