Duration: 10/01/2012 to 09/30/2017

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

NEEDS. The long-term goal of this collaborative project is to prevent and control enteric diseases of cattle, swine and chickens with a mandate to decrease food and waterborne illness in the USA. Foodborne illness has been a prominent public health concern for over two decades yet the Centers for Disease Control (CDC) still list many enteric foodborne pathogens as leading causes of morbidity and mortality in the US. Despite many concerted efforts to use hygiene and sanitation measures to control these pathogens in food animals pre- and postharvest, incidence of many food and waterborne pathogens remains high and some are increasing. Nevertheless, a broad range of educational, scientific and practical controls have succeeded in decreasing the incidence of five key foodborne pathogens. Over the last 10 years, our enteric diseases group has been a part of that effort and we are dedicated to prevent and control animal and human disease due to enteric pathogens. Our collaborative efforts harmonize with national efforts established this year under the FDA Food Safety Modernization Act to ensure the US food supply is safe by shifting the focus of federal regulators from responding to contamination to preventing it. A main avenue for prevention is decreasing carriage and disease due to enteric pathogens in food animals.


Foodborne outbreaks. Foodborne illness is a major public health concern in the US due to the occurrence of many large-scale outbreaks with intense media scrutiny(9). From 2009 to 2011, outbreaks of Salmonella occurred from exposures to animal products (Italian style meats, ground turkey, bologna, shell eggs), produce (alfalfa sprouts, cantaloupes, fresh papayas, pistachios), processed foods (peanut butter, frozen entrees, frozen fruit pulp), animals (ducklings, frogs, chicks), restaurant chains, and microbiology laboratories. These included many strains of Salmonella including: Enteritidis, Chester, Typhi, Hartford, Baildon, Newport, Montevideo, Typhimurium, Heidelberg, Agona, Altona, Johannesburg, Hadar, Panama, Saintpaul and several untyped Salmonella. Outbreaks of disease due to Shiga toxin-producing (STEC) E. coli have also occurred during this time after exposures to beef, Lebanon bologna, cheese, prepackaged cookie dough, romaine lettuce, hazelnuts, and travel to Germany. In these outbreaks, strain types were mainly STEC E. coli O157:H7 but, non-O157 strains e.g.O104 also caused outbreaks. Other multistate foodborne outbreaks included Listeria linked to cantaloupes and Campylobacter linked to raw milk. Most exposures involved contaminated meats, milk or cross contamination of other foods from meat or animals. Moreover, the strain diversity and allelic variation expressed by bacterial isolates from these outbreaks demonstrates the complexity of sources contributing to these problems.


Sporadic foodborne illness. In 1995, improved surveillance for foodborne illness was instituted when Food-Net was established; this is a collaborative program among CDC, 10 state health departments, USDA FSIS, and FDA. Food-Net conducts surveillance for bacterial (Campylobacter, Listeria, Salmonella, STEC E. coli O157 and non-O157, Shigella, Vibrio, and Yersinia) and parasitic (Cryptosporidium, Cyclospora) infections diagnosed by laboratory testing of patient samples. Initial work by Food-Net demonstrated a huge US burden of disease with >75 million cases of foodborne illness annually, resulting in 325,000 hospitalizations and 5,000 deaths. By 2011, FoodNet reports 48 million illnesses, 128,000 hospitalizations, and 3,000 deaths annually from foodborne infections. Despite encouraging declines, annual rates of disease due to enteric pathogens are high: Campylobacter (2 million cases), Salmonella (1.4 million cases), STEC E. coli O157 and non-O157 (73,480 cases), Listeria (2,797 cases), Shigella (1,780 cases), Vibrio (193 cases), Yersinia (159 cases), Cryptosporidium (1,290 cases), and Cyclospora (28 cases)(58). It is not surprising that the President, Congress, and USDA have made food safety a high priority. Statistics from FoodNet surveillance highlight three main 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. Recent recognition of norovirus induced food and waterborne illness is a striking reminder that agents of major importance can go unrecognized for years. 2) Most of the known bacterial, viral and parasitic foodborne disease agents are primarily zoonotic. Thus, investigation and control in animal reservoirs are required to understand their epidemiology and biology to maximize opportunities for control. 3) Several of these agents are also severe pathogens of animals or have close relatives that are animal pathogens. Thus, investigation of the host-parasite relationship in animal models or in animal populations could solve these problems in humans.


