Duration: 10/01/2007 to 09/30/2012

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

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.

Related, Current and Previous Work

Enteric viruses

NC1007 stations have contributed significantly to the identification and understanding of enteric viral infections of humans and animals. The only enteric calicivirus that has been successfully cultivated is a porcine enteric calicivirus (Cowden SaV), related to human enteric caliciviruses, and which causes diarrhea and viremia in gnotobiotic pigs (Saif, 1980; Guo, 2001). Bile acids present in the duodenum (where caliciviruses replicate) induce a cell signaling pathway that down-regulates STAT1 (IFN-induction) promoting viral growth (Chang, 2004). This novel concept of caliciviral replication is related to both in vitro viral adaptation and in vivo viral pathogenesis and will be explored in future NC-1007 collaborative studies. The recent development of an infectious clone of porcine Cowden SaV permits future studies of the genetic basis for enteric calicivirus virulence and cell adaptation applicable to porcine, bovine and human enteric caliciviruses (Chang, 2005). Recognition that ABOH blood groups function as NoV receptors in humans and the presence of similar A and H type antigens on pig tissues, suggests that they may mediate genetic susceptibility to NoV infections in pigs. The role of these antigens in pig susceptibility to NoV infections will be examined in future NC-1007 collaborative studies. Members of NC-1007 have developed RT-PCR and other assays for enteric caliciviruses, have identified, sequenced and typed caliciviruses in bovine and porcine fecal samples and have confirmed their ability to cause diarrhea (Smiley, 2003; Han, 2004, Wise, 2004; Wang, Han, Cheetham, 2005; Wang, Han, Funk, 2005; Wang, 2006). Genomic sequence data indicates that porcine NoVs and SaVs are most closely related to human strains (Smiley, 2003; Han, 2004, Wise, 2004; Wang, Han, Cheetham, 2005; Wang, Han, Funk, 2005; Wang, 2006; Saif, 1980; Guo, 2001). The zoonotic potential of bovine and porcine enteric caliciviruses, as well as their role and prevalence as pathogens of calves and piglets, will be investigated in future collaborative studies. Recent work identified enteric caliciviruses of both human and animal origin from shellfish, raising concerns for their transmission to humans, an issue to be addressed by future NC-1007 studies (Costantini, 2006).

Porcine respiratory coronaviruses (PRCV) are natural deletion mutants of transmissible gastroenteritis virus (TGEV) that display altered tropism for the respiratory tract and cross-react immunologically with TGEV. NC-1007 researchers have improved vaccines for these agents, and have developed methods to differentiate TGEV and PRCV, important for the sale of breeding stock (Sestak, 1999; Kim, Chang, 2000). NC-1007 research also revealed that TGEV and PRCV strains co-circulate on pig farms masking typical clinical signs of TGEV and complicating TGEV diagnosis (Kim, 2000). Furthermore consistent sequence differences in the S gene of these viruses suggest the presence of coronavirus quasispecies with different tissue tropisms within single hosts, important for the evolution of coronaviruses (Costantini, 2004). Sequence analysis of the genomes of TGEV and PRCV revealed that all attenuated TGEV and PRCV strains had a conserved mutation in the S gene (nt 1753) compared to virulent TGEV strains (Zhang, 2006). Continued work on differentiation, pathogenesis and evolution of TGEV and PRCV in the field is needed and planned in this proposal.

Bovine torovirus (BoTV) causes diarrhea in cattle and it was recently associated with pneumonia and reduced growth (with BCoV) of feedlot cattle (Hoet, 2002). Because it cannot be grown in cell culture and it is difficult to identify by electron microscopy, it has been recognized by diagnostic labs only infrequently. Recently, using RT-PCR, BoTV was detected in 36% of 118 fecal samples from Canadian cattle with diarrhea (Duckmanton, 1998), 24% of fecal samples from veal calves (Hoet, Smiley, 2003) and 26% of diagnostic samples from diarrheic calves (Hoet, Nielsen, 2003). Members of this project have developed and are refining RT-PCR and ELISA procedures for BoTV (Hoet, Chang, 2003) and collaborative research is planned to determine the prevalence of BoTV in the NC-1007 area as well as to further characterize and study the pathogenic potential of BoTV enteric and respiratory strains.

In spite of extensive research and the availability of vaccines, rotaviruses continue to be one of the most common causes of diarrhea in calves and pigs. One of the more promising developments is the recent identification and characterization of an intestinal receptor for group A rotaviruses (Rolsma et al 1998, Bergner et al., 2001) by NC-1007 collaborators. This has resulted in the development of an orally administered synthetic neoglycolipid (SLPE) that blocks virus attachment, and, in preliminary experiments, dramatically attenuates diarrhea and virus shedding by orally infected pigs. Furthermore, a possible synergistic effect of SLPE and specific porcine and human milk oligosaccharides, as well as soybean derived flavonoids, which we have found to exhibit anti-rotavirus activity via inhibition of both virus attachment as well as virus replication is being investigated. Accordingly, continued multistation collaborative research in these areas is planned. Results from these studies suggest that specific oligosaccharide/isoflavone mixtures or profiles may be engineered to provide a deliverable nutriceutical for the treatment and prevention of rotavirus disease across susceptible animal species and humans (Andres, et al., 2005; Ochonicky et al., 2005). Other research includes collaboration by multiple stations to determine the incidence of non-group A rotaviruses, and to determine the genotypes of group A rotaviruses from calves and pigs with diarrhea. In complementary studies, IL investigated the kinetics of overland transport and survival of microbial pathogens (rotavirus and cryptosporidium) responsible for frequent contamination of agricultural watersheds (Trask et al., 2004).

