Food-borne disease is an increasingly growing health concern with the WHO stating that "Governments all over the world are intensifying their efforts to improve food safety" in a response to the rising problem of food safety concerns and an increase in consumer rights legislation (WHO factsheet no. 237, 2007). This rise is of particular concern in developed regions such as Europe and the US, with the latter estimated to have 76 million cases annually, resulting in approximately 5,000 deaths, although many of these cases go both undiagnosed and untreated (Mead et al., 1999). These food-borne illnesses, commonly known as "food poisoning" result from eating contaminated food and can result from the wide range of industrial production processes food is subjected to before reaching the consumer, as well as through consumer neglect in relation to food safety (Tauxe 1997).
More than 200 known diseases are transmitted through food, with causes ranging from viruses, bacteria and prions to toxins and metals, causing conditions such as cholera and shigellosis (Mead et al., 1999). Bacteria, to which the focus of my study Campylobacter belongs are particularly prevalent and can colonise a range of food preparation surfaces, utensils and cleaning apparatus, from which they can then be transferred to food (Rusin et al., 1998). Refrigeration practices can also lead to cross contamination of foodstuffs through improper maintenance of temperature and thorough cleaning, with this believed to account for 28% of food-borne disease outbreaks (Ryan et al., 1996).
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Disease from food is not a new phenomenon, such as the numerous typhoid fever outbreaks in the US in the early 20th century, but it is changing with typhoid control achieved through the disinfecting of drinking water and sanitation of shellfish beds in addition to milk pasteurisation, similarly cholera and bovine tuberculosis have been controlled in most developed countries (Tauxe 1997). In the modern era however, there are new microbial threats, such as the rise of nontyphoid strains of Salmonella post World War II, with Salmonellosis now a problem in most countries and is derived from meat and dairy products, causing nausea, vomiting and diarrhoea (WHO factsheet no. 237, 2007).
In addition to these many other food-borne pathogens have become prevalent in the last 20 years, and some known pathogens have recently been found to be predominately food-borne for example Listeria monocytogenes, which was long known as a cause of meningitis was identified through a number of studies to be most commonly found in poultry (Jackson and Wenger 1993). It has also been realised that these now common pathogens have a number of similar characteristics, virtually all having an animal reservoir from which to transfer to humans, making them food-borne zoonoses, but unlike many of the already established zoonoses they do not necessarily cause infection and disease in the host animal, appearing healthy, leading to a need to include testing of "healthy" animals and their diet (Tauxe 1997).
The historic widespread use of antibiotics on farmyard communities has also posed a significant problem, leading to many pathogens developing antibiotic resistance. One study shows that Campylobacter species isolated from human and poultry in Europe have increasing resistance to fluoroquinolones, after this antimicrobial was used in animal populations (Endt et al., 1991). Another important complication of such pathogens is that the foodstuffs usually smell, look and taste as they would normally and that the pathogens can survive many cooking processes (Tauxe 1997). Salmonella Enteritidis in eggs for example, has been show to survive in potentially hazardous amounts even when the egg is prepared and cooked into an omelette (Humphrey et al., 1989), which illustrates the need for more radical means of testing seemingly "safe" produce.
In conjunction with this, new food manufacturing principles, such as the combined of prepared ingredients from more than one country into a "ready to eat" product and the global transportation of food with a relatively short shelf life, make the process of tracking an outbreak a much more difficult practice (Tauxe 1997). Along with the significant health risks posed by such bacterium, the economic cost to the producer must also be analysed, with the slaughtering and incinerating of infected animals the usual process, in 2007 the recall of almost 22 million pounds of ground beef within the United States due to E. coli O157:H7 contamination resulted in the manufacturer, Topps Meats going into liquidation (Nugen and Baeumner, 2008). Similarly the WHO reports that an outbreak of cholera in fishing waters cost the Peruvian government an estimated $500 million in fish exports, with the estimated medical costs and loss of productivity caused by such major pathogens resulting in the US losing $35 billion annually (WHO factsheet no. 237, 2007).
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Therefore screening and analysis of produce rapidly and effectively is of vital importance and in conjunction with education of food safety protocols within the home environment to consumers will help minimise cases of "food poisoning" allowing for a safer human environment with the distinct possibility of a breakthrough in how to eradicate such pathogens from their animal hosts.
The Campylobacter genus:
The Campylobacter genus, to which a rapid identification tool is the main aim of my project is an important emerging pathogen in developed countries, with Campylobacter jejuni being the predominant cause of bacterial gastroenteritis worldwide (Champion et al., 2005).