Chronic disease. While most foodborne pathogens cause acute disease, many of them can cause severe complications or chronic diseases. Severe manifestations include hemorrhagic colitis, septicemia, meningitis, joint infection, hemolytic uremic syndrome with kidney failure, paralysis and miscarriage, among other diseases. Autoimmune disorders are rapidly increasing in incidence(3) and a number of these syndromes are documented to be triggered by enteric pathogens. For example, C. jejuni is a leading cause of bacterial gastroenteritis that can trigger serious autoimmune diseases. The acute neuropathies Guillain Barré Syndrome and Miller Fisher Syndrome, and Inflammatory Bowel Disease and Reiters Arthritis have all been associated with recent Campylobacter infection(44).


Waterborne illness. The CDC, EPA, and Council of State and Territorial Epidemiologists run the Waterborne Disease and Outbreak Surveillance System for collecting and reporting data on waterborne disease outbreaks associated with drinking and recreational water(72). A report from 2005-2006 showed 78 waterborne disease outbreaks in 31 states with 4,412 persons affected resulting in 116 hospitalizations and five deaths. 61% of these were enteric illness with the majority caused by parasites, viruses and bacteria. The agents of highest prevalence were Cryptosporidium, Vibrio, Campylobacter and Naegleria. Conclusions were that there was a substantial increase in number of recreational water-associated diseases and outbreaks compared to previous years. Recent increases in disease in marine animals exposed to ground water runoff containing animal and human enteric pathogens points out the need to manage both human and animal wastes(63). A major long-term goal of our group is to 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, Sapoviruses) cause over 9 million cases of foodborne illnesses yearly, making them the most common cause of acute foodborne gastroenteritis in the US(40). 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(10, 11, 23, 36, 60, 66, 68-70). Moreover, shellfish approved for human consumption contain both animal and human enteric caliciviruses(10).


Food animals harbor enteric pathogens. STEC. Cattle are important reservoirs of E. coli O157:H7 because the organism colonizes the colon without causing disease and exhibits a tissue tropism for the rectum of adult cattle(46, 55). In contrast, other serotypes (O5, O26, O111) colonize the entire large intestine of young calves(61). Intestinal colonization by E. coli O157:H7 and other EHEC requires the formation of A/E lesions, mediated through proteins secreted by a type III secretion system and the outer membrane protein known as intimin(12, 41, 47, 52, 67). Colonization of the colon in cattle results in shedding of the organism in feces(41, 54, 62). 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(1, 15, 62). Although post-harvest interventions have been implemented and significantly reduced E. coli O157 contamination of ground beef(2), the organism is still highly prevalent in US cattle and pre-harvest interventions are needed to reduce carriage levels(7, 33).


