An exploratory study on the presence of Helicobacter heilmannii and Helicobacter billis in the feces of companion dogs

Abstract Background Companion animals like dogs play an important role in the lives of many people and are often considered to be members of families, but definitely, any contact with them poses an inherent risk of transmitting zoonotic pathogens. One of these pathogens is the genus Helicobacter which is linked to many disorders in human and animal. Objectives The aim of this study was to investigate the presence of some zoonotic species of genus Helicobacter in companion dogs. Results Through culturing in a special medium, nine samples (9%) were detected as infected (two pure and seven mixed culture). Based on multiplex‐PCR, 13 samples (13%) were infected by Helicobacter spp. although none of them were infected by H. pylori. Species‐specific PCR indicated that 38.5% or 5/13 of the samples were infected with H. heilmannii, while 15.45% or 2/13 of the samples were infected by H. billis. Multivariate logistic regression analysis showed that the age factor had a significant effect on Helicobacter spp. infection (odds ratio [OR] = 2.42, p = 0.01). Conclusion This study revealed the negligible faecal transmission of H. pylori. Moreover, due to the detection of H. Heilmannii and H. billis in feces and their association with human gastric diseases, dog owners should be educated about the risks and transmission modes of zoonotic bacterial infections of dogs.

adenocarcinoma and gastric mucosa associated lymphoid tissue (MALT) lymphomas. In 1994, the International Agency for Research on Cancer (IARC) categorized H. pylori as a class I carcinogen and reported it in 75% of patients with MALT lymphoma and in 60% of those with an increasing risk of gastric cancer Morgner et al., 2000). Also, H. pylori infection could be related to some important diseases in humans such as the reduction of the ferritin and iron levels in patients with coronary artery disease and changes in lipid profiles and inflammatory factors, such as pre-eclampsia as a result of impairing the placental development, and glucose homeostasis in patients with type 2 diabetes (Bonfigl, 2016;Di Simone et al., 2017;Fallah et al., 2016). Furthermore, the infection is directly related to the metabolic syndromes such as high values of triglycerides, body mass index and systolic blood pressure and low HDL (Upala et al., 2016).
Following the discovery of H. pylori, other spiral bacteria have been observed in many other considered animal species (Dent et al., 1987).
In further research employing molecular analysis of the 16rRNA, it was elucidated that these spiral bacteria belonged to the Helicobacter genus (Solnick et al., 1993). At present, this genus, with at least 35 species distributed worldwide, can infect human and animals by colonizing in different anatomical regions of the gastrointestinal system such as oral cavity, stomach, intestine and liver. Depending on the place of colonization, it is also divided into gastric and enterohepatic species (Fox & Wang, 2002;Mladenova-Hristova et al., 2017;Recordati et al., 2009). Gastric Helicobacter such as Helicobacter heilmannii, Helicobacter felis, Helicobacter salomonis, Helicobacter bizzozeronii, Helicobacter cynogastricus and Helicobacter pylori and enterohepatic Helicobacter such as Helicobacter canis, Helicobacter billis, Helicobacter cinaedi and Helicobacter rappini were isolated from dogs. These species, which are associated with hepatobiliary and gastrointestinal diseases, are transmitted either directly through oral-oral and anal-oral contact or indirectly through water and food in dogs (Buczolits et al., 2003;Ekman et al., 2013;Haesebrouck et al., 2009;Jalava et al., 1997;Jankowski et al., 2016a;Jankowski et al., 2016a;Kubota-Aizawa et al., 2017;Mladenova-Hristova et al., 2017;Recordati et al., 2007;Recordati et al., 2009;Rossi et al., 2008;Van den Bulck et al., 2005). Helicobacter spp.
The diagnostic methods include invasive and non-invasive means; the former with high sensitivity and specificity encompasses gastroscopy and collecting a biopsy sample of the gastric mucosa for rapid urea test, histopathological examination, direct Gram staining, microbiological culture, electron microscopy, fluorescent in situ hybridization and PCR. Furthermore, regardless of the risks of gastroscopy and the associated anaesthesia, it should be noted that many veterinarians and clinics do not have the facilities and ability to perform gastroscopy. Therefore, non-invasive methods, including serology, culture and PCR on blood, saliva and faecal samples are preferred (Haesebrouck et al., 2009;Hong et al., 2015;Pohl et al., 2019;Shinozaki et al., 2002).
Therefore, the aim of this study was to determine the prevalence of Helicobacter spp. in canine feces by culture and molecular methods.
Another objective was to confirm the excretion of H. pylori from feces in companion dogs with and without gastrointestinal disorders. The results of the present study may be useful to clarify the epidemiology of Helicobacter spp. in dogs and humans.

