Detection of Helicobacter pylori in Bovine,Buffalo,Camel,Ovine,and Caprine Milk in Iran

Abstract

Helicobacter pylori infection in humans is one of the most common infections worldwide. However, the origin and transmission of this bacterium has not been clearly explained. One of the suggested theories is transmission via raw milk from animals to human beings. This study was conducted to determine the prevalence rate of H. pylori in bulk milk samples from dairy bovine, buffalo, camel, ovine, and caprine herds in Iran. In the present study, 447 bulk milk samples from 230 dairy bovine, buffalo, camel, ovine, and caprine herds were collected in four provinces and tested for H. pylori by cultural method and polymerase chain reaction (PCR) for the detection of the ureC (glmM) gene. The animals whose milk samples collected for this study were clinically healthy. Using the cultural method, three of 447 milk samples (0.67%), including two sheep (2.2%) and one buffalo (1.6%) milk samples, were found to be contaminated with H. pylori. H. pylori ureC gene was detected in 56 (12.5%) of milk samples, including 19 cow (14.1%), 11 sheep (12.2%), nine goat (8.7%), two camel (3.6%), and 15 buffalo (23.4%) milk samples. Using PCR method, there were significant differences (p<0.05) in the level of contamination with H. pylori between milk samples collected from different species. The present study is the first report of the isolation of H. pylori from raw sheep and buffalo milk in Iran and the first demonstration of H. pylori DNA in camel and buffalo milk.

Introduction

H elicobacter pylori is a major human pathogen causing chronic gastritis and has been associated with several serious diseases of the gastrointestinal tract, including duodenal ulcer, gastric cancer, and primary gastric lymphoma (Hatakeyama and Brzozow, 2006; Das and Paul, 2007). The organism can be found in 70–90% and 25–50% of the population in developing and developed countries, respectively (Guillermo et al., 2004; Sykora et al., 2006; Vale and Vitor, 2010). However, this pathogen is a cohabitant of the host (Cremonini and Gasbarrini, 2003), and it does not always cause illness in infected people (Santos et al., 2009). Several risk factors for H. pylori infection have been highlighted. These include poor social and economic development (Lehours and Yilmaz, 2007), low education level, poor hygiene practices during childhood, crowded families, lack of a household bath, absence of sanitary drinking water, absence of a sewage disposal facility during childhood (Nouraie et al., 2009), and food handled inappropriately (Van Duynhoven and de Jonge, 2001). Most of these factors are a consequence of and associated with socioeconomic development. The improvement of general hygienic conditions decreases the prevalence of the infection (Fujimoto et al., 2007).

The transmission pathways of H. pylori remain unclear. The most commonly acknowledged hypothesis is that infection takes place through fecal–oral route (Vale and Vitor, 2010), and contaminated water and food may play an important role in transmission of the microorganism to humans (Van Duynhoven and de Jonge, 2001; Gomes and De Martinis, 2004; Vale and Vitro, 2010). Indeed, H. pylori has been detected in drinking water (Queralt et al., 2005) and foods of animal origin such as milk (Dore et al., 2001; Fujimura et al., 2002; Quaglia et al., 2008). Therefore, the existence of animal reservoirs of the microorganism has been hypothesized (Dore et al., 2001; Fujimura et al., 2002). This hypothesis is further supported 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 (Dore et al., 2001), suggesting that these animal species may act as reservoirs and spreaders of H. pylori. H. pylori survives for long period in complex foodstuffs such as milk and ready-to-eat foods (Poms and Tatini, 2001; Quaglia et al., 2007). Therefore, food may serve as a vehicle for H. pylori infection.

Direct sale of unpasteurized milk and dairy products from producers to the consumer is not uncommon in many rural regions in Iran. Therefore, this study was conducted to determine the occurrence of H. pylori in bulk milk samples from dairy bovine, buffalo, camel, ovine, and caprine herds in Isfahan, Chaharmahal va Bakhtiari, Fars, and Khuzestan provinces, by means of a conventional bacteriological procedures and polymerase chain reaction (PCR).

Methods

Sample collection

Overall, 230 bovine, ovine, caprine, camel, and buffalo herds were randomly selected in Isfahan, Chaharmahal va Bakhtiari, Fars, and Khuzestan provinces, Iran. From September 2010 to July 2011, a total of 135 bovine bulk milk samples were collected from 86 commercial dairy herds, 90 ovine bulk milk samples were collected from 51 sheep breeding farms, 103 caprine bulk milk samples were collected from 45 goat breeding farms, 55 camel bulk milk samples were collected from 17 camel breeding farms, and 64 buffalo bulk milk samples were collected from 31 buffalo breeding farms. The animals whose milk samples collected for this study were clinically healthy, and the milk samples showed physical (color, pH, and density) consistency. The samples were immediately transported to the laboratory in a cooler with ice packs and were processed within an hour of collection.