ETEC. Enterotoxigenic E. coli cause diarrhea in neonates (piglets, calves and lambs) and young animals (piglets) by adhering to intestinal epithelial cells and producing enterotoxin(45). ETEC cause death of 10.8% of all pre-weaning pigs and 1.5-2% of all weaned pigs(25, 65). Incidence of neonatal diarrhea has been reduced substantially using vaccines, but post-weaning ETEC diarrhea remains economically significant for the swine industry. Common ETEC diarrheal strains in piglets produce K88 (F4) or F18 fimbriae(18). These fimbriae bind to glycoconjugates that serve as receptors in porcine enterocyte brush borders. Absence of the respective glycoconjugate renders animals resistant to bacterial colonization and diarrheal disease(16, 17, 19). ETEC strains produce several types of enterotoxins, including heat labile enterotoxin (LT), heat stable enterotoxin-a (STa), heat stable enterotoxin-b (STb)(45), and enteroaggregative E. coli produce heat stable enterotoxin 1(39, 57, 71). ETEC must produce both enterotoxin and fimbriae in order to cause severe dehydrating diarrheal disease(4, 19, 59). 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 correlates with expression of an intestinal mucin-type glycoprotein receptor for K88+ fimbria(19, 22). K88+ ETEC strains are extremely virulent because of high intestinal colonization, severe dehydrating diarrhea, post-diarrheal septicemia, and death(42). Post-diarrheal septicemia involves the development of severe dehydration, hypovolemic shock, and ischemia of the intestinal mucosa(4, 42).


Salmonella. Foods of animal origin are commonly contaminated with Salmonella spp. including the food animals that we study (cattle, swine, chickens). Salmonella enterica is a common cause of systemic and diarrheal disease in livestock with economic losses estimated to be $12 billion; it causes ~2 million cases of diarrhea per year in humans in the US with up to 1000 deaths. Many strains of Salmonella have recently emerged. In 2000-2005, the CDC noted a rapid increase in laboratory confirmed Salmonella Newport infections (126%). Concern was raised because: 1) the spectrum of illness due to this serovar tends to be more severe, and 2) an increasing number of Newport isolates are 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 was attributed to various factors including intensive farming practices, movement of cattle between farms and overuse of antimicrobial agents on dairy operations(24).


Campylobacters. Incidence of foodborne disease due to C. jejuni remains very high worldwide; chicken is the most common source(20, 28). Yet, a recent study showed using multilocus sequence typing that water and milk are commonly contaminated with C. jejuni and that sporadic infections in humans may arise from sources other than chickens(32). It is well known that cattle and swine shed C. jejuni, C. coli and often other campylobacters at slaughter. Raw milk from dairy cattle has caused outbreaks of C. jejuni enteritis (29, 30, 50); recent molecular testing showed C. jejuni, E. coli, Salmonella Typhimurium, Listeria and Yersinia in raw milk(21). Besser et al showed transmission of C. jejuni among feedlot cattle during the feeding period that resulted in a high prevalence of excretion of this pathogen by cattle slated to go to slaughter (5). Unfortunately, chlorination of water did not decrease this problem. C. jejuni has also been associated with weaning age diarrhea in swine(35, 56). Pigs harbor both C. coli and to a lesser extent C. jejuni, but recent data show that both cause human enteritis; in fact C. coli predominates as a cause in some regions and other campylobacters can also causes disease(53, 64). Another growing problem is the increasing prevalence of antibiotic resistant campylobacters. A disturbing trend is that enhanced fitness in the host was observed for fluoroquinolone-resistant C. jejuni in the absence of antibiotic selection pressure(34). So, despite the removal of fluoroquinolones as feed additives for chickens, this antibiotic resistance has persisted and increased.


Lawsonia. Proliferative enteropathy (ileitis) is a common enteric disease of weaned pigs and other animals caused by an intracellular bacterium, L. intracellularis(37). Infections are common and estimates of annual economic losses are ~$100 million for the US swine industry(38). Clinical signs include diarrhea, weight loss, and melena(38).Characteristic lesions in all species are marked proliferation of immature epithelial cells in crypts of the ileum or colon, or both, leading to thickening and branching of crypts and gross mucosal thickening with intracellular bacteria(38). Little is known of the pathogenesis and sensitive and specific methods for diagnosis are not universally available. Genomic characterization of the organism will help to identify genes responsible for virulence and to develop diagnostic reagents and recombinant vaccines. A molecular typing database will enable studies on ecology and epidemiology of L. intracellularis.


