NC1041: Enteric Diseases of Swine and Cattle: Prevention, Control and Food Safety
Statement of Issues and Justification
Food-borne illness has been a prominent public health concern in the United States in recent years due to the occurrence of large-scale outbreaks which receive intense media scrutiny (for example, the Jack-In-The-Box outbreak of E. coli O157:H7 infection associated with undercooked ground beef, and the Chicago area Salmonella Enteritidis outbreak associated with post-pasteurization contamination of milk) as well as to the enormous incidence of sporadic food-borne disease problems revealed through the use of improved surveillance mechanisms such as the Food-Net program. The latter resulted in estimates of >75 million cases of infectious and noninfectious food borne illness annually in the United States during the 1990s, resulting in 325,000 hospitalizations and 5,000 deaths. It is not surprising that the current and immediate past presidential administrations, Congress, and the USDA have made food safety a high priority. The statistics arising from the FoodNet surveillance programs highlight three very important points:1) Most food-borne illness events are of undefined etiology, stressing the need for identification and characterization of novel, emerging, or previously unrecognized agents, which undoubtedly account for many of these cases. The relatively recent recognition of norovirus-associated food- and water-borne illnesses is a striking reminder of the potential for agents of major importance to remain unrecognized for years.
2) Most of the known bacterial, viral and parasitic food-borne disease agents are primarily zoonotic in nature. Therefore, investigation and control in the animal reservoir are required to fully understand their epidemiology and biology in order to maximize the opportunities for their control.
3) Several of these agents are also severe pathogens of animals or have close relatives that are animal pathogens, such that investigation of the host-parasite relationship in animal models or in fact in the animal populations themselves will be informative regarding the host-parasite interactions in humans.
The USDA Economic Research Service estimates released in 2000 for medical costs, productivity losses, and costs of premature deaths for diseases caused by five foodborne pathogens including Campylobacter (all serotypes), Salmonella (nontyphoidal), E. coli O157, E. coli non-O157 Shigatoxin-producing E. coli (STEC), and Listeria monocytogenes. total $6.9 billion per year (USDA Economic Research Service, 2000). In addition, the cost of just E. coli O157:H7 to the beef industry from 1993-2003 was estimated to be $2.671 billion (Kay, 2003. Recent reports from Food-Net indicate significant progress in the control of infectious diseases caused by several infectious agents noted above including Campylobacter spp., Salmonella (nontyphoridal), E. coli O157:H7, Listeria moncytogenes, Cryptosporidium parvum and Yersinia spp. These declines likely are the result of diverse actions such as pre-harvest and post-harvest interventions and education of producers and consumers. While the incidence of disease caused by some of these agents (Campylobacter, Listeria, STEC O157) approaches the 2010 targets, these rates can be further reduced with additional knowledge and new detection procedures developed through research such as that proposed in this collaborative project.
In addition to human health considerations, animal diarrheal disease due to both food-safety related pathogens as well as many other animal-specific pathogens, remains an economically very important cause of production loss to livestock producers. Recent surveys and studies from the National Animal Health Monitoring System (cattle and swine) and the National Pork Producers Council (swine) indicate the continuing importance of enteric diseases as major sources of morbidity, mortality, and associated economic costs in these industries. As the production systems utilized by these industries continue to evolve toward larger sizes and complexity, antibiotics in feed become banned and at the same time, as natural , grass-fed , and organic production systems emerge and expand (with additional constraints on types of acceptable production facilities and the available therapeutic armamentarium), continued research in support of food safety and of control of diarrheal diseases of livestock will be necessary to optimize animal health and welfare in the production of safe foods.
American agriculture is noted worldwide for its high productivity, quality, and efficiency in delivering goods to the consumer. However, when improperly managed, agricultural activities can affect water quality. Animal agriculture farms, especially dairy and cattle farms are potential sources of microbial contamination of the water supply. The2000 National Water Quality Inventory reports that agricultural -non-point - source (NPS) pollution is the leading source impacting water quality in surveyed rivers and lakes, the third largest source of impairments to surveyed estuaries, and also a major contributor to ground water contamination and wetlands degradation. Microbial pathogens present in agricultural runoff impose a significant hazard on human health when acquired directly via the fecal-oral route or indirectly as a waterborne contaminant. A major long-term goal of the NC -1007 committee is to develop implement strategies derived from basic research efforts to control microbial contamination of water resources and provide a safe and sustainable environment for animal production facilities. Based on CDC estimates, enteric caliciviruses (Noroviruses, NoV; Sapoviruses, SaV) cause over 9 million cases of food-borne illnesses in the U. S. yearly, making them the most common cause of acute food-borne gastroenteritis in the U. S. (Mead, 1999) Recently, caliciviruses that are genetically more closely related to human caliciviruses than to other animal caliciviruses have been identified in fecal samples from swine and cattle. (Guo, 1999; Van der Poel, 2000; Smiley, 2003; Han, 2004, Wise, 2004; Wang, Han, Cheetham, 2005; Wang, Han, Funk, 2005; Wang, 2006). Moreover, shellfish approved for human consumption contain both animal and human enteric caliciviruses (Costantini, Loisy 2006). These observations raise concerns of whether swine or cattle could be reservoirs for enteric caliciviruses transmissible to humans, an issue that urgently needs to be addressed by further research.