ETEC

Much of the current understanding of the pathogenesis of ETEC had come through collaborative investigations by researchers in NC-1007 participating Experiment Stations. SD recently conducted a survey of virulence genes found in ETEC isolates from cases of post-weaning diarrhea (PWD) in swine using PCR. A broad array of virulence factors was observed to be associated with PWD in pigs. The role(s) of some are understood, but much remains to be done before cause and effect relationships between virulence factors can be interpreted. SD and NE conducted several collaborative studies, which showed that LT promotes colonization by ETEC. These studies were done using isogenic constructs and experimentally infected F4ac+ (K88ac+) -receptor-positive gnotobiotic piglets All K88ac+ isogenic strains colonized the small intestines of piglets exhibiting the K88ac receptor, but only LT and STb positive strains caused diarrhea. Expression of LT, but not STb enhanced ETEC colonization in 5-day-old gnotobiotic piglets. These novel findings may provide new insight into the evolutionary advantage of toxin production by the pathogen and identification of new control measures.

Results from SD also suggest LT is able to enhance the ability of ETEC to colonize the porcine small intestine. Strain of ETEC expressing LT bound to IPEC-J2 cells (neonatal piglet jejunal cells) with a higher affinity than LT-negative strains. A non-toxigenic construct also blocked adherence of a wild-type K88ac+ LT+ E. coli strain (3030-2) in gnotobiotic piglets. The mechanism by which LT enhances the colonization by K88+ E. coli remains to be determined, but is most likely explained by its receptor-binding ability instead of its enzymatic activity. These observations may facilitate development of either preventive or therapeutic strategies against an important animal pathogen.

The role(s) of enterotoxins in the pathogenesis of porcine ETEC infection were further studied by construction of LT-, STb-, and LT- STb- double deletion mutants and comparison of the ability of these mutants to induce fluid secretion and colonize the jejunum in ligated intestinal loops of 8-week-old pigs (NE). Strains expressing STb induced secretion in intestinal loops; however, those expressing LT did not induce secretion, suggesting that bacterial binding to the intestinal epithelium receptors via K88ac+ fimbriae is necessary for LT induction of fluid secretion, unless high quantities are delivered in the form of whole cell extracts or culture supernatants. Inoculation of K88+ac receptor-positive pigs will need to be done to test this hypothesis. Additional experiments will be needed to determine the potential role of EAST-1 and copy number for each individual contributory toxin gene (i.e., eltAB, estB and astA).

SD and KS have recently conducted experiments that suggest a potential role of EAST-1 as a virulence factor, based on evidence of an association between the copy number of the EAST-1 toxin gene (astA) and fluid stimulation in porcine ligated gut loops. This is the first experimental demonstration that EAST-1 contributes to diarrheal disease caused by any E. coli strain in its natural host species. The astA gene was cloned into single, medium and high copy-number plasmids in E. coli. Porcine ligated loop assays suggested that fluid secretion in the porcine loops was correlated with the astA gene copy number.

Substantial progress in understanding the biological activity of EAST-1 will require purified toxin and this has been a goal of work conducted by KS. KS has cloned astA into several high yield expression vectors, although none that produced amounts of EAST1 higher than wild-type strains of enteroaggregative E. coli (EAEC). However, KS now has an adequate source of EAST-1 for future studies with objectives 1) to quantify the effects of EAST-1 on electrolyte secretion, barrier function, and cell signaling, and 2) to determine whether EAST-1 modifies the enterotoxic activity of other E. coli toxins such as LT and STa. The hypothesis is that cells lining the intestine express more than one receptor-second messenger pathway that is stimulated by EAST-1 to account for the effects that have been reported. Electrolyte secretion rates were quantified under basal conditions and in the present of EAST-1, LT, and STa, alone or in combination to quantify their individual and combined effects on both electrolyte secretion and barrier function.

EHEC

NE has investigated the efficacy of vaccinating feedlot cattle with a bacterial extract containing E. coli O157:H7 type III secreted proteins in order to reduce the probability of intestinal colonization and fecal shedding of this organism. A series of clinical trials to test this vaccine was conducted in research and commercial beef feedlots in Nebraska. Results in different studies in the research feedlot have shown that: 1) three doses of vaccine administered to cattle reduced fecal shedding by 59%; 2) the efficacy of reducing fecal shedding using 1, 2, or 3 doses was 68, 67, and 73% respectively; 3) unvaccinated cattle housed in pens with vaccinated cattle were 59% less likely to shed E. coli O157:H7 than those housed with unvaccinated cattle; and 4) vaccinated cattle were 97% less likely to be colonized in the terminal rectum by E. coli O157:H7 compared with non-vaccinated cattle (Peterson et al., 2006a). A large-scale study (140 pens containing 20,556 cattle) in 19 commercial feedlots was conducted in which vaccinated pens of cattle (2 doses) were 27% less likely to test positive by rope environmental sampling than non-vaccinated pens. Vaccinated cattle were 76% less likely to be colonized in the terminal rectum compared to non-vaccinated cattle (Peterson et al., 2006b). In other studies, it was found that feedlot cattle treated with a direct-fed microbial consisting of Lactobacillus acidophilus strain NP51 had a 35% lower probability to shed E. coli O157:H7 in the feces than cattle in untreated pens (Peterson et al., 2006c).

Campylobacter spp. and Lawsonia intracellularis

Campylobacter spp. (primarily C. jejuni) cause an estimated 2.4 million cases of gastroenteritis per year in the U. S. (Mead and Dubreuil, 2002) yet their pathogenic mechanisms remain largely unknown. Several studies, including studies by participants in this project, have shown that Campylobacter spp. are commonly carried in the intestinal tract of asymptomatic swine and cattle. Recent findings by NC-1007 researchers resulted in: 1) identification of a gene, cibB, that encodes a type III secretion-like protein that is necessary for invasion of cultured mammalian cells and in vivo virulence; and 2) demonstration that the catalase gene, katA, is necessary for survival within macrophages (Day et al., 2000; Konkel et al., 1999; Ziprin et al, 2001). Collaboration on the roles of these and other genes in virulence of C. jejuni will continue.

