Many of the common enteric pathogens such as Salmonella, Escherichia coli O157: H7 and Campylobacter are carried in the intestinal tract of ruminants, including domestic animals used in milk production, e.g. cows, sheep and goats. Preventing faecal material contaminating the milk is an important step in reducing the prevalence of pathogens entering raw milk. Effective cleaning procedures, including removing faecal material from udders prior to milking, can reduce the risk, although heat treatment of raw milk is the most important process used to eliminate the risk from viable vegetative pathogenic bacteria and provide a safe product.
Raw milk can be a significant source of food-borne pathogens, and there have been numerous food-poisoning outbreaks associated with direct consumption of raw milk, milk that has been inadequately heat-treated, or milk that has been re-contaminated after heat treatment. The presence of pathogens in milk is likely to arise from contamination by faecal material during the milking process. Good hygiene, including the removal of faecal material from udders and ensuring a clean environment, is therefore important. Contaminated milking parlour equipment and floors can facilitate the spread of these pathogens to the udders; subsequently, milking equipment including teat cups, pipelines, filters and bulk storage vessels can become colonized (O’Loughlin and Upton 2001). Failure to implement effective cleaning and disinfection procedures can result in contamination of subsequent batches of product.
Besides the risks associated with consuming raw milk there are concerns over the safety of cheeses made from raw milk. Although cheese can be made safely with raw milk, there have been food-poisoning outbreaks linked to raw milk cheeses caused by Salmonella, Campylobacter, Staphylococcus aureus and E. coli O157: H7 (Zottola and Smith 1991; De Buyser et al. 2001). Nevertheless, raw milk remains popular for cheesemaking because of enhanced organoleptic properties, notably the flavour it imparts to the final product. In Europe, cheese manufacture is controlled by the Food Hygiene Regulations (European Commission 2004a, 2004b) and cheeses are generally considered to be one of the safest foods consumed. However, dairy products including cheese can occasionally contain pathogenic bacteria. The most dangerous of the vegetative pathogens associated with raw milk and raw milk cheeses are the Verocytotoxin-producing E. coli (VTEC), especially VTEC O157: H7, which is the topic of this review.
Verocytotoxin-producing E. coli
Escherichia coli is a common Gram-negative, nonspore-forming bacterium belonging to the family Enterobacteriaceae. It is found in the gastrointestinal tract of man and other animals, although it can be found in water, soil and food, often as a result of faecal contamination. Because of this association, E. coli is used as an indicator of potential faecal contamination of food and water (Baylis and Petitt 1997). Most E. coli strains are harmless commensal organisms; however, pathogenic strains have evolved which are responsible for distinct types of clinical disease in man.
There are two recognized groups of pathogenic E. coli. Extraintestinal pathogenic E. coli (ExPEC) represent E. coli associated with urinary tract infections and newborn meningitis, whilst the intestinal pathogenic E. choli (IPEC) are responsible for a range of diarrhoeal diseases. Within the IPEC there are currently six distinct groups of E. coli that are associated with food-borne disease: VTEC, enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAggEC) and the diffusely adherent E. coli (DAEC). These diarrhoeagenic E. coli, the associated virulence mechanisms and their role in disease are described elsewhere (Sussman 1997; Nataro and Kaper 1998; Donnenberg 2002; Baylis et al. 2006).
The VTEC which are also commonly referred to as Shiga-toxin producing E. coli (STEC), comprise over 400 serotypes of E. coli (Scheutz and Strockbine 2005). The common feature which defines this group is their ability to produce a distinct range of toxins termed Verocytotoxins (VT) which show potent cytotoxicity against Vero cells (Konowalchuk et al. 1977). Owing to the similarity in structure and biological activity of these toxins and the toxin produced by Shigella dysenteriae type 1 (O’Brien and Holmes 1987), they are also termed Shiga toxins (Stx). Two antigenically distinct types of VT (VT1 and VT2) were initially identified (Scotland et al. 1985), although there are now at least three subtypes of VT1 (VT1, VT1c and VT1d) and six subtypes of VT2 (VT2a, VT2c, VT2d, VT2e, VT2f and VT2g) recognized (EFSA 2007). The genes for these toxins (vtx/stx) are generally encoded by prophages of the lambda (λ) family, and VTEC strains may produce either VT1 or VT2 alone or together. The most common combination of toxin types associated with severe human disease are VT2 and VT2c (Friedrich et al. 2002; Persson et al. 2007). In contrast, VT1c and other toxin types (e.g. VT2e, VT2f and VT2g) are commonly associated with milder disease or are rarely associated with human disease (Friedrich et al. 2003; Beutin et al. 2007).
Clinical manifestations of infection with VTEC range from asymptomatic carriage through mild diarrhoea to life-threatening conditions such as haemorrhagic colitis (HC) and the severe complications of haemolytic uraemic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP), which can result in kidney damage, renal failure and even death (Tarr et al. 2005). Some VTEC, notably those belonging to serotype O157: H7, are associated with severe disease. Besides toxin production, some VTEC have the ability to cause attaching and effacing (A/E) lesions on epithelial cells using similar mechanisms to those found in EPEC. The process of A/E is mediated by a pathogenicity island termed the locus of enterocyte effacement (LEE) which is an important cluster of genes in the bacterial chromosome involved in this process. The LEE encodes a type III secretion system and the eaeA gene codes for the outer membrane protein intimin, which mediates binding of the bacterium to the host cell surface. Strains carrying eaeA are often associated with more severe forms of disease. Additional virulence factors can be carried on large plasmids found in some VTEC. In VTEC O157, a large (ca 92 kb) plasmid termed pO157 carries the gene hly/ehxA which encodes for enterohaemolysin, katP for catalase-peroxidase and espP which codes for an extracellular serine protease (Burland et al. 1998). These and other genes have all been associated with pathogenicity, although their exact role in disease is not yet fully understood. Some VTEC strains lacking eae and ehxA genes have, on occasion, been shown to cause human illness, so these genes may not necessarily be essential for a VTEC to cause disease (Neill 1997).