The USDA Economic Research Service estimates released in 2000 for medical costs, productivity losses, and costs of premature deaths for diseases caused by five food-borne pathogens including Campylobacter (all species), Salmonella (nontyphoidal), E. coli O157, E. coli non-O157 Shigatoxin-producing E. coli (STEC), and Listeria monocytogenes total $6.9 billion per year (USDA Economic Research Service, 2000). In addition, the cost of just E. coli O157:H7 to the beef industry from 1993-2003 was estimated to be $2.671 billion (Kay, 2003). These costs were attributed to a reduced demand for beef ($1.584 billion); a reduction in boneless beef prices ($0.1720 billion); packer expenditures ($0.850 billion); and, research investments ($0.0650 billion).
Cattle are an important reservoir of E. coli O157:H7 because the organism colonizes the large intestine, without causing disease, and exhibits a tissue tropism for the terminal rectum of adult cattle (Naylor et al., 2003; Rice et al., 2003). In contrast, other serotypes such as O5, O26 and O111 colonize the entire large intestine of young calves in a diffuse fashion (Stevens et al., 2002b). Intestinal colonization by E. coli O157:H7 and other EHEC requires the formation of A/E lesions, which are mediated through proteins secreted by a type III secretion system and the outer membrane protein known as intimin (Dean-Nystrom et al., 1998; Moxley, 2004; Naylor et al., 2005; Potter et al., 2004; Vlisidou et al., 2006). Colonization of the large intestine in cattle results in shedding of the organism in feces (Moxley, 2004; Renter & Sargeant, 2002; Stevens et al., 2002a). Control of fecal shedding of E .coli O157:H7 by cattle and other animal reservoirs is imperative since it represents the primary contamination source of food and water and can also infect humans via direct contact (Armstrong et al., 1996; Elder et al., 2000; Stevens et al., 2002a). Although post-harvest interventions have been implemented and significantly reduced E. coli O157 contamination of ground beef (Arthur et al., 2004), the organism is still highly prevalent in cattle in the United States and pre-harvest interventions are needed to reduce carriage levels (Callaway et al., 2004; Loneragan & Brashears, 2005)
Enterotoxigenic E. coli (ETEC) cause diarrhea in neonatal (piglets, calves and lambs) and young animals (piglets) through adherence to intestinal epithelial cells and production of enterotoxins, which induce water and electrolyte loss from the small intestine (Nataro & Kaper, 1998). It has been reported that ETEC are responsible for the death of 10.8% of all pre-weaning pigs and 1.5-2% of all weaned pigs (Tubb et al., 1993; Hampson, 1994). The incidence of neonatal diarrhea has been reduced substantially by the use of vaccines. However, post-weaning ETEC diarrhea, continues to be economically one of the most important diseases for the North-American swine industry.
The most common ETEC strains associated with diarrhea in piglets produce K88 (F4) or F18 fimbriae (Francis, 2002). These fimbriae bind to glycoconjugates which serve as receptors in porcine enterocyte brush borders. Absence of the respective glycoconjugate renders the animal resistant to bacterial colonization and consequential diarrheal disease (Erickson et al., 1992; Erickson et al., 1994; Francis et al., 1998). ETEC strains produce several types of enterotoxins, including heat labile enterotoxin (LT), heat stable enterotoxin-a (STa), heat stable enterotoxin-b (STb; Nataro & Kaper, 1998), and enteroaggregative E. coli heat stable enterotoxin 1 (EAST1; McVeigh et al., 2000; Savarino et al., 1996; Yamamoto & Echeverria, 1996). An individual ETEC strain may produce one or more enterotoxins. However, ETEC must produce both enterotoxin and fimbriae in order to cause severe dehydrating diarrheal disease (Berberov et al., 2004; Francis et al., 1998; Smith & Linggood, 1971). Piglet enterocyte susceptibility to K88+ ETEC adherence is inherited in a simple Mendelian fashion as a dominant trait, and susceptibility to K88+ ETEC mediated disease is correlated with expression of an intestinal mucin-type glycoprotein (IMTGP) receptor for the K88+ fimbria (Francis et al., 1998; Grange et al., 1998). The high virulence of K88+ ETEC strains in susceptible swine is evidenced clinically by their tendency to cause extensive intestinal colonization, severe dehydrating diarrhea, post-diarrheal septicemia, and death (Moxley et al., 1998). The pathogenesis of post-diarrheal septicemia involves the development of severe dehydration, hypovolemic shock, and ischemia of the intestinal mucosa, the last presumably a consequence of the shock-induced low-flow state (Moxley et al., 1998; Berberov et al., 2004). Shock-induced ischemia causes sloughing of the intestinal epithelium, and ETEC bacteria gain entrance to the general circulation following adherence to exposed intestinal basement membranes (Moxley et al., 1998; Berberov et al., 2004).
