Dog as Potential Source of Helicobacter Pylori in Egypt: Public Health Signicance

Background: Helicobacter species is a group of taxonomically related Gram negative, microaerophilic bacteria, which are known to colonize the gastrointestinal and biliary tracts of human and different animal species. The objective of the present study was to determine the occurrence of H. pylori in owned dog and its role in transmission of H pylori to the dog owners. Method: Human gastric biopsies (n=80) and Canine stool (n=60) were collected and examined by nested PCR. The PCR positive samples from human and dog isolates were further subjected to partial 16s sequencing. Result: H pylori was proved in 62.5% and 91.6% of owned dog and dog owners, respectively. The 16s sequencing of the isolates from human and dog were homologous. Conclusion: This study showed higher occurrence of H pylori in both human and dogs in Egypt. Zoonotic transmission between dogs and human is probable and reects a public health concern.


Background
Helicobacter pylori is a spiral, Gram-negative microaerophilic bacteria which prefer commonly the acidic medium, because of its ability to produce urease [1]. It was considered to be the second predisposing cause of cancer associated deaths and the fourth cancer causing factor all over the world [2]. It was reported that above half of the world's people with higher percentage from adults are affected by Helicobacter pylori infection especially in developing countries [3].
Helicobacter species were recorded to inhabit the gastric and intestinal mucosa of humans, pet animals as (dogs, cats), avian species, as well as wild animals as monkeys [4,5]. In human, H. pylori mainly invade the mucosa of human stomach. The majority may develop to asymptomatic gastritis, nevertheless 10% of infections may progress to cause gastric or duodenal ulcers, and 1% may develop to gastric carcinoma [6]. It is well known that there is a close interaction between human and companion animals which provide a great chance for sharing of many zoonotic diseases. Most of these infections in humans initiate from animals, including dogs, through direct contact [7,8].
Domestic animals especially dogs were charged to be a common source of infection of Helicobacter to human since it was founded a similarity between helicobacteria isolated from both canine stomach and affected human cases with gastritis [9,10]. Many studies globally reported that the majority of dogs affected with gastric ulcer may act as a reservoir of H. pylori moreover; it can be the initiator host of that pathogen. These studies were reported in many countries as Belgium [11], Thailand [12], Italy [13], Iran [14], and Egypt [15].The prevalence of gastric Helicobacter infection in dogs was reported to range between 61-100% without known route of transmission [16][17][18][19].
In dogs, spiral-shaped bacteria are commonly found in the stomach. They are present in 67 to 86% of clinically healthy dogs and in 61 to 100% of dogs presenting chronic vomiting [13,20]. Helicobacter spp. was also isolated from saliva, dental plaque, and feces of dogs which is considered a good prove for the suggestion of transmission by these animals, oro-oral or oro-fecal [21] and proposed to be reservoir and source of H. pylori affection. The same condition in people with H. pylori infection because oral-oral and faecal-oral are considered possible routes of transmission [22].
The relationship between pet ownership or frequent exposure to dogs and infection with different gastric Helicobacter species was assessed [23]. The evidence of Helicobacter pylori transmission from dogs to humans, enhance the need for Helicobacter detection and treatment in accompanying animals [24]. Therefore, eradication of Helicobacter infections in dogs that have close contact with humans should be considered as one of the methods to control this zoonotic infection. This work was aimed to study the occurrence of H. pylori infection in dog and its role in human transmission.

Sample collection
Human gastric biopsies (n = 80) were collected from different private laboratories with the history of dog owner after signed approval of patients who were informed with the obtained results. Canine stool (n = 60) was collected from hospital of the faculty of veterinary medicine, Cairo university and other private veterinary clinics. Biopsies were collected in sterile tubes containing BHI broth [Merck-Germany] and 5% non-activated fetal calf serum and transferred on ice to the laboratory. Molecular identi cation of Helicobacter pylori by nested PCR targeting 16S rRNA gene DNA was extracted from stool samples using [QIAamp DNA Stool Mini Kit] according to the manufacturer's instructions. While for gastric biopsies, DNA was extracted by using a modi cation of a previously described method [25]. Brie y, the thawed biopsy samples were homogenized in Gri th's tubes  The PCR products were puri ed using Qiaquick puri cation kit (Qiagen) and were sequenced using Big Dye Terminator V3.1 sequencing kit (Applied Biosystems, Waltham, MA, USA). The obtained nucleotide sequences were compared with those in the Public Database using the NCBI-BLAST server and were deposited in the GeneBank under accession number (MN901212 and MN901172).
The obtained nucleotide sequences were compared with those available in public domains using the NCBI-BLAST server. Sequences were downloaded and imported into the BioEdit program version 7.0.1.4 for multiple alignments using the BioEdit Clustal W program. Phylogenetic analysis was performed with the MEGA program version 7 using the neighbor-joining approach.