Spirochetes. 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 Brachyspira hyodysenteriae and Brachyspira pilosicoli, respectively(26, 27, 51). SD has devastating economic impacts on pig production, but changes in management designed to eliminate SD has produced a declining prevalence in US swine. Yet, in most pig producing countries SD continues to be a major health challenge. In contrast, porcine CS, a less severe form of diarrheal disease of grower pigs, has become more widely recognized. While SD is restricted to pigs and rodent vectors, CS affects a wide range of hosts including human and non-human primates, dogs, horses, and birds(13, 14, 43). Although limited epidemiological investigations suggest a zoonotic potential with public health significance for B. pilosicoli (6, 48, 49), the role of this spirochete in human colitis is uncertain.


Cryptosporidium. Cryptosporidium is the most common enteric pathogen of calves and is responsible for significant economic losses to the dairy and cattle industries. It is also a significant threat to water resources and human health. In 1991, a National Dairy Heifer Evaluation Project 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 can excrete up to 10 7 oocysts per gram feces. Damage to host intestinal epithelium during infection results in a decrease in absorptive surfaces, gut mucosal inflammation, secretion of electrolytes into the lumen, and persistent diarrhea. In 2011, there are no effective treatments for humans or livestock with Crypto.


IMPORTANCE & CONSEQUENCES. In 2000, USDA ERS estimated $6.9 billion/year for medical costs, productivity losses, and costs of premature deaths for diseases caused by five common foodborne pathogens.


Besides human health risks, animal diarrheal disease due to food-safety related pathogens and other animal-specific pathogens remain an economically important cause of production loss to livestock producers. Recent surveys from NAHMS and the National Pork Producers Council indicate the continuing importance of enteric diseases as major sources of morbidity, mortality, and economic costs. The cost of just E. coli O157:H7 to the beef industry from 1993-2003 was estimated at $2.671 billion(31). The 2000 National Water Quality Inventory reports that agricultural non-point-source pollution is the leading source impacting water quality in surveyed rivers and lakes. It is also a major contributor to ground water contamination, wetlands degradation and human illness from waterborne pathogens. Yet, Food-Net indicates significant progress in control of illness caused by Campylobacter, Salmonella, E. coli, Listeria, Cryptosporidium and Yersinia. These declines are the result of diverse actions such as pre- and post-harvest interventions and education of producers and consumers. While the incidence of disease caused by some of these agents approaches 2010 targets, rates can be further reduced with new knowledge and new detection procedures developed through this collaborative research. As production systems for food animals evolve toward larger sizes and complexity, antibiotics in feed become banned and natural, grass-fed, and organic production systems emerge and expand, continued research in support of food safety and control of diarrheal diseases of livestock is needed to optimize animal health and welfare and to produce safe foods. Consequences of inaction are increases in disease and costs.

FEASIBILITY. Progress has been made in preventing and controlling foodborne illness. A recent report by CDC based on 1996-2010 data concluded that the incidence of several high impact pathogens has declined based on targeting them for control and prevention(8). Declines in incidence occurred in Campylobacter, Listeria, STEC O157, Shigella, and Yersinia infection, while Salmonella and Vibrio increased. CDC believes this success demonstrates the feasibility of preventing foodborne illnesses; research, collaboration and dissemination of successful innovations will be important to continue this trend.

MULTISTATE EFFORTS. The magnitude of this problem dictates a team-based approach to devising and implementing preventatives. People with markedly varied areas of expertise are needed to devise scientific strategies for pathogen control, for education of agricultural experts and producers and for applying the strategies on farms. 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 we offer. The NC-1041 group has bacteriologists, virologists, molecular biologists, pathologists, and immunologists with a history of collaboration and productivity in developing innovative strategies.


