Isolation of verocytotoxin-producing Escherichia coli O157 from water buffaloes (Bubalus bubalis) in southern Italy

VEROCYTOTOXIN (VT)-producing Escherichia coli O157 (VTEC O157) is a zoonotic pathogen that causes severe disease in human beings, such as haemorrhagic colitis and haemolytic uraemic syndrome (Griffin and Tauxe 1991). The gastrointestinal tract of cattle is considered to be the main natural reservoir of this organism, and the consumption of products of bovine origin has been frequently associated with large epidemic outbreaks of human disease (Armstrong and others 1996). However, the ecological niche of E coli O157 seems to include other ruminant species; sheep (Kudva and others 1997) and goats (Synge 1999) appear to be frequently colonised by VTEC O157, and the pathogen has also been isolated from wild ruminants (Rice and others 1995). VTEC have also been detected in water buffalo (Bubalus bubalis). Strains belonging to serogroups other than O157 were first reported in buffalo calves with diarrhoea in Sri Lanka by Mohammad and others (1985). In a previous study, Galiero and others (1997) showed that six of 110 E coli strains (5·4 per cent) isolated from water buffalo calves reared in Italy produced VT, but none of them was E coli O157. The Mediterranean water buffalo is intensively reared in areas of central and southern Italy, particularly in the Campania region, and its fat-rich milk is used unpasteurised to produce a type of mozzarella cheese very popular with consumers. This short communication describes a survey to investigate Italian water buffalo herds for the presence of VTEC O157 by examining sixto 18-month-old animals, which, in cattle, usually have the highest prevalence of VTEC O157 carriage (Hancock and others 1998), using an O157-specific and sensitive immunomagnetic separation (IMS) enrichment technique (Wright and others 1994). The survey was conducted between September 2002 and October 2003, and included 65 herds located in the Campania region. The herds ranged in size from 10 to 950 animals, including between four and 70 animals aged six to 18 months per herd, and comprised a total of 13,788 animals (1364 between six and 18 months of age), representing approximately 5 per cent of all the water buffaloes reared in the region. None of the farms held cattle together with the buffaloes. Each farm was visited once, and in each sampling visit fresh faecal samples were collected from two to 13 healthy animals, depending on the size of the herd. From each sample, 1·0 g of faeces was added to 9 ml buffered peptone water, homogenised in a stomacher bag and incubated at 37°C for six hours; IMS was then performed using Dynabeads anti-E coli O157 (Dynal) following the manufacturer’s recommendations. The magnetic beads were inoculated on to sorbitol MacConkey’s agar (SMAC; Oxoid) and on to SMAC supplemented with cefixime and tellurite (Oxoid). After incubation at 37°C for 18 to 24 hours, sorbitol-nonfermenting colonies were tested for agglutination using an E coli O157 latex test kit (Oxoid). Isolates that gave positive results were confirmed as E coli by using biochemical assays, and serotyping was confirmed by tube agglutination of heattreated cultures. VT production was assessed by the Vero cell assay, and the presence of VT genes and the intimin-coding eae gene was determined by PCR analyses (Bonardi and others 1999). A total of 289 animals was examined, approximately 21 per cent of all the animals aged from six to 18 months on the farms visited. VTEC O157 strains were detected in 42 animals (14·5 per cent), from 21 (32·3 per cent) of the farms surveyed. On the positive farms, the average prevalence of VTEC O157positive animals was 35·6 per cent, ranging from 15·3 per cent (two positive of 13 examined) to 100 per cent (two positive of two examined). Among the 42 VTEC O157 strains isolated, three carried both the vt1 and vt2 genes, and 39 carried only vt2. All of the strains harboured the eae gene. The results of this survey indicate that young water buffaloes reared in this region of Italy frequently carry VTEC O157. The strains isolated were biochemically typical of E coli O157 and harboured the genes encoding VT and intimin, and therefore have to be considered as potential human pathogens. The carriage rates observed (32·3 per cent and 14·5 per cent of the herds and the animals sampled, respectively) are comparable with those reported in cattle in Italy (Conedera and others 1997, Bonardi and others 1999) and in other countries (Armstrong and others 1996), and appear to be higher than those reported in sheep (Chapman and others 1997, Heuvelink and others 1998), which are also regarded as an important natural reservoir of VTEC O157. As has been reported for cows (Armstrong and others 1996) and sheep (Rubini and others 1999), buffalo milk could be contaminated by VTEC O157. Since the traditional production process of buffalo mozzarella cheese requires the use of raw milk, the risk of VTEC O157 entering the production chain should be considered. However, the manufacturing process involves a curd-stretching step of five minutes, performed in water kept at 90 to 95°C, and this should effectively reduce the number of VTEC O157 that may be present. In a recent survey performed on Italian traditional dairy products, E coli O157 was not found in any of 500 samples of buffalo mozzarella cheese, despite the use of the sensitive IMS enrichment procedure (Conedera and others 2004). In conclusion, the results of this survey indicate that water buffaloes should be considered as a natural reservoir of VTEC O157. Since water buffalo farming is an important economic activity in some areas of southern Italy, with a total of approximately 250,000 animals reared, further studies are needed to understand better the ecology of VTEC O157 in buffalo farms and to evaluate the risk of contamination of buffalo milk and mozzarella cheese produced from it.