Sample collection
The sample studied in this exploratory study included 50 companion dogs with and 50 dogs without gastrointestinal disorders (existence of diarrhoea, vomiting, constipation and loss of appetite) that were referred to the only veterinary hospital in Ahvaz city of Khuzestan province. Also, on the basis of the history and clinical examinations, the dogs infected or suspected of having other body systems diseases were excluded from this study. In addition, there was no history of drug therapy such as antibiotics and proton pump inhibitors (e.g. omeprazole, lansoprazole and pantoprazole) in any of the dogs (at least 2 weeks before sampling). The city of Ahvaz with a population of about 1,300,000 and approximately 4000 domestic dogs is the capital of Khuzestan province (Census, 2016). Companion dogs in this city are often kept only at homes and have no close contact with other dogs and cats (Didehban et al., 2020). In line with the research, the purpose of the study was explained to the dog owners, and informed consent was taken. Sterile cotton swabs were used for scraping the rectal mucosa, and then placed in a 1.5 ml sterile microtube. Swabs were quickly sent to the microbiology lab and cultured in the selective medium. To isolate the Helicobacter spp., a minimum time interval between sampling and culture is crucial due to the possible negative effects on Helicobacter viability. In addition, the age, sex, breed, and habitat (the keeping place) of each dog were recorded.

Bacterial isolation
To prepare the selective medium, sheep blood agar and several additional antibiotics were required. Therefore, blood agar base (Biolab Diagnostics Laboratory Inc., Hungary) was prepared and autoclaved for TA B L E 1 Goal gene, sequence and size of used primers

Name (gene) Primer sequence Size (bp) Reference
Helicobacter genus ( Before the culturing process, 200 μl of sterile phosphate-buffered saline (PBS) was added to each microtube, and then the swab was inoculated in the first region of the plate and streaked by loop in another region. The cultured medium was incubated in the microaerophilic condition at 37 • C for 3-5 days.
Subsequently, the suspected colonies were detected and purified in the blood agar medium under the previous condition. Meanwhile, the microscopic examination was conducted via preparing the smear and Gram staining. Finally, the suspected isolates inoculated with skim milk were stored at −70 • C until being retrieved for further analysis.

DNA extraction
In order for the genomic study of isolates and swab samples, first the DNA extraction was done using the GeneAll DNA kit (GeneAll Biotechnology Co., South Korea) following the manufacturer's instructions.
Then, based on genus-specific and different species-specific primers, the polymerase chain reaction (PCR) test was carried out for the detec- of Helicobacter genus and pylori species by multiplex PCR, the thermal cycles were set as follows: 1 min at 94 • C for denaturation, 2 min at 55 • C for annealing and 2 min at 72 • C for extension. Thirty-five repetitions were considered for this stage with an early denaturation at 94 • C for 5 min and a final elongation at 72 • C for 10 min (Farshad et al., 2004).
Each PCR reaction tube contained 25 μl of the reaction mix (consisting of 12.5 μl of Mastermix [Ampliqon, Denmark] with 2 mM MgCl2, 1 μl [10 pmol/μl] of each primer and 5 μl of chromosomal extracted DNA). In order to decrease the negative effects of inhibitory factors, 400 ng/ml of bovine serum albumin (BSA) was added to each reaction (Kreader, 1996).
The protocols of Fox et al. (1995) and Neiger et al. (1998)

Statistical analysis
The statistical analysis of the data was performed using SPSS (

Prevalence of Helicobacter spp
In the total number of 100 samples, there were 56 female and 44 male dogs. The mean score and standard deviation of age were 1.55 and 0.84 years, respectively. The relative frequency of Terrier, Doberman, Dachshund, German Shepherd, Spitz, Siberian Husky, Pitbull and Rottweiler breeds was 25%, 17%, 15%, 13%, 10%, 9%, 8% and 3%, respectively. Fifty-nine percent of dogs were kept in the apartment and the rest were kept in the yard.
According to the culture and biochemical test, nine of the samples (9%) were positive, but two of them were pure and the rest were a Besides, kappa statistic was 0.8 (p < 0.001). The molecular prevalence rate of Helicobacter spp. was 13% (95% CI: 6.4%-19.6%); however, none of them was detected as H. pylori by multiplex-PCR (Figure 1)