The name Campylobacter means "twisted bacteria," characterised by their spiral appearance. The genus contains 14 species of gram-negative bacteria, which are microaerophilic, meaning they require oxygen to survive, but generally at lower concentrations than present in the atmosphere (optimum 5-10% oxygen), and possess uni- or bi-polar flagella, used for propulsion. (Ryan and Ray 2004)
Campylobacter were first discovered in 1886, with Escherich observing Campylobacter like organisms in stool samples of children with diarrhoea, in 1913 McFaydean and Stockman noticed similar organisms in fetal samples of aborted sheep. In 1957, this developed further with King isolating Campylobacter species from blood samples of children with diarrhoea. Much progress was made in the 1970s with selective growth media techniques and in 1976 Campylobacter was isolated from stool samples of infected patients in Belgium. (Kist 1985) By 1996, 46% of laboratory confirmed bacterial gastroenteritis was caused by Campylobacter species (Campylobacterosis) (Altekruse et al., 1999), estimating that it cause around 2.2 million cases per year in the USA alone (Tauxe 1992).
Campylobacterosis infections range in severity from loose stools to dysentery and usually presnt themselves after an incubation period of approximately 7 days. It is common for symptoms to include diarrhoea, abdominal pains and a fever, with around half of patients having a history of bloody diarrhoea, Campylobacter infections can also present septic arthritis, producing bacteremia and other complications extraintestinally (Peterson, 1994). Campylobacter species are particularly pathogenic and although dependant on age and health of the host, one study found infection occurred after ingestion of just 800 C.jejuni organisms (Black et al., 1988), Patients with HIV are most at risk, with the rate of Campylobacterosis aprroximately 39 times higher in HIV patients when compared to the general population (Sorvillo et al., 1991).
Campylobacter species injure tissues in the jejunum, the ileum, and the colon, with C.jejuni appearing to destroy epithelial cells, becoming attracted to bile mucus. C.jejuni in particular adheres to epithelial cells and mucus, containing a superficial antigen PEB1 which acts as an adeshin, promoting colonisation in the gut. Survival from the immune system may be species specific, with C.fetus giving resistance to phagocytosis by possessing a surface S-layer protein which disrupts c3b binding. (Javid and Ahmed 2009)
Transmission to humans of Campylobacter is most frequently through the ingestion of contaminated water, foodstuffs or unpasteurised milk, although more rarely from fecal-oral or person-to-person sexual contact. Interaction with ill pets, especially dogs has also been associated with Campylobacter outbreaks (Javid and Ahmed 2009). It is estimated that around 50-70% of all human cases of Campylobacterosis cases are accountable to chickens. Campylobacter ecology is most prevalent in wild bird populations such as geese and ducks, but also includes domesticated bird species as well as rodents and can be carried insect exoskeletons (Jacobs-Reitsma 1995). In respect to commercial farming the intestines of young calf and poultry can be easily colonised, with transmission rates declining with age, vertical transmission had been suggested as a method of contraction but is not widely accepted with the most common routes through unchlorinated drinking water, exposure to infected beetles and farm workers (Altekruse et al., 1999). Controls of the infection rates before the consumer are twofold; controlling rates on the farm and in the processing plant. In the farmyard environment, intestinal level of Campylobacter species may be significantly reduced by the drinking of chlorinated water and the immunisation of birds. In the abattoir, the maintenance of clean working surfaces is of vital importance and in England, chlorinated sprays are used on the carcasses to reduce bacterial levels (Altekruse et al., 1999).
The ability for these species to survive in the environment is poor, with those most associated with diarrhoea growing best at 40-420C, although tolerant to the 370C conditions found in humans (NHS, BSOP ID 23, 2007), and is sensitive to freezing, drying and acidic conditions (pH â‰¤ 5.0) (Altekruse et al., 1999).
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Treatment of Campylobacterosis is usually through the replacement of fluids and electrolytes lost, with the duration of the illness usually between 5-10 days. If antibiotics are used generally erythromycin is prescribed. Antibiotic resistance in Campylobacter species is on the rise, with a 1994 study finding that most clinical isolates of C.jejuni from US troops in Thailand were resistant to ciprofloxacin with a third resistant to azithromycin (Murphy et al., 1996). Similarly cases of fluoroquinolone resistant Campylobacter strains are on the rise in correlation with its approval for use on chickens, with a UK study finding fluoroquinolone resistant species in 22% of poultry and three quarters of pig farms (Taylor et al., 2008).
With the above information in mind, it is clear to see that bacterium in the Campylobacter genus are an emerging and serious pathogen to humans worldwide, the leading cause of bacterial gastroenteritis and pose a significant risk to human health, becoming harder to treat when infection takes hold due to increased antibiotic resistance. This therefore illustrates the need for a quick and genus specific testing procedure of foodstuffs, to detect the levels of this genus present, so as to enable the consumer to a safe product.
Comparison of current diagnostic techniques:
The British Standard technique