Multiple NC-1007 stations contributed to the identification of Lawsonia intracellularis, reproduction of proliferative enteropathy (PE) in pigs using isolates from the U.S., and the development of molecular test to identify Lawsonia intracellularis in diagnostic samples. More recently two stations worked to improve molecular diagnostics (Zhang et al., 2000) and that work continues. Traditionally recognized as a disease of pigs and hamsters, PE has recently been recognized by collaborative NC-1007 researchers in horses, ratites and primates (Lawson and Gebhart, 2000; Klein et al, 1999; Lavoie et al, 2000). In swine, PE has become especially important in high health and nucleus herds (Lawson and Gebhart, 2000). Thus, it is important to determine if strains that affect different animal species are the same or different, in order to ascertain if other animals are sources of infection for pigs, and visa versa. Because it is an obligate intracellular bacterium that can be grown only in cell culture, almost nothing is known about its virulence mechanisms (Zhang et al., 2000). This work provide a strong basis for the p to explore cross-reactivity between isolates, virulence mechanisms, improved diagnostics, and development of a mouse model to study pathogenesis.

Salmonella

The main focus of NC1007 research was to understand how S. enterica can persistently colonize the intestinal tract of pigs, to survey antibiotic resistances among S. enterica isolates from animals, and to study the epidemiology of S. enterica in shell fish. A clinical isolate of S. enterica serovar Typhimurium strain 798, which exists as two distinct phenotypes designated adhesive (ON) and non-adhesive (OFF) was used to study the mechanism of persistence. The phenotypes interconvert at a fairly frequent rate consistent with phase variation and can be correlated with adherence to porcine enterocytes and different colony morphologies on Tryptone Phosphate Agar. The strain is not highly virulent in either a mouse model or oral challenge of pigs. Two highly virulent strains in the mouse model were tested for this same phenotypic variation. Both transitioned to OFF phase cells at a rate comparable to strain 798, but reverted to the ON phase at a ~100-fold higher rate, and hence mainly are found in the virulent ON phase. The ability to invade intestinal epithelial cells also was measured and it was found that while strain 798 is less virulent than the other two strains, cells in the ON phase more readily invaded HENLE 407 cells. Using a cDNA based microarray, it was found that cells in the ON phase over-expressed genes encoded on Salmonella pathogenicity island 1, which are known to be involved in virulence, and down regulate the stationary phase RNA polymerase sigma factor gene (rpoS).

Salmonella and Antibiotic Resistance.

In an attempt to find alternatives to antibiotics in production of food animals, KS examined the effects of mannanoligosaccharides (MANN) and sodium chlorate (SC) on growth of pigs before and fecal shedding after challenge with S. Typhimurium (Markey et al. 2004). MANN and SC depressed feed intake and average daily gain. After challenge, the weight gain and feed efficiency of pigs fed carbadox, but not MANN or SC, were significantly better than those fed the control diet. The S. Typhimurium fecal shedding scores of the MANN group were significantly decreased in comparison to the other groups at 7 days; however, 14 days after challenge there was no difference between the groups.

Research by WA NC-1007 workers demonstrated the emergence of epidemic S. Typhimurium DT104 in the United States and documented the important role of cattle in the occurrence of human diarrheal disease with this pathogen (Besser et al., 2000). Subsequently WA has developed novel tools for the analysis of R-plasmids from MDR Salmonellae by comparative genetic content analysis by mixed plasmid microarray (Call et al., 2006). WA workers also used the genetic content data derived from this microarray to characterize the specific genetic element encoding the AmpC class beta-lactamase cmy-2 that is encoded by the currently epidemic S. Newport as well as a diverse array of other Salmonella serovars and commensal E. coli (Kang et al, 2006). WA workers have also demonstrated the emergence of cmy-2 mediated cephalosporin resistance in S. Dublin, of particular significance as it indicates that the selection that resulted in the emergence of this resistance most likely occurred in cattle, the nearly exclusive host of this host adapted serovar (Davis et al., 2006).

Salmonella and Shellfish:

In recent studies conducted on market oysters by Arizona, Salmonella was isolated at a prevalence rate of 7.4%. The majority of the isolates were of the S. Newport serovar with one genotype comprising 98% of the Newport isolates. S. Newport is currently ranked third by CDC in causing human intestinal infections, with an increasing number of S. Newport isolates being highly multi-drug resistant. The genotype isolated from oysters (PulseNet JJPX01.0014) represented 3.1% of all the S. Newport isolates in the National Database and was the first MDR Salmonella AmpC identified within the PulseNet (1999); CDC has isolates of this genotype from salmonellosis cases in 39 states and Canada. This genotype was also identified in dairy cattle. However, recent (2006) follow-up experiments conducted by WA demonstrated the apparent absence of this strain type in WA estuary oysters, and on-going studies will continue this surveillance for a 12 month period. If the strain reappears in WA oysters, follow-up studies will be conducted to identify the likely source of the contaminating strain.

Pathogenic Brachyspira spirochetes

NC1007 researchers documented that, concurrent with the decline of SD, porcine colonic spirochetosis (CS), a less severe form of diarrheal disease of grower pigs associated with B. pilosicoli 1) has became more widely recognized (Hampson and Duhamel, 2006), 2) demonstrates intimate epithelial attachment and invasion of the colonic mucosa by B. pilosicoli resulting in colitis (Duhamel, 2001), 3) infects a wide range of hosts in addition to pigs that are susceptible to colonic infection by B. pilosicoli (Duhamel, 2001; Smith, 2005) and 4) in a macaque model, interacts with certain enterohepatic Helicobacter species to cause colonic epithelial barrier breakdown and host inflammatory and immune response activation similar to ulcerative colitis (Duhamel, et al., 2003). To properly evaluate the role of B. pilosicoli in colitis of humans and animals, further investigations into the epidemiology and host-pathogen interactions of this pathogenic intestinal spirochete are urgently needed.