Not all VTEC are associated with severe human disease, however, there are many VTEC serotypes that have been isolated from animals and foods which have rarely or never been associated with cases of human infection. Therefore, the clinical significance of these VTEC serotypes or the potential risk they pose has not yet been fully elucidated. Some studies have shown differences between VTEC from dairy products, including cheese, and those commonly isolated from humans. In a study by Pradel et al. (2008), VTEC strains from dairy food were predominantly carrying vtx1 and few were harbouring the eae, espP and katP, which are commonly associated with disease-causing strains isolated from patients.
Dairy animals as a source of VTEC
Cattle have long been identified as a major reservoir for VTEC, including VTEC O157: H7 (Chapman et al. 1993a; Hancock et al. 1994). Cattle are known to shed a range of VTEC, including VTEC O157: H7, and others are associated with human disease. In a study of 514 VTEC isolates from diarrhoeic and healthy cattle in Spain, 20% carried vtx1, 54% carried vtx2 and 26% carried both toxin genes (Blanco et al. 2004). In addition, enterohaemolysin and intimin genes were detected in 63% and 29% of the isolates, respectively. Overall, 85% of the cattle isolates were identified as those associated with human disease and 54% were serotypes associated with human HUS.
Although raw meat and undercooked meat products have been commonly associated with outbreaks of VTEC O157: H7 infection, raw milk and dairy products have also been implicated in cases of human infection, including HUS. The prevalence of VTEC in dairy cattle and their products has been reviewed by Hussein and Sakuma (2005). In this review the prevalence of VTEC in faeces of dairy cattle was reported to be between 0.2% and 48.8% and 0.4% and 74.0% for E. coli O157 and non-O157 VTEC, respectively. Verocytotoxin-producing E. coli serotypes isolated from dairy cattle and associated with disease include O2: H29, O22: H8, O26: H11, O103: H2/H−, O111: H8/H−, O113: H21 and O145: H− (Hussein and Sakuma 2005).
Besides cattle, other domestic animals used to produce milk, such as goats and sheep, also harbour these bacteria and shed them in their faeces. Compared with cattle, higher prevalence of E. coli O157 and other VTEC in sheep and goats has been reported (Beutin et al. 1993; Sidjabat-Tambunan and Bensink 1997; Fagan et al. 1999). In Germany, reports indicate prevalence rates for non-O157 VTEC in sheep, goats and cattle of 67%, 56% and 21%, respectively (Beutin et al. 1993). Similar results were found during another German study which reported VTEC prevalence rates of 32%, 75% and 18%, respectively in sheep, goats and cows (Zschock et al. 2000).
In addition to the high prevalence of VTEC in sheep and goats, these animals appear to harbour a broad range of VTEC serotypes, including VTEC associated with human infection. In a study by Rey et al. (2003), comparison of 253 VTEC strains isolated from sheep in Spain revealed 43% to be carrying stx (vtx 1) gene, 4% carried vtx 2 and 53% carried both toxin genes. In addition, the virulence-associated genes ehxA and eae were present in 47% and 4% of strains, respectively. The majority of these VTEC strains belonged to six serogroups (O6, O91, O117, O128, O146 and O166) and 71% belonged to nine serotypes (O6: H10, O91: H−, O117: H−, O128: H2, O128: H−, O146: H21, O166: H28, O76: H19 and O157: H7). Rey et al. (2003) also reported the discovery of 10 new VTEC O: H serotypes, not previously described.
In dairy herds, faecal contamination of udders poses a risk of pathogens entering the raw milk. Preventing faecal material from contaminating the milk is therefore an important step in reducing the prevalence of VTEC and other pathogens in raw milk. The implementation of effective cleaning procedures to remove faecal material from udders prior to milking can reduce the risk but not eliminate it entirely. The risk of transferring pathogens, including VTEC, to milk can be further reduced by hygiene precautions during milking.
Good animal husbandry on the farm reduces udder infections, which can allow pathogenic bacteria to be introduced into the milk. Bovine mastitis can be caused by E. coli (Jones 1990) but there is little if any published evidence that mastitis can be caused by E. coli O157. Turutoglu and Madul (2002) reported the isolation of E. coli from 50 (13%) of 382 mastitic milk samples in the Burdur province of Turkey, but none of these were identified as belonging to serogroup O157. There is also little published information to indicate that VTEC strains are responsible for mastitis, although they could potentially gain access from the udder to the milk in mastitic cows (Stephan and Kühn 1999). Both VTEC and other E. coli come from the same faecal origin and they can readily be isolated from the cow’s environment, so there is no reason to assume that VTEC cannot enter milk by the same routes used by other enteric bacteria.
Pasteurization is an effective treatment for removing vegetative pathogens, including VTEC, from milk. Without this step there is always the risk of pathogenic bacteria being present in the milk. Raw milk and dairy products produced using unpasteurized milk, such as cheeses, have been responsible for outbreaks of food-borne illness (Djuretic et al. 1997). With the increased popularity of products such as cheeses made from unpasteurized milk, strict hygiene, good quality and process control during their manufacture are essential.