Isolates of K88+ porcine commonly produce both LT and STb (Nagy et al., 1990; Wilson & Francis, 1986), are often PCR-positive for the EAST1 gene (Choi et al., 2001), and frequently cause death in natural infections (Moxley et al., 1998; Nagy et al., 1990). EAST1 expression has been shown to occur with porcine ETEC, but this has only been studied with one strain, which also expressed LT and STb (Berberov et al., 2004). While the ability to produce multiple enterotoxins is a rational hypothesis for explaining why some ETEC strains are more virulent, there is a lack of information concerning the contribution of the different enterotoxins to virulence, especially in light of defined fimbrial type and host susceptibility.
Several enterotoxins produced by ETEC have been shown to activate secretory pathways in enterocytes mediated by the cyclic nucleotides, cAMP and cGMP. Heat labile enterotoxin (LT) activates adenylate cyclase in enterocytes, which causes increased intracellular concentrations of cAMP (Nataro & Kaper, 1998). Increased cAMP levels result in stimulation of Cl- secretion from crypt epithelial cells and inhibition of NaCl absorption by villous enterocytes with resultant osmotic diarrhea (Nataro & Kaper, 1998). Heat stable enterotoxin (STa ) activates guanylate cyclase in enterocytes, resulting in increased intracellular concentrations of cGMP, Cl- secretion, and inhibition of NaCl absorption in the small intestine (Nataro & Kaper, 1998). STb stimulates secretion of HCO3- from enterocytes, which causes its accumulation, along with increased concentrations of Na+ and Cl-, in the intestinal lumen (Dubreuil, 1997). Enteroaggregative E. coli toxin (EAST-1) has significant homology with the enterotoxic domain of STa, and with guanylin, a mammalian analog of STa (Menard & Dubrueil, 2002). EAST-1 was reported to elicit enterotoxic activity via stimulation of increased cGMP concentrations within enterocytes (Menard & Dubrueil, 2002). However, EAST-1 has not yet been purified to homogeneity (Menard & Dubrueil, 2002), and preliminary results by the KS station in the NC-1007 group have found results that do not agree reported observations (see below). Synthetic EAST-1 peptide was found to induce a significant rise in short-circuit current in Ussing chambers that was similar, but not identical to the electrogenic response evoked by STa (Savarino et al, 1993). The effects of EAST-1 on induction of electrolyte loss from the porcine small intestine have not yet been determined.
Salmonella enterica is a common cause of systemic and diarrheal disease in livestock and causes approximately 2 million cases of diarrhea per year in humans in the US with up to 1000 deaths and an economic loss estimated to be $12 billion. Over the past few years, the CDC has noted a rapid increase in the number of laboratoryconfirmed Salmonella Newport infections (126%). This raises concern for two reasons: 1) the spectrum of illness due to this serovar tends to be more severe, and 2) an increasing number of Newport isolates have recently been shown to be multi-drug resistant. Multi-drug resistance prevalence increased from 1% in 1998 to 26% in 2001, with some isolates being resistant to ceftriaxone, a drug commonly used to treat invasive Salmonella infections. The emergence of a multi-drug resistant S. Newport has been attributed to various factors including intensive farming practices, the movement of cattle between farms and the overuse of antimicrobial agents in dairy operations (Gupta et al., 2003).
Proliferative enteropathy (ileitis) is a common enteric disease of weaned pigs and other animals caused by the obligately intracellular bacterium, Lawsonia intracellularis (McOrist et al, 2005a). The characteristic pathological feature in all species is marked proliferation of immature epithelial cells in the crypts of the ileum or colon, or both, which leads to thickening and branching of the crypts and gross mucosal thickening (McOrist et al, 2005b). The bacteria are invariably present in the apical cytoplasm of the proliferative enterocytes. The clinical signalment in affected weaned animals can include diarrhea, weight loss, and melena (McOrist et al, 2005b). Infections resulting in these signs are common on swine farms, and estimates of the annual economic losses are generally around $100 million for the US swine industry alone (McOrist et al, 2005b). The 2000 NAHMS (NAHMS, 2000) report described proliferative enteropathy as the most common disease present in grower/finisher pigs in the U.S.
