vacA Genotype Status of Helicobacter pylori Isolated from Foods with Animal Origin

According to controversial theories and results of studies, foods with animal origins play an important role in the transmission of H. pylori to human. The aim of this study was to determine the distribution of vacA genotypes of H. pylori, isolated from milk and meat samples of cow, sheep, goat, camel, and buffalo. Eight hundred and twenty raw milk and meat samples were collected from various parts of Iran. Samples were cultured and those found positive for H. pylori were analyzed for the presence of various genotypes of vacA gene. Out of 420 milk and 400 meat samples, 92 (21.90%) and 105 (26.25%) were positive for H. pylori, respectively. The most commonly detected genotypes in the vacA gene were s1a (86.80%), m1a (79.18%), s1b (69.54%), and m1b (63.45%) and detected combined genotypes were mostly m1as1a (68.52%), m1as1b (60.40%), m1bs1b (55.83%), and m1bs1a (53.29%). High presence of bacteria in the milk and meat samples of sheep represents that sheep may be the natural host of H. pylori. High presence of H. pylori strains in milk and meat samples similar to vacA genotypes in human being suggests that milk and meat samples could be the sources of bacteria for human.


Introduction
Although Helicobacter pylori (H. pylori) has been accepted as a major cause of gastrointestinal disorders and especially gastric adenocarcinoma, type B gastritis, mucosa associated lymphoid tissue lymphoma, and peptic ulcer disease, its route of transmission, sources, and also the role of foods are still unknown. H. pylori is a Gram negative, coccoid flagellated bacterium with 2 to 4 m in length and 0.5 to 1 m in width, which is the first formally recognized bacterial carcinogen and one of the most successful human pathogens [1][2][3][4][5]. High prevalence of H. pylori in the stomach of domestic animals, milk, meat, and gastric biopsies suggests that food with animal origin, and also domestic animals, may be its reservoirs [1][2][3][4][5]. Higher prevalence of H. pylori in meat eaters than vegetarians which was achieved in the previous investigation supports the significant role of foods with animal origins in the transmission of bacteria to humans [6]. Appropriate condition of meat and milk including acidic PH, nutritional values, salt concentration, and also high amount of activated water (AW) facilitate the growth and survival of H. pylori and provide adequate setting for transmission of H. pylori to human [7]. High prevalence of antibodies against H. pylori in the serum samples taken from veterinarians, butchers, and staffs of the slaughterhouses and milking rooms can support the zoonoses aspects of this bacterium [1,8].
To evaluate the pathogenicity of H. pylori, apprising virulence factors is requisite. The most commonly identified virulence factor among H. pylori strains is vacuolating cytotoxin (vacA) [9,10]. VacA belongs to the group of genes with mutable genotypes associated with damage to gastric epithelial cells. This gene exists in practically all strains of H. pylori. This gene is polymorphic and comprises variable signal regions (type s1 or type s2) and midregions (type m1 or type m2) [9,10]. The s-region is classified into s1 and s2 and the m-region is categorized as m1 and m2. The s1 type is further subtyped into s1a, s1b, and s1c and the subcategories of m1 are m1a and m1b, respectively. Higher cytotoxicity and acuity have been done by this mosaic pattern [11,12]. Genotyping using vacA alleles is considered as one of the best methods to study the associations of H. pylori strains in various samples.
Industrialized information designated the fact that closely 50% of the world population and also 60-90% of Iranian people are infected with virulent strains of H. pylori [4,13]. According to the high prevalence of H. pylori in Iran and other parts of the world, and also with respect to the indistinct situation of H. pylori in foods with animal origins, the present investigation was carried out in order to study the vacA genotype status of H. pylori isolated from Iranian raw milk and meat samples.

Sample Collection.
In all, 420 raw milk samples were collected: cow ( = 120), sheep ( = 120), goat ( = 80), buffalo ( = 50), and camel ( = 50) raw milk samples were collected from farm bulk tanks and milk collection centers from several geographic regions of Iran, from March 2013 to March 2014. Cow and buffalo milk samples were collected throughout this time period. Because the lactating periods of ewes and goats in Iran are seasonal (from March through May and September to November of the subsequent year), goat and sheep milk samples were only available through these months within the fore-mentioned time frame. At each site, sampling of milk was performed according to the International Dairy Federation guidelines (IDF 1995). Samples (100 mL, in sterile glass containers) were transported to the laboratory at ca. 4 ∘ C within a maximum of 6-12 h after sampling. For raw meat samples, 100 cow, 100 sheep, 100 goat, 50 buffalo, and 50 camel meat samples were purchased from butchers of various parts of Iran. All samples were kept under refrigeration in plastic bags; information about dates of production and assigned shelf lives was not presented. Meat samples were collected over a period of eight months from August 2013 to February 2014, and they were analyzed on the day of acquisition. Samples were transported under refrigeration (4-6 ∘ C) in thermal boxes containing ice packs and were tested immediately after collection.

