High occurrence of Helicobacter pylori in raw goat, sheep and cow milk inferred by glmM gene: A risk of food-borne infection?

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Abstract

Helicobacter pylori is an organism widespread in humans and sometimes responsible for serious illnesses, such as gastric and duodenal ulcers, MALToma and even gastric cancer. It has been hypothesized that the infection route by H. pylori involves multiple pathways including food-borne transmission, as the microorganism has been detected from foods such as sheep and cow milk. This work reports the results of a survey conducted in order to investigate the presence of H. pylori in raw goat, sheep and cow milk produced in Southern Italy, employing a Nested Polymerase Chain Reaction (Nested-PCR) assay for the detection of the phosphoglucosamine mutase gene (glmM), as screening method followed by conventional bacteriological isolation. Out of the 400 raw milk samples examined, 139 (34.7%) resulted positive for the presence of glmM gene, but no strains were isolated. In this work H. pylori DNA has been firstly detected from 41 (25.6%) raw goat milk samples. The results deserve further investigations on the contamination source/s of the milk samples and on the major impact that it may have on consumers.

Introduction

Helicobacter pylori is considered a serious problem impairing the public health in both developed and developing countries (Brown, 2000, Olivares and Gisbert, 2006) because it colonizes the gastric mucosa of about half of the world population (Torres et al., 2000, Guillermo et al., 2004, Frenck and Clement, 2003, Olivares and Gisbert, 2006). It has been acknowledged as a major cause of gastritis and it is associated with gastric and duodenal ulcer disease, low grade B-cell mucosa associated lymphoid tissue lymphoma of the stomach (MALToma) and gastric cancer (Edit et al., 1994, Wotherspoon et al., 1993, Parsonnet et al., 1994, Hatakeyama and Brzozow, 2006). Despite the high incidence of the infection, the reservoirs and the transmission pathways of H. pylori to humans are still unclear, although multiple routes of transmission have been suggested (Goodman and Correa, 1995, Allaker et al., 2002). The current literature suggests that the transmission of H. pylori occurs by person to person both via the oral–oral and fecal–oral routes (Versalovic and Lewandrowski, 1998, Parsonnet et al., 1994, Cellini et al., 2001). Furthermore, many authors suggested that the human infection may occur by contaminated foods (Velàzquez and Feirtag, 1999, Gomes and De Martinis, 2004). Indeed, H. pylori has been detected from drinking water (Hegarty et al., 1999, Lu et al., 2002, Glynn et al., 2002, Queralt et al., 2005), sea water (Cellini et al., 2004a) and foods of animal origin, such as sheep and cow milk (Dore et al., 2001, Fujimura et al., 2002). Therefore, the existence of animal reservoirs of the microorganism has been hypothesized (Dore et al., 2001, Fujimura et al., 2002). Besides, the histopathology of lesions by H. pylori in humans differs from that of many other gastric helicobacters causing mild or absent inflammatory responses in their natural hosts (Thompson et al., 1994). These data suggest that H. pylori may have not originally evolved as a human pathogen, but it has been likely introduced into the human population from a mammalian reservoir sometime in the distant past (Dore et al., 2001). This hypothesis is further supported by the demonstration of H. pylori in the gastric mucosa of calves, pigs and horses (Dimola and Caruso, 1999) 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 like milk and ready-to-eat foods such as lettuce, tofu, and chicken (Fan et al., 1998, Poms and Tatini, 2001, Quaglia et al., 2007); this aspect lends credit to the hypothesis that primary contamination of food (animal reservoir) or secondary contamination due to unfit handling (human reservoir) may occur that make food a vehicle for H. pylori infection (Quaglia et al., 2007).

Isolation of H. pylori from food samples, particularly when they present high loads of accompanying microflora, is exacting and time-consuming since it requires the use of selective media added with numerous antibiotics, microaerophilic conditions and long incubation periods (7 days) (Stevenson et al., 2000). Furthermore, under adverse environmental conditions such as desiccation, lack of protection against oxygen, exposure to antimicrobial agent, H. pylori may produce viable nonculturable form (VNC) (Dunn et al., 1997, Cellini et al., 2004b). Detection of H. pylori in food samples by means of conventional microbiological techniques, unable to detect the VNC, may yield false negative results and thus underestimate the presence of the bacterium in food; however, it has been hypothesized that VNC forms may be still infective (Bode et al., 1993, Cao et al., 1997) thus representing a potential microbiological risk for consumers. Hence, several investigators have settled up molecular assays in order to detect H. pylori in water and different foodstuff (Velàzquez and Feirtag, 1999, Quaglia et al., 2005).

The aim of this study was to determine the occurrence of H. pylori in raw goat, sheep, and cow milk produced in Southern Italy, by means of a Nested Polymerase Chain Reaction (Nested-PCR) followed by conventional bacteriological procedures for the isolation of the microorganism.

Section snippets

Milk sampling

A total of 400 bulk raw milk samples (160 samples of goat milk, 130 of sheep milk, and 110 of cow milk) from farms of Apulia, Sardinia and Calabria region (Southern Italy) were collected and analysed from October 2004 to October 2006.

Each milk sample (about 100 ml) was collected in sterile condition and put into sterile refrigerated containers (4 °C), transferred to our laboratory within 4 h for the milk samples collected in Apulia region and about 3 days for those collected in Sardinia and

Results

H. pylori glmM gene was detected from 139 (34.7%) out of the 400 milk samples examined.

In particular, 41 of the 160 goat milk samples (25.6%), 43 of the 130 sheep milk samples (33%) and 55 of the 110 cow milk samples (50%) examined, resulted positive (Table 2, Table 3).

Fifty of the 139 positive samples (36%) were collected in Apulia region, 55 (39,5%) in Sardinia region and 34 (24,5%) in Calabria region (Table 3).

No statistically significant differences were observed in the prevalence of

Discussion

H. pylori has been estimated to infect about 50% of the world population at early age (Torres et al., 2000, Frenck and Clement, 2003, Guillermo et al., 2004). Foodstuffs, and in particularly milk, have been considered as a probable source of human infection (Wesley, 1997, Meng and Doyle, 1997, Velàzquez and Feirtag, 1999, Dore et al., 2001, Fujimura et al., 2002) since H. pylori DNA was found from raw sheep milk samples and sheep gastric tissue (Dore et al., 2001). Recently in a study conduced

Acknowledgements

This research was funded by Fondi Ateneo, University of Studies of Bari (ex 60%) 2005 and 2006. Assegno di ricerca research program n. 07.06, Veterinary and Pharmaceutics Science, SSD n. VET/04 titled “Helicobacter pylori and foods of animal origin: development of detection and characterization method”.

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