IMPACTS, INNOVATION, OUTCOMES. 1)Emerging diseases. We expect to identify, characterize and develop improved detection and prevention methods related to newly recognized, novel or emerging causes of zoonotic enteric disease and enteric pathogens of food animals. 2)Developing preventions & interventions. We expect to develop and improve preventative measures and interventions to reduce the incidence and prevalence of infections of food animals with enteric and foodborne pathogens. 3)Disseminating knowledge. We will provide training or continuing education to disseminate new information to students, producers, veterinarians, diagnostic labs and others to implement interventions and preventative measures. Expected outcomes will be increased understanding of mechanisms of initiation of acute and chronic enteric infections for known and emerging enteric pathogens. This will provide science-based best practices and implementation strategies for preventive measures and interventions for the major enteric diseases of food animals. The new NC-1041 project addresses critical cross-cutting research areas and objectives that will enhance food safety while maintaining efficient pork, beef and chicken production.

Related, Current and Previous Work

Our NC1041 working group is focused on bacteria, viruses and parasites that cause enteric disease in food animals. Many of these pathogens are zoonotic and cause foodborne illness. In some, fecal carriage without disease still poses a significant risk to human health. Over 5 years, we published over 281 scientific manuscripts, held annual NC1041 meetings collaborating with CRWAD, held a Rushmore meeting and trained people at all levels of sophistication to control these diseases. This section shows a small selection of projects to demonstrate our productivity and show our follow-up strategies.

BACTERIA:

Campylobacter jejuni colonization factors (Rajashekara Lab).

C. jejuni is a major foodborne pathogen and exhibits a remarkable capacity for survival and adaptation in humans, animals and the environment (61). Chickens and cattle are major sources of C. jejuni. Thus, understanding physiological and genetic properties that allow C. jejuni to survive and adapt to various stress conditions is crucial for therapeutic interventions. Critical gaps exist in our understanding of mechanisms used by C. jejuni to colonize chickens, which limits ability to design and implement effective control measures. We showed that the twin-arginine translocation (TAT) pathway is an important virulence mechanism in many bacterial pathogens including C. jejuni (16, 27, 42);the TAT system is an important C. jejuni pathogenicity/colonization determinant. Experimental identification of proteins that are translocated through the TAT system is critical to define the basis for its contribution to C. jejuni physiology and virulence. Similarly, polyphosphate and its associated enzymes (PPK1 and PPK2) play important roles in stress survival and adaptation and may contribute to genome plasticity, and spread and development of antimicrobial resistance in C. jejuni (7, 18, 19, 26). Absence of homologs of TAT, PPK1 and PPK2 proteins in humans and animals show the potential of these proteins as novel targets for therapeutic interventions.

C. jejuni epidemiology in cattle (Rajashekara Lab).

Although it is known that chickens constitute a major reservoir for Campylobacter, the occurrence of these pathogens in cattle and their potential impact on human health remain largely uncharacterized. Campylobacter associated with cattle also pose indirect risks, including the contamination of surface and ground waters with waste run-off from cattle farming and processing, which further stresses the importance of understanding the role of cattle in the dissemination of these pathogens (39, 47, 56). Our recent studies suggest that cattle are a reservoir for clinically important Campylobacter. Thus, identifying genetic relatedness will enhance understanding of the epidemiology of Campylobacter in cattle, resulting in better risk assessment for humans consuming contaminated cattle products.

Epidemiology of ruminant C. jejuni clone SA (Zhang Lab).

Poultry are a major food vehicle for transmission of C. jejuni. However, our recent work has identified a ruminant C. jejuni clone that is emerging as a significant foodborne hazard in the US. (45). This clone (named clone SA) was first identified in sheep by our group and has recently become the predominant cause of ovine abortion in the US (45). This clone is highly pathogenic in pregnant ruminant animals and is able to spread systemically as a result of oral ingestion. Our ongoing collaboration with the CDC has revealed that C. jejuni clone SA is increasingly associated with outbreaks (related to raw milk) and sporadic cases (unknown sources) of foodborne gastroenteritis in humans. The increasing importance of C. jejuni clone SA in sheep abortion and foodborne diseases underscores the need for enhanced efforts to reduce its prevalence and transmission. Additionally, a rapid detection and identification method is needed to facilitate the control of this zoonotic clone.