Isolation of Helicobacter pylori

Twenty-five milliliters of each sample was added to 225 mL of Wilkins Chalgren anaerobe broth (Oxoid, UK) supplemented with 5% horse serum (Sigma, St. Louis, MO) and colistin methanesulfonate (30 mg/L), cycloheximide (100 mg/L), nalidixic acid (30 mg/L), trimethoprim (30 mg/L), and vancomycin (10 mg/L) (Sigma) and incubated for 7 days at 37°C with shaking under microaerophilic conditions. Then, 0.1 mL of the enrichment selective broth was plated onto Wilkins Chalgren anaerobe agar (Oxoid) supplemented with 5% defibrinated horse blood and 30 mg/L colistin methanesulfonate, 100 mg/L cycloheximide, 30 mg/L nalidixic acid, 30 mg/L trimethoprim, and 10 mg/L vancomycin (Sigma) (Poms and Tatini, 2001) and incubated for 7 days at 37°C under microaerophilic conditions. Suspected colonies were identified as H. pylori on the basis of the morphology of the colonies, Gram staining, and oxidase, catalase, and urease production (Dunn et al., 1997). The isolates were identified as H. pylori by using conventional bacteriological methods and were also positive using the PCR assay. For comparison, a reference strain of H. pylori (American Type Culture Collection [ATCC] 43504) was employed.

Antimicrobial susceptibility testing

Pure cultures of H. pylori isolates were used for antibiotic susceptibility test. One strain from each H. pylori-positive sample was selected for susceptibility tests. Antimicrobial susceptibility testing was performed by the Kirby-Bauer disc diffusion method using Mueller-Hinton agar (HiMedia Laboratories, Mumbai, India) supplemented with 5% defibrinated sheep blood and 7% fetal calf serum, according to the Clinical Laboratory Standards Institute) CLSI, 2006). The following antimicrobial impregnated disks (HiMedia Laboratories) were used: metronidazole (5 μg), clarithromycin (2 μg), erythromycin (5 μg), tetracycline (30 μg), amoxicillin (10 μg), and furazolidone (1 μg). After incubation at 37°C for 48 h in a microaerophilic atmosphere, the susceptibility of the H. pylori to each antimicrobial agent was measured, and the results were interpreted in accordance with interpretive criteria provided by CLSI (2006).

Detection of Helicobacter pylori using PCR method

DNA from 1 mL of each milk samples was extracted by a DNA isolation kit for cells and tissues (kit 11814770001; Roche Applied Science, Germany) according to the manufacturer's instructions, and its density was assessed by optic densitometry. Extracted genomic DNA was amplified for the ureC (glmM) gene and detected with the following specific primers:

HP-F: 5′-GAATAAGCTTTTAGGGGTGTTAGGGG-3′

HP-R: 5′-GCTTACTTTCTAACACTAACGCGC-3′

The gene product was 294 bp. PCR reactions were performed in a final volume of 50 μL containing 5 μL 10×buffer + MgCl₂, 2 mM dNTP, 2 unit Taq DNA polymerase, 100 ng genomic DNA as a template, and 25 picomole of each primer. PCR was performed using a thermal cycler (Eppendorf Co., Germany) under the following conditions: an initial denaturation for 10 min at 94°C; 35 cycles for 1 min at 94°C, 1 min at 55°C, 1 min at 72°C, and a final extension at 72°C for 10 min. The PCR products were electrophoresed through 1.5% agarose gels (Fermentas Co., Germany) containing ethidium bromide. A DNA ladder (Fermentas Co.) was used to detect the molecular weight of observed bands under an ultraviolet (UV) lamp. All tests were performed in triplicate. Samples inoculated with H. pylori were used as positive controls.

Statistical analysis

Data were transferred to an Excel spreadsheet (Microsoft Corp., Redmond, WA) for analysis. Using SPSS 16.0 statistical software (SPSS Inc., Chicago, IL), Chi-square test and Fisher's exact two-tailed test analysis was performed; differences were considered significant at values of p<0.05.

Results

Using traditional bacteriologic methods, three of 447 milk samples (0.67%) were found to be contaminated with H. pylori, of which two were sheep milk samples (2.2%) and one was a buffalo milk sample (1.6%). There was no significant difference in the level of contamination with H. pylori between different milk samples.

Overall, H. pylori ureC gene was detected from 56 (12.5%) of milk samples examined. In particular, H. pylori ureC gene was detected in 19 cow milk samples (14.1%), 15 buffalo milk samples (23.4%), 11 sheep milk samples (12.2%), nine goat milk samples (8.7%), and two camel milk samples (3.6%) (Table 1). Statistically significant differences (p<0.05) were observed in the prevalence of H. pylori ureC gene in milk samples collected from different provinces (data not shown) and dairy species (Table 1).

Table 1.

Prevalence of Helicobacter pylori Detected in Bovine, Buffalo, Camel, Ovine, and Caprine Milk Samples in Iran by Polymerase Chain Reaction

Milk sample	No. of samples	No. of H. pylori-positive samples
Bovine	135	19 (14.1%)^a
Buffalo	64	15 (23.4%)^b
Camel	55	2 (3.6%)^c
Ovine	90	11 (12.2%)^a
Caprine	103	9 (8.7%)^a
Total	447	56 (12.5%)

Results are expressed as the number of H. pylori-positive samples per number of samples analyzed (%).

a–c

Values with different superscripts are significantly different (p<0.05).

