Serologic evidence and risk factors for Helicobacter pylori infection in animals and humans

Introduction: Helicobacter pylori (H. pylori) is one of the most common bacterial infections among humans worldwide. Although many records imply its interfamilial acquisition, the role of animals remains poorly understood. This study was undertaken to investigate the seroprevalence of H. pylori in animals and their human contacts in Cairo and Giza governorates, Egypt. Methodology: Commercial enzyme-linked immunosorbent assay (ELISA) kits were used to detect IgG antibodies to H. pylori in dogs, cattle, and humans. Results: Seropositive dogs (35/94; 37.2%), cattle (24/80; 30%) and humans (40/90; 44.4%) were found. Seroprevalence in animals significantly varied in different areas of sample collection, but there was no association with sex or age. Human seropositivity rates were associated with increasing age; moreover, seropositive dog owners (51.7%; 15/29), had seropositive dogs. However, infection was not associated with subject's sex, occupation, or history of animal contact. Conclusions: Our findings indicate H. pylori is widely distributed in cattle and dogs and their human contacts in Cairo and Giza, Egypt. Further studies to determine infection in other occupational groups are needed. This study provides baseline information on the seroprevalence of H. pylori, which may be required to begin prevention control programs in our area.


Introduction
Helicobacter pylori (H.pylori) is one of the most common bacterial infections among humans, and has worldwide distribution.The causative agent is a helical, microaerophilic, flagellated Gram-negative bacterium that inhabits and adapts to the acidic human gastric mucosa [1].The organism colonizes about 50% of the world population's upper gastrointestinal tracts [2], which makes the infection a public health concern worldwide [3].Although infected individuals usually never encounter clinical symptoms except chronic gastritis, acute infections can cause acute gastritis with abdominal pain or nausea [4,5].The ability of the organism to hydrolyze urea leads to gastric ulcers and increases the risk of developing duodenal and stomach cancer to the level that the World Health Organization (WHO) classifies as a class I carcinogen, where the infection is found in 80%-90% of patients with gastric ulcers [6,7].
The prevalence rate of H. pylori varies greatly by the geographic area, age, and socioeconomic status.Infection appears to be more common in developing than in developed countries [8,9].Modes of infection are yet not clearly understood.Although spread can occur through the environment or via reservoirs or vectors, little is known about the main route of dissemination from an infected individual [10].Transmission from person to person, through gastrooral, oral-oral, fecal-oral routes, and through exposure to contaminated food or water is highly controversial; furthermore, close and intense human contact with animals has been identified as a risk factor [11][12][13].The possibility of zoonotic transmission from animals has been previously suggested [14].Moreover, detection of anti-H.pylori antibodies in abattoir workers in France and in northern Sardinian shepherds, at higher levels than their siblings inhabiting the same house [15,16], demonstrates the zoonotic importance of the organism.However, the exact role of animals as a reservoir of infection remains unclear.
Most data about the rates of H. pylori infection in different geographical and demographic populations comes from seroprevalence studies [17].Serological tests using enzyme-linked immunosorbent assay (ELISA) are preferred as a noninvasive alternative method to endoscopy and biopsy for rapid diagnosis of H. pylori.Thus, serological tests have been used extensively in screening humans in clinics and for epidemiologic studies [18].Unfortunately, in Egypt, very little epidemiological data are available about the situation of H. pylori infection in humans and animals.To provide further information, the present study was undertaken to address the occurrence of H. pylori infection in apparently healthy humans and animals, including their owners.

Ethics statement
Protocols for the collection of samples were reviewed and approved by the Scientific Research Committee and Bioethics Board of Suez Canal University, Faculty of Veterinary Medicine, Ismailia, Egypt (No. 2016086).

Sample collection
Whole blood was collected from convenience samples of apparently healthy cattle and dogs.Cattle were randomly sampled from farms in El-Badrasheen and Mazghona and Gameeit Ahmed Orabi of Giza and Cairo governorates of Egypt, respectively.Domestic dogs were those mostly kept indoor and admitted with their owners to small animal veterinary clinics in El-Haram and Heliopolis and El-Maadi for other purposes.Stray dogs were those had been captured roaming in rural and suburban areas in Giza and Cairo governorates.Human whole blood samples (n = 90) were collected from consenting apparently healthy people (n = 61) attending the Giza and Cairo hospitals for routine health examinations and from owners of some of the sampled dogs (n = 29).Demographic data on age and sex were also obtained.
Following collection, samples were transported on ice box to Cairo University, Faculty of Veterinary Medicine, where whole blood samples were centrifuged, and aliquots of sera were separated and stored at -20°C for ELISA.

Serological assay
The canine, bovine, and human H. pylori IgG (Hp-IgG) indirect ELISA kits (MyBioSource, San Diego, USA) were used according to the manufacturer's instructions to detect IgG antibodies against H. pylori in sera of dogs, cattle, and humans, respectively.
The sample optical densities (OD) were measured using a microplate reader (CLINDIAG, Orange, USA) at 450 nm, and the sample-to-negative ratio was determined.As recommended by the manufacturer, for human kits, samples were considered to be ELISA positive if the OD sample/OD negative ≥ 2.1, while if OD sample/OD negative less than 2.1, the sample was considered as negative.For canine and bovine H. pylroi IgG ELISA kits, the cut-off was calculated based on the following formula: average of negative control + 0.15.Samples exceeding the calculated cut-off value were considered positive.