Routine demonstration of pathogenic Brachyspira species in diagnostic specimens involves immunohistochemical staining of colon with a Brachyspira species-specific mouse monoclonal antibody developed by the KS in collaboration with NE (Fisher, et al., 1997). Bacteriological culture of colon or feces using anaerobic culture on selective agar media (Duhamel and Joens, 1994), and confirmation by amplification of Brachyspira species-specific DNA sequences by the polymerase chain reaction are available only in a few specialized research and diagnostic laboratories in the US (La et al., 2003). To develop improved diagnostic methods, including molecular-based serological tests for rapid detection and removal of sub-clinical carrier pigs and replacement with pigs free of SD, increased understanding of the molecular biology, host immune response, and epidemiology of pathogenic Brachyspira is needed.

Although several potential virulence determinants (motility, hemolysin, and NADH oxidase) have been show to play a role in disease caused by pathogenic Brachyspira species (Hampson et al., 2006; Hampson and Duhamel, 2006), no practical vaccines have resulted from these studies. However, dietary modulation, probably through alterations in synergistic commensal bacterial flora components, can significantly diminish the ability of pathogenic Brachyspira species to colonize and cause disease.

Cryptosporidium parvum and other parasites

Currently, there are no effective treatments for Cryptosporidium parvum infection of humans or livestock. As part of this project, IL has discovered a long-chain unsaturated fatty acid (LCUFA), purified from bovine intestinal mucosa (Johnson et al, 2004) and colostrum, blocks sporozoite infectivity in vitro. This lipid inhibits parasite microneme secretion and resultant gliding motility (Wetzel et al., 2005), processes required for sporozoite invasion of host cells. We will investigate the molecular mechanisms responsible for LCUFA-mediated inhibition of microneme secretion and gliding motility and develop this natural product (or its active derivatives) as a preventative/therapeutic agent for C. parvum induced diarrhea for use in both animals and people. LCUFAs or their active derivatives will be tested in both in vitro and in vivo infectivity experiments that employ novel tissue culture assays as well as bovine intestinal-nude mouse xenografts and whole animal model systems, respectively.

Objectives

1. Focus on emerging diseases- Identify, characterize and develop improved detection methods related to newly recognized, novel or emerging causes of zoonotic enteric disease and enteric pathogens of cattle and swine.
   
2. Focus on effective interventions- Develop and improve interventions and preventative measures to reduce the incidence and prevalence of infections of cattle and swine with enteric and food borne disease agents.
   
3. Focus on disseminating knowledge- Provide training and continuing education opportunities and dissemination of information to students, producers, veterinarians and diagnostic laboratories.
   