In this study, the antimicrobial resistance pattern of three H. pylori isolates was tested to five antimicrobial agents. All three isolates were resistant to metronidazole, and one isolate was resistant to clarithromycin. No resistance to amoxicillin, furazolidone, or tetracycline was observed.

Discussion

There is indirect evidence of H. pylori transmission through milk, similar to that obtained for water, but less extensive (Dore et al., 2001; Fujimura et al., 2002; Quaglia et al., 2007, 2008, 2009; Ghasemian et al., 2011). These studies led to the hypothesis of H. pylori infection being considered a zoonosis.

H. pylori changes to three different forms under environmental stress (including viable spiral, viable coccoid, and nonviable degenerative forms). The viable spiral forms are culturable, virulent, and infectious, and induce inflammation in experimental animals. The viable coccoid forms are nonculturable, less virulent, and less likely to colonize, and induce inflammation in experimental animals. The nonviable degenerative forms are dying forms of H. pylori (Oliver, 2005). In the present study, only three milk samples (0.67%) were found to be contaminated with H. pylori using traditional bacteriologic methods. H. pylori has rarely been isolated from raw milk samples (Dore et al., 2001; Fujimura et al., 2002; Angelidis et al., 2011). In several studies, no H. pylori was found in raw sheep milk samples (Turutoglu and Mudul, 2002; Quaglia et al., 2008), and raw cow or goat milk samples (Quaglia et al., 2008). This could be attributed to the fact that H. pylori can survive for short periods of time in milk (Quaglia et al., 2007), which is also a food product with a short shelf life. Moreover, the method employed for H. pylori isolation may lack sufficient sensitivity to recover very low numbers of H. pylori (Angelidis et al., 2011).

In this study, ureC gene of H. pylori was detected in cow, sheep, goat, camel, and buffalo milk samples. In a study conducted in Italy, H. pylori DNA was detected in 50%, 33%, and 25.6% of raw cow, sheep, and goat milk, respectively (Quaglia et al., 2008). In a study in Japan, H. pylori DNA was detected in 72.2% of raw cow milk samples (Fujimura et al., 2002). Recently, in a study reported from Iran, IgG antibody against H. pylori was detected in serum samples of 27% of cows. H. pylori antigen and the ureC gene were found in 16% of milk and 40% of feces samples collected from these seropositive cows (Ghasemian Safaei et al., 2011).

The PCR assay employed in this work specifically targets a region of the ureC (glmM) gene which has been shown to be unique and essential for the growth of H. pylori. It has been previously reported that detecting this gene improves sensitivity and specificity of recognition of H. pylori in samples containing prokaryotic cells as well as many organic impurities (Quaglia et al., 2008, 2009; Ghasemian Safaei et al., 2011). However, because the PCR assay detects ureC (glmM) gene of H. pylori, we are unable to speculate on the viability of organisms in milk samples. Further studies will be needed to determine survivability of these ruminant and other H. pylori strains in raw milk.

Ruminants may serve as a natural reservoir of H. pylori. Previous reports have described detection and isolation of H. pylori from the gastric mucosa of cattle and sheep without injury (Dore et al., 2001). It has been shown that H. pylori can be eliminated from the stomach of animals as viable forms in feces and then could potentially contaminate milk during milking process (Haesebrouck et al., 2009). The high prevalence of H. pylori isolated from healthy human carrier (Guillermo et al., 2004; Olivares and Gisbert, 2006) suggests that milk contamination due to poor hygiene management during milking, chilling, and storage may be the sources for H. pylori infection in human. Therefore, the consumption of raw milk or dairy products made with raw milk would be a potential risk of H. pylori infection for the consumer. Additional studies are required to investigate the human virulence potential of the H. pylori strains isolated from sheep and buffalo milk. Genotypic characterization of these isolates using molecular techniques such as multilocus sequence typing (MLST) and whole-genome DNA microarray will be needed to determine the zoonotic potential of H. pylori.

In conclusion, the results of this study show the importance of raw milk as a potential source of transmission of H. pylori to humans. To the author's knowledge, the present study is the first report of the isolation of H. pylori from raw sheep and buffalo milk in Iran and the first demonstration of H. pylori DNA in camel and buffalo milk. Further studies will be necessary to determine the prevalence of H. pylori in raw milk in Iran and to explore the potential risk of human infection with H. pylori via consumption of raw milk.

Footnotes

Acknowledgments

We would like to thank Dr. Hassan Momtaz, Dr. Amir Shakerian, Manouchehr Momeni, and Majid Riahi for help in performing technical parts of the project. We are also grateful to Dr. Ali Moosavi, Dr. Mazear Rafei, and Dr. Morteza Mosavean for assistance with sampling and Dr. Mehrdad Ameri for help in preparation of this manuscript.

Disclosure Statement

No competing financial interests exist.

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