Statistical analysis
PASW Statistics, SPSS version 18.0 software (SPSS Inc., Armonk, USA) was used to analyze the data.Chi-squared (χ 2 ) and Fisher's exact tests were performed to analyze H. pylori antibody positivity between various groups.Differences were considered statistically significant if the P value was < 0.05.

Serologic detection of anti-H. pylori antibodies in cattle
Cattle from three different farms were examined: one in Cairo (Gameet Ahmed Orabi) and two in Giza governorates (Badrashen and Mazghona), in addition to sporadic cases bred by individual farmers (Table 2).Thirty percent of the total examined cattle were seropositive (24/80).Prevalence significantly varied by the area of sample collection, with the highest found in Badrashen in Giza governorates (52%, 13/25) and Gameet Ahmed Orabi in Cairo governorate (30%, 6/20); the lowest was in Mazghona in Giza governorate (14.3%, 5/35) (p < 0.05).Seropositivity was not associated with age or type of animal production (p > 0.05) (Table 2).

Serologic detection of anti-H. pylori antibodies in humans
A total of 44.4% (40/90) of humans were found to have antibodies to H. pylori.There was no statistically significant difference in the seropositivity in men (17/44) and women (23/46), while there was significant difference among different age groups (p < 0.05) (Table 3, Figure 1).Although 15 seropositive dog owners had seropositive dogs, there was no statistically significant difference in seropositivity in dog owners (51.7%; 15/29) and people with no history of animal contact (41%; 25/61) (p = 0.338) (Table 4).

Discussion
This study was undertaken as an attempt to evaluate the distribution and possible zoonotic relationship of H. pylori infection in Giza and Cairo governorates, Egypt.Overall, the differences of seroprevalence in the examined dogs, cattle, and humans was not statistically significant (p = 0.151).Seroprevalence in dogs was 37.2% (Table 1).Generally, serodiagnosis in dogs represents a big challenge, since they might be infected with several Helicobacter species [19][20][21].
Thirty percent of the examined cattle were seropositive (Table 2), whereas lower seroprevalence Figure 1.Seroprevalence of H. pylori in people by age and gender using ELISA.Seropositivity was associated with increasing age but not with gender.Individuals in the third decade of life were more likely to have a higher seroprevalence than those under 20 years of age.was reported in other studies [22,23].Previous detection of H. pylori from bovine feces [23,24] and/or milk [23,25] and seroconversion in farm workers [26,27], however, might suggest the probable role of this animal species in transmission of infection to humans in case of inappropriate farm management practices.
Results showed an association between seroprevalence and increasing age of the examined subjects (Table 3, Figure 1).Similarly, this was reported in other studies [29,35].Individuals in the third decade of life were more likely to have a higher seroprevalence than those under 20 years of age.This may be because Egyptian youth in such age groups like to have food away from home during their outside activities in trips or camps; thus, they might be infected from consumption of contaminated food or water sources.Generally, it is estimated that colonization of H. pylori in gastric mucosa is associated with old age, male sex, and low socioeconomic status [11,12].
The controversial finding is that around fifty percent (51.7%; 15/29) of the dog owners who reacted on ELISA had seropositive dogs (Table 4).Although there was no significant difference in the seropositivity between dog owners and the other group, contact with dogs was identified as a risk factor for acquiring H. pylori infection in other studies [36,37].Similar to our findings, in other seroepidemiological studies, the exact relationship between pet ownership and human seropositivity cannot be clearly established [38,39].
It is worth mentioning that an association had been reported between animal contact and seropositivity in abattoir workers and veterinarians working in abattoirs and meat processing plants in New Zealand [40,41].However, the cross-reactivity with antibodies to other gastrointestinal organisms which might be acquired from slaughtered animals was not excluded in those studies.Thus, these findings are questionable, and it was suggested that this higher prevalence was due to cross-reactivity to Campylobacter jejuni [14].In another study, high seroprevalence was reported in shepherds from northern Sardinia in comparison to their siblings inhabiting the same house who had no contact with sheep.Authors claimed that contact with sheep and sheepdogs was a risk factor for infection [16].However, other authors failed to isolate H. pylori and/or to detect its antibodies due to natural infection from stray and pet cats [42,43] and pigs in abattoirs [44].These questionable data suggest doubtful zoonotic transmission of such agent, and whether animals are true reservoir hosts for H. pylori or not is still not obvious.It seems that infection might be contracted from a common source (e.g., drinking water, consumption of raw vegetables) or might suggest that H. pylori infection could be an anthroponosis (infections maintained mainly in humans that may be transmitted to animals) [42].It was very difficult in this investigation to implicate or rule out infection of H. pylori to animal contact alone since combinations of other factors, including hygienic conditions, environmental factors, and socioeconomic status, contribute to the spread of the disease.

Conclusions
H. pylori is common in humans and animals in Cairo and Giza governorates, Egypt.Epidemiology of H. pylori is complex, and the zoonotic risk has not been clearly identified in this study.Further investigations with greater numbers of samples are essential to study the mechanism of disease transmission and potential risks for acquisition of infection.The present results provide a background and baseline data that may be required for commencing Helicobacter control programs in the studied area.

Table 1 .
Seroprevalences of IgG antibodies to H. pylori by ELISA in dogs from Cairo and Giza governorates.

Table 2 .
Seroprevalences of IgG antibodies to H. pylori by ELISA in cattle from Cairo and Giza governorates.

Table 3 .
Seropositivity of IgG antibodies to H. pylori by ELISA among humans of different age groups.

Table 4 .
Seropositivity of IgG antibodies to H. pylori by ELISA in dog owners and subjects with no history of animal contact.