Methods

Objective 1: Focus on emerging diseases  Identify, characterize, and develop improved detection methods related to newly recognized novel or emerging causes of enteric diseases and enteric pathogens of cattle and swine A. Enteric viruses OH, KS and MI will identify bovine enteric calicivirus (BEC) and porcine enteric calicivirus (PEC) isolates from bovine and porcine fecal samples or waste lagoon or processed manure samples supplied by NC-1007 diagnostic laboratories (AZ, KS, SD, NE, IA, WA). OH will genetically characterize these isolates and investigate their ability to cause diarrhea and viremia, and to cross-protect against other strains. OH and KS will attempt to adapt the BEC, other PEC or human isolates to cell culture using techniques previously successful for PEC Cowden. OH will use microarray analysis to assess gene expression during in vitro caliciviral replication to identify the various pathways involved in virus adaptation to cell culture. In collaboration with IL, OH will identify and characterize the ABOH histo-blood type antigens on porcine and bovine tissues and assess different calicivirus VLP genotypes for binding to these ABOH types, comparing various NoV strains including human NoVs to predict or confirm the potential for interspecies transmission. OH and KS will use infectious clones of PEC (wild type and cell culture adapted) to determine the molecular basis for calicivirus cell culture infectivity and virulence. OH, AZ and IL will survey shellfish, environmental water sources or unprocessed foods for potential foodborne enteric pathogens and use sequence analysis to infer their host species origin. OH will obtain fecal and nasal samples from NC-1007 diagnostic laboratories (KS, SD, NE, IA, WA) from animals with respiratory and/or enteric disease for identification and/or isolation of new strains of bovine-like toroviruses (BoTV) and bovine-like coronaviruses (BCoV). OH will compare genome sequences and cross-protection among these isolates originating from different syndromes or different hosts to explore their genetic and antigenic relatedness and to identify conserved or host specific mutations that affecttissue or host tropism. OH with input from NC-1007 collaborators will examine the association between BoTV and the various above clinical disease entities. OH will collaborate with NC-1007 members in characterizing strains of porcine coronaviruses from outbreaks of atypical TGEV. OH will determine if the viruses are atypical strains of TGEV with reduced virulence or strains of PRCV that are shed in the feces or newly emerging TGEV/PRCV recombinant strains. OH will study the pathogenicity of selected porcine coronavirus isolates in gnotobiotic or conventional TGEV/PRCV seronegative pigs, and will analyze a portion of the S or other genes to identify the genetic changes that are associated with the biological differences seen clinically. New enteric viruses will be detected in fecal, nasal, lagoon, water, oyster or food samples by a combination of electron microscopy, ELISA and RT-PCR using antisera or degenerate primers, respectively designed by OH or other NC-1007 collaborators. Products will be partially sequenced to confirm their identity and relationships to one another and to reference virus strains to study their potential origin (human vs. animal). In addition, using gnotobiotic pigs or calves, OH will examine the pathogenicity and disease mechanisms for selected new or newly emerging virus strains. Through its characterization of a porcine rotavirus receptor, IL has synthesized a relatively inexpensive neoglycolipid carbomimetic analogue of the active epitope of the intestinal receptor for group A rotavirus, which blocks rotavirus binding to and infection of host cells in vitro. Preliminary results indicate this analogue protects pigs from challenge by the OSU strain of rotavirus. IL and OH will collaborate to test the therapeutic efficacy of this receptor analogue in protecting against various strains of porcine rotavirus. OH will provide a variety of porcine and bovine rotavirus strains and rotavirus-like particles (VLP) with known P and G types to define the receptor specificity and for potential vaccine applications B: Bacterial diseases The incidence of enteric diseases caused by specific pathogens and the genetic make-up of the pathogens themselves evolve over time. Similarly, constant changes occur in livestock management practices, availability and use of vaccines, and animal genetics. Strategies for prevention or management of disease must evolve to keep abreast of disease changes. For example, strains of enterotoxigenic E. coli expressing 987P and STa were very frequent from pigs with colibacillosis during the 1980s (Wilson and Francis, 1986) but have since disappeared while F18/STa/STb±Stx2e strains first recognized in the 1990s have emerged as very common isolate from colibacillosis cases (Francis, 2002; Zhang et al, submitted). Documentation of prevalence changes is critical to understand the underlying processes in order to make management adjustments and modify preventive products licensed or in development appropriately. NC-1007 workers will 1) distribute pathogen typing tools for enteric agent surveillance among cooperating stations; 2) monitor pathogen genotypes to identify trends in prevalence and to detect the emergence of new types; 3) archive strains for comparison with future isolates and for historic assessment of prevalence of virulence determinants yet to be discovered. These efforts will be distributed among stations as follows: SD: -enterotoxigenic E. coli; NE, WA: E. coli O157:H7; MN: L. intracellularis; KS, AZ, WA: S. enterica; AZ: C. perfringens; and WA: C. jejuni and coli. These stations will also archive strains of these species by storage in untracold freezers, liquid nitrogen or lyophilization. Strain source, clinical association, geographical location, and date of collection will be recorded along with genotype information, and the archive content will be published along with the Committees reports. 2. EHEC  on farm control, transmission, runoff and other environmental issues KS will focus on dietary interventions that influence hindgut fermentation to reduce prevalence and shedding of E. coli O157 in cattle, as this site is now recognized as the principal site of persistence or colonization. The working hypothesis for this work is that dietary factors influencing the ecology of the hindgut will have a significant effect on the ability of E. coli O157 to survive and colonize. Grains that are less extensively digested within the rumen may present more starch to the hindgut, and increase fermentative activity among competing species of flora, resulting in increased acid production and a gut environment that is inhospitable for the growth and survival of E. coli O157. Similarly, certain dietary ingredients could make the hindgut more hospitable to E. coli O157. For example, new data suggests that adding distillers grains in cattle diets increases fecal prevalence of E. coli O157. In order to develop intervention strategies to reduce or eliminate pathogen load associated with feeding distillers grains, we need to understand the reasons for the positive association. To evaluate dietary effects on prevalence of E. coli O157: 1) calves will be orally inoculated with NalR E. coli O157 to evaluate the dietary effects on duration and level of fecal shedding; 2) a prevalence study will be conducted in beef cattle in KS in conjunction with ongoing nutritional studies; 3 ) a natural prevalence study will be done using known E. coli O157 infected cattle; and 4) in vitro batch culture fermentations will be performed with rumen microbial or fecal microbial inoculum to determine ingredient and metabolite (fermentation products, pH, etc.) effects on the growth of E. coli O157. Objective 2: Focus on effective interventions  Develop and improve interventions and preventive measures to reduce the incidence and prevalence of infections of cattle and swine with enteric and food borne disease agents A. ETEC, EAEC, and EHEC 1) Enterotoxins, colonization, virulence and secretory response NE, SD and KS will collaborate in efforts to further assess and define the role of EAST1, heat labile enterotoxin (LT) and heat stable enterotoxin-b (STb) in facilitating bacterial colonization of the intestine. E. coli constructs and deletion mutants of each of the relevant toxins (LT, STa. STb, EAST-1) will be tested in IPEC-J2 cells, ligated intestinal loops and orally-inoculated gnotobiotic piglets for the ability to colonize, and cause intestinal secretion and diarrhea using previously utilized methods (Berberov et al, 2004; Zhang et al, 2006). 2) ETEC and EHEC vaccines  role of LT Development of vaccines using attenuated live vaccine strains or mucosal adjuvants capable of stimulating production of mucosal antibodies to pathogen subunit antigens may effectively elicit mucosal immunity in neonatal animals. SD will pursue development of live vaccine constructs for porcine ETEC utilizing reagents and technologies developed by SD, NE and KS. Several stations will pursue subunit vaccine development utilizing E. coli LT as an adjuvant, including SD (ETEC); AZ (C. jejuni) and MN (L. intracelularis). Methods for the purification of sufficient amounts of LT for this purpose developed by SD will be applied by MN and AZ in these efforts. B: Pathogenesis and molecular typing of Lawsonia Infection and transmission of L. intracellularis will be investigated VNTR genetic typing. Though Lawsonia is antigenically conserved, genetic variation exists among isolates. Slight variation exists between isolates from different geographic locations, but this variations is no greater between continents than between Midwestern U.S. pig farms. Marked variation exists between isolates from pig and certain non-pig sources. These variations may be used to track an outbreak occurring in pigs, horses, or other animals. A database of this genetic variation will be generated to provide a basis for further epidemiologic studies, providing bioinformatics data and will further enhancing our understanding of the transmission dynamics and epidemiology of proliferative enteropathy. Serological tests are the most economical tools for diagnosis of proliferative enteropathy in pigs. There is currently no basis for assessing which protein antigens are the best for use in Lawsonia serological testing. MN will characterize virulence and pathogenic factors in the Lawsonia sequence identifiable by sequence homology, and clone and express potential pathogenic or immunogenic genes from Lawsonia in bacteria. Resultant recombinant proteins will be purified and evaluated for use in ELISAs, singly or in combination, in order to improve Lawsonia serologies used in veterinary diagnostic laboratories. In vitro and in vivo studies will be conducted by MN and SD to evaluate factors involved in infectivity of Lawsonia in cell cultures and germ-free pigs, respectively. Resultant models of proliferation will be used to evaluate the mechanism of proliferation of L. intracellularis- infected cells. C: Campylobacter intervention strategies AZ has discovered a novel pilus expressed by C. jejuni upon colonization of abiotic and biotic surfaces during biofilm formation. This pilin (Pil) protein has been sequenced and the gene identified in the genomic sequence of NCTC11168 and a mutant with disrupted pilA