Results and Discussion
All of the milk and meat samples were examined using the culture and PCR techniques.  Table 2). The most commonly contaminated milk and meat samples were raw sheep milk (29.16%) and raw sheep meat (37%). There were no statistically significant differences among the incidence of bacteria in milk and meat samples. There were statistically significant differences in the incidence of H. pylori between sheep and camel milk ( = 0.033) and between sheep and camel meat ( = 0.048). Distribution of vacA genotypes of the H. pylori strains of meat and milk samples is shown in Table 3.
Results of the gel electrophoresis of PCR products for amplification of various genotypes of vacA gene are shown in Figures 1-3.
Results of current study showed that raw milk and meat samples were reservoir for H. pylori. Total prevalences of H. pylori in raw cow, sheep, goat, buffalo, and camel milk samples of our survey were 20.83%, 29.16%, 18.75%, 24%, and 10%, respectively. Rahimi and Kheirabadi [3] reported that the incidence of H. pylori in raw cow, sheep, goat, buffalo, and camel milk samples of Iranian herds was 1.41%, 12.20%, 8.70%, 23.4%, and 3.6%, respectively, which was lower than our results. In a study carried out in Italy, H. pylori was detected in 50%, 33%, and 25.6% of raw cow, sheep, and goat milk, respectively, which was higher than our results [16]. In  a study conducted in Japan, H. pylori was detected in 72.2% of raw cow milk samples [17]. Total distribution of H. pylori in the milk samples of Greek [18] and American [4] herds was 20% and 60%, respectively. Recent clinical investigation among Iranian cows showed that 16% of milk and 40% of feces samples of seropositive herds were infected with H. pylori [2]. Total prevalence of H. pylori in cow, sheep, goat, buffalo, and camel meat samples of our survey was 25%, 37%, 22%, 28%, and 14%, respectively, which was entirely contrary to the results of Stevenson et al. [19]. They suggested that transmission of H. pylori from beef and beef products is not a primary factor in the high prevalence of this bacterium in humans. Moreover, Mhaskar et al. [20] reported that the prevalence of peptic ulcer and H. pylori infection were entirely higher in those patients who have used meat and meat products (Odds Ratio (OR): 2.35, 95% and Confidence The possibility that H. pylori may be a zoonosis first rose the publication of two epidemiological studies that exhibited that the prevalence of H. pylori infection in abattoir and meat workers was significantly increased in comparison with the subjects that were not involved in handling animals or meat [21,22]. This hypothesis is further reinforced by the demonstration of H. pylori in the gastric mucosa of calves, pigs, and horses and its isolation from sheep's gastric tissue and milk [4], suggesting that these animal species may act as reservoirs and spreaders of H. pylori. Findings of Momtaz et al. [1] have conclusively proved the zoonotic aspects of H. pylori. They showed that the vacA s1a/m1a was prominent H. pylori genotype in all cow, sheep, and human beings clinical samples. They showed 3.4-8.4% variability and 92.9-98.5% homology between sheep and human samples [1].
High prevalence of H. pylori in the milk (29.16%) and meat (37%) samples of sheep of our study suggested that sheep may be the natural host of H. pylori. Dore et al. [4] reported that H. pylori DNA was demonstrated in 60% (38/63) of milk samples and in 30% (6/20) of sheep tissue samples. They showed that the vacA gene was amplified in five of 38 milk samples, and in two of six sheep tissue samples, respectively. Sequence analysis of 16S rRNA PCR products from H. pylori strains [4] investigation demonstrated 99% identity with H. pylori.
Genotyping using vacA virulence marker gene is considered as one of the best approaches for studying of the correlations between H. pylori isolates from different samples [1]. H. pylori strains of milk and meat samples of our study showed similar status in the distribution of vacA genotypes. Totally, the most commonly detected genotypes in milk and meat samples were s1a (86.  [25]. The high presence of m1as1a and m1as1b genotypes has been reported previously from Iran [13] and Germany [25] but far different results have been reported from Thailand [23] and Mexico [24].
According to the high prevalence of pathogenic strains of H. pylori in milk and meat samples especially in those collected from sheep and also based on the considerable consumption of milk and meat in their raw forms in some areas of the world [26][27][28], consumption of these food products in their raw forms should be stopped. On the other hand, thorough cooking of meat and pasteurization of milk can prevent the presence and also transmission of pathogenic bacteria like H. pylori.

Conclusions
H. pylori which is harbored from milk and meat samples are similar in genotype of the vacA allele with isolates recovered from human. Also, since there was a high similarity in the genotyping pattern of H. pylori DNA among milk and meat samples and human specimens of other investigations, it is suggested that raw milk and meat samples are the sources of the bacteria and that they entered the human population in period of time. On the other hand, diversity of H. pylori genotypes between milk and meat samples with the clinical isolation of other studies suggested that consumption of contaminated milk and meat with H. pylori strains may be a threat to human health.