Discussion
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 [28,29 ].
Recorded ndings revealed that dogs may play an imperative impact in transmission of H. Pylori to human [13,15].Gastrointestinal H. pylori infections are routine in dogs [10,19].
Invasive methods for the detection of Helicobacter spp. are still commonly used, involving gastroscopy and collection of a biopsy sample for the performance of rapid urease test and histopathological exam. Currently, the use of the detection of bacterial DNA by PCR has been suggested for the identi cation of Helicobacter spp. [30] .
In the current study, the occurrence of H pylori recovered from dog stool was 62.5%. In investigation carried by [31] in China, dogs are the main home-reared animals, and it was found that the positive rate of H. pylori infection was higher in populations rearing dogs. Epidemiological data revealed that rearing dogs are a risk factor for H.pylori infection and it was inferred that dogs may be the potential storage source of bacteria [32]. Obviously a study conducted by [26] showed that the higher rates were obtained from dogs gastric biopsies 76.6% (46/60), 89.1% (41/46) in PCR and cultivation respectively.
The H. pylori infection rate is lower in developed countries than in developing countries [33], the rate of H. pylori infection is generally lower than 30% in developed countries, while its infection rate might be as high as 50-70% in developing countries. Some studies have pointed out that the poor economic status and a lower degree of culture may induce higher H. pylori infection rates [34].
Low levels of hygiene used for maintenance of dogs, their close contact with stray animals could be a risk factor in increasing the prevalence of Helicobacter. Moreover, a research was done by [35] in household dogs raised under hygienic conditions and fed with cooked food revealed a low prevalence (8.66%). Although there is a signi cant presence of helicobacteria in dogs, it is not possible to relate it with gastric alterations in these animals [36][37][38].
The virulence of Helicobacter in human is established by [39] who mentioned that this bacterium adheres to the gastric mucosa by an adhesine found on its surface called BabB that facilitate the entrance of antigenic products inside the host.On other hand, there is another mechanisms for its pathogencity by production of cytokines Cag A and VacA [10].
In this study, H pylori was proved in 91.6% of dog owners.
The prevalence of helicobacter was investigated in 290 patients from Macau, China [40]. In Egypt, serological detection of H. pylori was done by [27] in dog and human revealed that 37.2% (35/94), 44.4% (40/90) respectively was positive. Twenty -nine serum samples which taken from owners of infected dog revealed 51.7% were found to have antibodies to H. pylori. This indicates the zoonotic importance and the possibility of transmission of disease between dog and its owner.
It is clear that some animals including cats, dogs, sheep, may be transitory infected by H. pylori, but their roles in the route of transmission to humans are not proved [41].
The investigations revealed that dogs, especially those suffering from gastric ulcer, may be the reservoir of H. pylori and/or might be the original host of this bacterium [14]. This nding has been documented previously from all-around the world including Egypt where [15] suggested that Helicobacter colonizes the stomachs and intestines of humans and several animal species, such as cats, dogs and might have jumped quite recently from animal hosts to people. Because it has been possible to transfer from humans to animals it is reasonable to suppose that animals might have been the original source of the bacterium.
In this study, the phylogenetic relation between human and dog samples were showed 100% homology.
Human infection by H. pylori likely post-dated the evolution of humans and resulted from a host jump from a different animal [42]. Host jumps are not necessarily unlikely, because the stomachs of multiple animals are infected by diverse Helicobacter species, whose phylogeny is incongruent with that of their hosts. Indeed, the closest known relative of H. pylori is H. acinonychis, which infects large felines and seems to have arisen by a host jump from humans [43].

Conclusion
The occurrence of H pylori in the high percentage in dog owners proved the role of the owned dogs in the transmission of this pathogen. The evidence of Helicobacter pylori transmission from dogs to humans, enhance the need for Helicobacter detection and treatment in accompanying animals.

Authors'contributions
All authors contributed to the collection of samples, isolation of strains, performing the molecular detection of target genes, analysis and interpretation of the data as well as writing the manuscript. All authors read and approved the nal manuscript.
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