has been prepared. Gene complementation in C. jejuni is difficult but possible (e.g., Day et al. (2000). AZ will complement the ?fla ?pilA mutant in trans to restore piliation using a published method (Mixter et al., 2003). To obtain purified protein for further characterization of the pilus and to produce antisera specific for the pilin protein, the pilA gene will be over-expressed in an E. coli expression vector (Dabrowski and Kur, 1999). AZ will examine the ability of the pilus mutant to adhere to epithelial cells and to colonize chicks. NCTC 11168, a human clinical strain, nevertheless has been shown (Gaynor (2004) to be able to colonize chicks. Finally, we will identify genes involved in biofilm formation using expression arrays. Genes up-regulated during biofilm formation will be mutated and pilus expression will be examined by electron microscopy and through in vitro biofilm formation to allow us to fully characterize the pilus formation by identifying genes involved in regulation and pilus expression. D: Brachyspira spp. In light of the re-emergence of SD in the US swine herd, the potential zoonotic role of B. pilosicoli in human colitis, and potential loss of effectiveness of antimicrobial agents for control of pathogenic Brachyspira, NE will 1) closely monitor the antimicrobial sensitivity of clinical isolates, 2) improve methods for identification of pathogenic Brachyspira in clinical specimens, and 3) determine the complete genomic nucleotide sequences of representative B. hyodysenteriae and B. pilosicoli. To monitor antimicrobial resistance in B. hyodysenteriae, the susceptibility of clinical isolates obtained from pigs from different regions of the US to the five most commonly used antimicrobial agents, NE in collaboration with the IA, MN, and NC will use a newly validated broth dilution susceptibility method (Karlsson et al., 2003). Molecular epidemiologic studies of B. hyodysenteriae will utilize a multilocus sequence typing (MLST) method recently developed by the NE will be used for allelic profiling of pathogenic Brachyspira obtained from different hosts in different regions in the US. E. Viral receptors and intervention OH will collaborate with other NC-1007 members to improve the efficacy of current rotavirus vaccines and to develop new rotavirus and calicivirus vaccines (virus-like particles and new adjuvant and delivery systems) for use in animals to reduce the presence of animal rotaviruses and caliciviruses in unprocessed food (oysters, etc) or as environmental contaminants. OH will use monoclonal antibodies to porcine immunoglobulins provided by IA, and monoclonal antibodies to bovine immunoglobulins provided by NE, to define the humoral and cellular immune responses to various group A rotavirus serotypes and enteric caliciviruses from swine and cattle. Such vaccine studies will provide animal models for similar vaccine approaches in humans. Comparisons of innate and adaptive mucosal immunity will be done using monoclonal antibodies in ELISA and ELISPOT assays to measure B cell responses (antibodies, antibody secreting cells), and T cell responses (cytokines and cytokine secreting cells, and lymphoproliferative responses), respectively in normal, diseased, and vaccinated animals. A new emphasis will be on comparative studies of innate immune responses to different enteric viruses and their role in decreasing viral infection and inducing adaptive immunity. OH will collaborate with IA and MN in comparing immunologic reagents and assays to quantitate innate, humoral and cellular immune responses in swine. These studies will be invaluable in defining the role of the host immune responses in disease pathogenesis and for the evaluation of existing as well as novel rotavirus and calicivirus vaccine approaches. IL, in collaboration with OH, will determine the in vivo deliver parameters necessary for optimal therapeutic efficacy of the new synthetic neoglycolipid carbomimetic rotavirus receptor (SLPE). This will include: dosage, time, frequency of administration, delivery vehicle, intestinal survival, transport, and absorption kinetics, as well as activity against different field strains of porcine rotavirus. These studies will define the dosage parameters necessary for achieving maximum benefit of receptor therapy as a treatment for porcine rotavirus disease. IL will also continue to pursue possible synergistic therapeutic effects of SLPE in combination with milk/colostrum oligosaccharides and dietary isoflavones for the prophylaxis/treatment of porcine rotavirus diarrhea. IL also will continue its work on defining the kinetics of overland transport and fate of rotavirus under different environmental conditions including the use of vegetative filter strips to reduce or eliminate virus contamination of agricultural watersheds. F: Parasitology IL will continue its work defining the mechanism of Cryptosporidium parvum sporozoite interaction with and invasion of host cells using two approaches. One is to identify naturally occurring molecule(s) that mediate sporozoite recognition or invasion and the other focuses on defining the cellular and molecular mechanisms by which one such molecule, LCFA, block microneme secretion, gliding motility and host cell invasion. IL is also in continuing epidemiologic and overland transport studies with C. parvum in collaboration with the Agricultural Engineering Department at the University of Illinois. These studies are investigating the stability of Cryptosporidium parvum oocysts and in dairy fecal waste, its potential contribution as run-off to contamination of municipal drinking water and the use of vegetative buffer strips to reduce this overland transport. IL will investigate the use of LCFA as a therapeutic agent for treatment of cryptosporidiosis using whole animal and intestinal xenograft animal models (Thulin et al., 1994; Gelberg et al., 1997). IL has identified a set of colostrum- derived LCUFAs that can block binding of C. parvum, and they will test the ability of this lipid to prevent colonization by C. parvum in intestinal xenographs and newborn calves. IL will evaluate the impact of dietary distillers dried grains with solubles (DDGS) on young pigs experimentally infected with Escherichia coli and on young chicks challenged with the coccidial parasite species, Eimeria acervulina. The results will be directly relevant to pigs and chickens and to this particular feed ingredient (DDGS), but will also provide broader insight into the use of dietary fiber in the control of enteric diseases in other species, including people. If these results are promising, experiments will also be initiated to test the efficacy of DDGS as an anti-cryptosporidial dietary supplement for dairy calves. The anticipated diversion of huge amounts of U.S. corn to ethanol production and the associated availability of enormous quantities of DDGS for use in animal feeds is a dramatic trend expected to result in large-scale changes in U.S. agriculture. We must find means to replace the corn starch that is diverted from animal feeds to ethanol and we must find ways to use the DDGS. . Objective 3. Focus and dissemination of knowledge  Provide training and continuing education opportunities and dissemination of information to student, producers, veterinarians, and diagnostic laboratories In order to educate the public, animal care professionals, students and fellow scientists, this multi-state committee and its cooperating Experiment Stations will provide 1) training to college undergraduate and graduate students; 2) information to livestock producers and/or professionals ; 3) knowledge and continuing education to station representatives and collaborating scientists and 4) a forum for scientific exchange among colleagues of the international scientific community, and dissemination of knowledge to the biologics industry. Training to college students will occur through dissemination of knowledge through formal institutional course offerings as occasion permits in the several universities represented by this multistate committee. This is an ongoing activity participated in by many NC-1007 members, but not previously documented. Instruction would include discussions of etiologies, prevalence, mechanisms in pathogenesis of disease, and dissemination of organisms. Working in cooperation with institutional Agricultural Extension personnel, station representatives will also provide information for and/or participate in the the dissemination of knowledge regarding livestock enteric diseases to interested producers and/or animal health providers through continuing education and/or extension programs or bulletins. In augmentation of this activity, the NC-1007 committee will assemble a lay document describing major advances in knowledge and technology through research in emerging infectious diseases, with livestock producers, veterinary professionals, and consumers as the target audience. The document will be formatted for dissemination in printed form or over the World Wide Web. Representatives from each participating station will be responsible for submitting pertinent information to the Chair of NC-1007, who will appoint an editing committee to assemble information of the highest perceived public interest from individual stations for inclusion in the document. After completion, the document will be emailed to individual station representatives who will disseminate it and pertinent in-state research findings through their respective states Cooperative Extension offices and other producer/consumer education entities for their information and use. To meet continuing educational needs of NC-1007 members, interested students and colleagues, we also propose to sponsor a mini-symposium to be held in conjunction with the Conference of Research Workers in Animal Diseases. Speakers will be chosen from among Station representatives and/or will be solicited from among external experts. This symposium will provide knowledge and continuing education to station representatives and their students and other animal and food safety infectious disease researchers. The committee will solicit grant funding and sponsorship by industry partners to support attendance at the symposium and expand attendance at the annual meeting. Graduate students will be invited and encouraged to present their research findings and to participate in research discussions. This will serve as a training experience for young scientists, and ensure their preparation to assume leadership roles in infectious disease research of importance to animal agriculture. Finally, we will sponsor the Fourth International Rushmore Conference on Enteric Disease to be held in the fall of 2010 or 2011. The Rushmore Conferences, held in Rapid City, SD provide a forum for scientific exchange among colleagues of the international scientific community, and the dissemination of knowledge to the biologics industry. This very successful conference series draws investigators and other personnel from around the work and represents perhaps the only forum for the exclusive discussion of enteric diseases with counterparts in humans and animals. Topics will likely include mechanisms of pathogenesis, diagnosis, and strategies for management of diarrheal disease and the prevention of the dissemination of food-borne enteric pathogens. Funding for the conference will be sought through a USDA-NRI conference grant, and from industrial sponsors.