Risk factors of Helicobacter spp. infections
The statistical analysis showed that the infection was correlated with age orders and increased with aging (p < 0.05). Univariate logistic

This pioneering epidemiological survey, in addition to determining
Helicobacter spp. in feces, identified some of the factors associated with its occurrence in companion dogs in the southwest of Iran. Understanding the epidemiology of this organism is definitely an essential key to establishing appropriate prevention strategies.
In this study, using culture and multiplex-PCR methods to assess faecal samples, Helicobacter spp. was identified in 9% and 13% of the dogs, respectively; however, this observed difference was not statistically significant. According to other studies, prevalence of Helicobacter spp. DNA in feces was reported to be 100% by Ekman et al. (2013), 62.5% by Hong et al. (2015) and 23.3% by Jankowski et al. (2016a).
In gastrointestinal biopsies of dogs with gastritis by PCR, the prevalence of Helicobacter spp. in Japan and Poland was reported as 34.7% and 100%, respectively (Jankowski et al., 2016a;Kubota-Aizawa et al., 2017). The quantitative polymerase chain reaction (qPCR), histological, histochemical and immunohistochemical evaluations on gastric samples revealed that Helicobacter spp. was present in 47.8%, 65.2%, 75.4% and 82.6% of dogs, respectively (Amorim et al., 2015). The relative frequency of Helicobacter spp. in oral cavity (dental plaque and saliva) samples was 71.1%-100% (Ekman et al., 2013;Jankowski et al., 2016a;Recordati et al., 2007a). In Brazil, Helicobacter spp. infection has been reported as 94.7% and 100% by rapid urease test and histological analysis (Okubo et al., 2017). The large discrepancy in the frequency percentage of Helicobacter spp. may depend on the diagnostic method, specimen type, sample size and management and environmental factors (Chung et al., 2014;Falsafi et al., 2009;Hong et al., 2015;Kabir, 2001;Shinozaki et al., 2002;Smith et al., 2012). The detection of Helicobacter spp. in faecal samples is performed by microbiological culture, determination of anti-Helicobacter antibodies and bacterial DNA using PCR (Falsafi et al., 2009;Mishra et al., 2008;Smith et al., 2012).  (Mishra et al., 2008;Smith et al., 2012;Tonkic et al., 2012). According to Prachasilpchai et al. (2007), the per-  (Dunn et al., 1997). The recognition of the particular species or strains of Helicobacter is feasible via PCR due to being a straightforward, precise, time-saving, automatic and highly efficient method. PCR is applicable to a wide array of samples ranging from gastric biopsies, gastric juice and dental plaque to feces. The sensitivity of this method is considerably higher than that of histology, bacterial culture and urease evaluation although the type of primer used can alter its sensitivity (Prachasilpchai et al., 2007;Sabbagh et al., 2019;Simpson et al., 1999). Finally, not a single 'gold-standard' method exists for the detection of the infection induced by Helicobacter spp.; thus, its confirmation requires the joint application of at least two tests (Jankowski et al., 2017;Patel et al., 2014). Ekman et al. (2013) and Hong et al. (2015) used laboratory dogs that were kept together in the same ken- between the current infection and the previous exposure is regarded as the main shortcoming due to resulting in misinterpretation (Sabbagh et al., 2019). In addition, another source of misinterpretation is the cross-reacting antigens, especially flagellar proteins, residing between H. pylori and campylobacters (Mégraud & Lehours, 2007).
In the present study, age unlike gender, breed, gastrointestinal disorders and habitat was significantly related to the infection caused by Helicobacter spp. Regarding the age, the higher infection rate in older dogs was probably due to a greater exposure to the Helicobacter spp. over time. In the present study, the relative frequency of positive samples in dogs with gastrointestinal disorders was not statistically higher than those without gastrointestinal disorders, thus proving that Helicobacter infection could be asymptomatic in dogs. In several studies, no significant relationship was detected between infection and age, gender, gastrointestinal disorders and domestic habitat (Ekman et al., 2013;Elhariri et al., 2017;Okubo et al., 2017;Recordati et al., 2007). However, Kubota-Aizawa et al. (2017)