Little is known of the pathogenic mechanisms for this L. intracellularis and sensitive and specific methods for diagnosing proliferative enteropathy are not universally available. Characterization of the gene products from this organism will help to identify genes responsible for microbial virulence, or that will be useful in the development of diagnostic reagents and candidate recombinant vaccines against this organism. Development of a molecular typing database will enable further studies on the ecology and epidemiology of L. intracellularis.
Recent advances in phenotypic and genotypic characterization of pig intestinal spirochetes have increased our understanding of swine dysentery (SD) and porcine colonic spirochetosis (CS) caused by the pathogenic species Brachyspira hyodysenteriae and Brachyspira pilosicoli, respectively (Hampson et al., 2006;Hampson and Duhamel, 2006). Because of the devastating economic impact of SD on pig production and changes in the structure and management of the swine industry to eliminate SD, the prevalence of SD in the US swine herd has steadily declined over the last decade. Consequently, the levels of diagnostic capabilities and research expertise have been reduced considerably. This is in contrast to most pig producing countries of the world, where SD continues to be a major health challenge.
Concurrent with a decline in SD prevalence, porcine colonic spirochetosis (CS), a less severe form of diarrheal disease of grower pigs, has became more widely recognized. By contrast with SD which is restricted to pigs and rodent vectors, a wide range of hosts in addition to pigs are susceptible to CS, including human and non-human primates, dogs, horses, and many species of wild and domestic birds (Duhamel, 2001; Duhamel et al., 2003; Smith, 2005). Although limited epidemiological investigations suggest a zoonotic potential with public health significance for B. pilosicoli (Oxberry et al., 1998; Brooke et al., 2006), the role of this spirochete in colitis of humans remains uncertain.
Cryptosporidium. parvum is the most common enteric pathogen of calves and responsible for significant economic loss to the dairy and cattle industries due to both morbidity and mortality. In addition, C. parvum is a significant threat to water resources and human health. A National Dairy Heifer Evaluation Project estimate from 1991 indicated that this parasite was present on more than 90% of farms surveyed, with 22% of pre-weaned heifers shedding oocysts on any given day. A single calf at the peak of infection can excrete up to 107 oocysts per gram of feces. The damage sustained to the host intestinal epithelium during infection results in a decrease in absorptive surfaces, gut mucosal inflammation, secretion of electrolytes into the lumen, and persistent diarrhea. Currently, there are no effective treatments for either humans or livestock infected by C. parvum . The complexity and range of these enteric pathogens and of the food animal production systems in which they occur require collaborative research involving scientists with a wide range of expertise to work together in pursuit of solutions. No individual institution can match the range of scientific expertise offered by NC-1007 Technical Committee in this regard. The NC-1007 Committee consists of bacteriologists, virologists, molecular biologists, pathologists, and immunologists who have a productive history of working together in innovative research.
This NC-1007 project addresses critically important cross-cutting research areas and objectives that will enhance food safety while maintaining efficient pork and beef production by identifying and characterizing emerging and newly identified agents associated with enteric disease in cattle and swine with particular attention to those with zoonotic potential, developing interventions and preventative measures to reduce animal carriage and human transmission of these enteric zoonotic agents, including development of new and improved vaccines and non-antibiotic therapies in order to prevent and treat enteric infections while reducing antibiotic usage and selective pressures for development of antibiotic resistance, and effectively disseminating information from the committee to possible users.
A CRIS search was performed to detect overlap between this project and other multi-state projects. The key words used were 'enteric disease(s)' and each of the diarrheal pathogens proposed for investigation in this project. The search yielded 105 current and expired projects. Of these 105, only 13 projects are active (and several of those expire later this year). No duplication with any other multi-state project was detected by this search. In fact, that was no duplication between NC-1007 and any Hatch or 1433 project, other than those of investigators who are members of NC-1007 and this search detected all NC-1007 investigators. We conclude that this proposed project does not contain significant overlaps with other multi-state projects.
Summary of revisions from previous project: - Add as a new priority a focus on emerging or currently unrecognized agents that result in food-borne human illness or enteric diseases of domestic animals, proposing research on the identification, characterization and development of diagnostics for emerging or currently unrecognized agents.
- Add as a new priority the development of effective and practical interventions to reduce prevalence of agents already characterized to be of major importance in food safety and / or animal health, based on research on the ecology of the agents and their epidemiology and population dynamics in animal reservoirs.
- Provide training and continuing education to disseminate knowledge regarding new and emerging agents and interventions effective at reducing enteric disease agent incidence and prevalence.
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