Measurement of Progress and Results

Outputs

• Obj 1 Enteric viruses: Identify and characterize bovine and porcine enteric calicivirus isolates obtained from station diagnostic laboratories
• Obj 1 Enteric viruses: Survey shellfish, water sources, and unprocessed foods for foodborne enteric viral pathogens
• Obj 1 Enteric viruses: Characterize porcine coronaviruses from atypical TGE outbreaks
• Obj 1 Enteric viruses: Identify new enteric viruses in fecal, nasal, environmental and food specimens
• Obj 1 Enteric viruses: Test therapeutic efficacy of RV receptor analogues
• Output 6: Obj 1 Bacterial diseases: Surveillance for strain type turnover among enteric bacterial pathogens
• Output 7: Obj 1 Bacterial diseases: Archive collection of bacterial pathogens and establish database of strain information
• Output 8: Obj 1 EHEC: Evaluate feed components that affect hindgut fermentation for effect on EHEC O157 fecal shedding
• Output 9: Obj 2 ETEC: Assess and define the roles of enterotoxins in facilitating bacterial intestinal colonization
• Output 10: Obj 2 ETEC vaccines: Develop and evaluate vaccines using LT as an adjuvant
• Output 11: Obj 2 Lawsonia: Establish a database of genetic variation within L. intracellularis
• Output 12: Obj 2 Lawsonia: Develop and optimize L. intracellularis ELISA serology using recombinant antigens
• Output 13: Obj 2 Campylobacter: Evaluate pilA effects on Campylobacter adherence to epithelial cells and to colonize chicks
• Output 14: Obj 2 Brachyspira: Develop improved methods for Brachyspira diagnosis
• Output 15: Obj 2 Enteric viruses: Develop improved efficacy RV vaccines for domestic animals
• Output 16: Obj 2 Enteric viruses: Optimize use of a synthetic neoglycolipid carbomimetic rotavirus receptor for blocking RV infection
• Output 17: Obj 2 Parasitology: Identification and characterization of naturally occurring molecules that interfere with Cryptosporidium parvum infection
• Output 18: Obj 3: Train undergraduate and graduate students, livestock producers, and animal health professionals regarding enteric pathogens and food safety
• Output 19: Obj 3: Organize and conduct a symposium on enteric pathogens and food safety to be conducted in concert with the Conference of Research Workers in Animal Diseases
• Output 20: Obj 3: Sponsor, organize and conduct the Fourth International Rushmore Conference on Enteric Disease.

Outcomes or Projected Impacts

• Discovery and characterization of novel enteric calicivirus(es) of domestic animals offers a better understanding of the etiology of diarrheal disease in domestic animals potentially treatable or preventable with drugs or vaccines.
• Shellfish, water and foods surveillance may identify novel important routes of human exposure
• Atypical TGE may require novel current preventative methods and therefore may offer disproportionate costs.
• Emergence of novel enteric viruses will provide new targets for prevention and therapy.
• RV receptor analogues may provide alternative more effective treatment and preventative methods
• Outcome/impact 6: Strain turnover in enteric bacterial pathogens is hugely important in the epidemiology and impact of the resulting diseases; surveillance may identify emerging strains important for human disease along with their principal reservoirs which may allow novel interventions for prevention or treatment. Outcome/impact 7: An archive of pathogen isolates and a database of associated disease traits will allow retrospective research regarding strains, virulence factors, antimicrobial resistance traits, and other characteristics which has the potential to identify novel biologic processes and more effective interventions. Outcome/impact 8: Due to the emerging understanding of the importance of the recto-anal junction colonization site of E. coli O157:H7 (due in part to researchers in this multistate project), identification of feeds with specific effects at this colonization site may provide effective pre-harvest interventions that could significantly lower the load of this pathogen carried by cattle into abattoirs or shed into the environment, reducing infection pressure on humans. Outcome/impact 9: The role of enterotoxin in facilitating intestinal colonization with ETEC (due to researchers in this multi-state project), could result in key new understanding of the basis of intestianl colonization with this pathogen group. Outcome/impact 10: LT adjuvants may greatly increase efficacly or reduce dosing requirements of ETEC vaccines. Outcome/impact 11: The database of genetic variation in Lawsonia intracellularis is essential to furthering our understanding of this economically important pathogen due to the difficulty of obtaining isolates by culture techniques. Outcome/impact 12: ELISA serology with recombinant antigens will bypass the difficulties in obtaining and amplifying pure Lawsonia antigens by cultures. Outcome/impact 13: PilA, if confirmed important in adherence to cells and to colononize chick will validate this protein as a novel vaccine candidate. Outcome/impact 14: Improved diagnostic methods are required to determine the true impact of this pathogen. Outcome/impact 15: An improved rotavirus vaccine for domestic animals is required due to the repeated demonstration of the limitations of currently available vaccines and the prevalence of this pathogen as a cause of neonatal morbidity and mortality. Outcome/impact 16: Synthetic neoglycolipid carbomimetic rotavirus receptors, if effective, will provide a novel therapeutic approach where none currently exists and in addition offer novel prophylactic methods. Outcome/impact 17: Naturally occurring molecules that interfere with the intestinal binding and colonization of Cryptosporidium parvum will again offer a potentially highly effective preventative / treatment method where none currently exist. Outcome/impact 18: Training undergraduate and graduate students, livestock producers, and health care professionals will provide future biomedical researchers to address emerging novel infectious disease threats, ensure that pre-harvest food safety and animal health interventions will protect against animal health threats, improve marketability and reduce the risks of zoonotic diseases. Outcome/impact 19: Symposia on enteric pathogens and food safety in the context of the CRWAD meeting enhances cooperation and collaboration within this multistate project an

Milestones

(2008): Identify novel enteric caliciviruses. Demonstrate efficacy of rotavirus receptor analogues in cell culture systems. Determine the role of LT and other enterotoxins in ETEC intestinal colonization. Quantify the effect of pilA in Campylobacter adherence to epithelial cells. Identify naturally occuring molecules that block binding of Cryptosporidium parvum to intestinal cells.

(2009): Identify the frequency of enteric viral pathogens in shellfish, water and food. Identify strain turnover in enteric bacterial pathogens. Develop LT adjuvanted vaccines. Improve diagnostic methods for Brachyspira. Training of graduate and undergraduate students is in progress, and outreach activities regarding food safety are initiated and underway.

(2010): Determine the characteristics of porcine coronaviruses from atypical TGE. The archive collection of bacterial pathogens will be established and a database of strain information has been created. Describe the range of genetic variation in Lawsonia intracellularis. Develop improved efficacy rotavirus vaccines for domestic animals. Organize and present CRWAD symposia on enteric pathogens and food safety.

(2011): Discover novel enteric viruses in animal feces, nasal secretions, the environment and foods. Characterize hindgut fermentation effects of feed components that may affect E. coli O157:H7. Develop recombinant antigen based ELISA for Lawsonia intracellularis. Test use of synthetic neoglycolipid rotavirus receptors for blocking RV infections. Convene the 4th International Rushmore Conference on Enteric Disease.

Projected Participation

View Appendix E: Participation

Outreach Plan

The outreach plan of this committee is incorporated into this proposal as Objective 3.

Organization/Governance

The recommended Standard Governance for multistate research activities include the election of a Chair, a Chair-elect, and a Secretary. All officers are to be elected for at least two-year terms to provide continuity. Administrative guidance will be provided by an assigned Administrative Advisor and a CSREES Representative.

Literature Cited

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Andres A, Donovan SM,. KuhlenschmidtTB, and. Kuhlenschmidt MS. Genistin at the Concentration Present in Soy-Based Infant Formula Inhibits Rotavirus Infectivity in vitro Through Inhibition of Protein Tyrosine Kinase Activity. American Oil Chemists Society Sixth Annual International Symposium on the Role of Soy in Preventing and Treating Disease, Chicago, IL. October 30-Nov. 2, 2005.

An, H., J. M. Fairbrother, C. Desautels, and J. Harel. 1999. Distribution of a novel locus called Paa (porcine attaching and effacing associated) among enteric Escherichia coli. Adv. Exp. Med. Biol. 473:179-184.

Armstrong, G. L., J. Hollingsworth, and J. G. Morris. 1996. Emerging food-borne pathogens: Escherichia coli O157:H7 as a model of entry of a new pathogen into the food supply of the developed world. Epidemiol. Rev. 18:29-51.

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Attachments

Land Grant Participating States/Institutions

AZ, GA, IL, KS, KY, MI, MN, ND, NE, OH, TN, WA

Non Land Grant Participating States/Institutions

Iowa State University - College of Vet Med, Southeast Poultry Research Laboratory, Texas Tech University