C. jejuni virulence factors (Mansfield Lab).

Our lab developed several mouse models that are useful for studying how C. jejuni colonizes and causes disease(7, 28, 38-40). We have examined responses of C57BL/6 IL-10-/- mice to infection with 22 genetically varied C. jejuni strains originating from cows, chickens, and human cases having a variety of clinical presentations; strains in this group caused enteritis, bacteremia, meningitis, and enteritis followed in some by the neurological diseases Guillain Barré Syndrome (GBS) or Miller Fisher Syndrome (MFS)(7, 38, 39). Data on 7 strains is published (5); these included two non-colonizing strains, one strain that colonized but did not cause enteritis, and four strains that both colonized and caused enteritis. Furthermore, mice shifted to a lower fat diet two weeks prior to infection with most C. jejuni strains were shown to experience more severe disease(5). Twelve strains, including strain 11168, were able to cause enteritis severe enough to require early euthanasia. Only strains from patients with GBS or MFS produced neurological disease in mice. In separate experiments, anti-ganglioside antibodies and/or neurological signs were observed in NOD, NOD IL10-/-, and NOD CD86-/- mice inoculated with GBS strains HB93-13 and 260.94, while C3H/HeJ IL-10-/- mice exhibited extraintestinal spread of C. jejuni 11168 (31). Thus, we can distinguish the complete range of human disease phenotypes in various strains of mice inoculated with an array of C. jejuni strains: no colonization, colonization without enteritis, colonization with mild enteritis, colonization with severe enteritis, colonization with disseminated infection, and colonization/disease with neurological sequelae. By working with these models, we discovered the importance of contingency genes as virulence factors during host infection (23). Future CRIS work will allow screening in mouse models to validate important C. jejuni virulence genes discovered by various NC1041 group members to inform new vaccines to prevent colonization in food animals.

STEC and Salmonella control and antimicrobial resistance (Renter Lab).

The Renter Lab and collaborators are an interdisciplinary research team working towards controlling E. coli O157:H7, other STEC, Salmonella, and antimicrobial resistance in enteric bacteria. The focus is primarily in beef cattle production systems, but also in swine and food production environments. We have developed diagnostic tools (multiplex PCR procedures) to identify, quantify and/or characterize important enteric bacteria (STEC). Our unique research outcomes on Salmonella in feedlot populations may facilitate approaches to controlling Salmonella in commercial feedlots. We have shown that management of insects may be important to prevent the spread of antibiotic-resistant and virulent enterococci in animal feed and feed manufacturing environments and that dispersal behavior and capacity to transport antibiotic-resistant bacteria make house flies a potential threat to public health. Thus, area wide insect management is needed to mitigate health risks. Also, we have shown that combinations of preharvest interventions for foodborne bacteria may be particularly important for supplementing harvest interventions during periods of high and variable exposure periods (fecal shedding of E. coli O157:H7 in summer). We have also had success with viral pathogens. Specifically, we have a focus on evaluating transmission of Bovine Viral Diarrhea Virus (BVDV) in cattle and on design and implementation of control programs for this virus. We have also considered potential antiviral strategies aimed at control of human norovirus infections.

Association of STEC with insects in confined beef cattle environment (Zurek Lab).

Insects, especially muscoid flies such as house and stable flies, commonly build up very large populations in the confined beef cattle environment that provides unrestricted supply of fly larval habitat (cattle manure) and food for adult flies (cattle feed). House flies are now recognized as more than a simple mechanical vector (2, 3, 29, 38, 49). STEC (O157) proliferated in the house fly mouthparts and were excreted for at least 3 days after bacterial feeding(49). We showed that house flies harbor antibiotic resistant bacteria that are an increasing public health concern. House flies in urban fast-food restaurants carried a large and genetically diverse population of enterococci with antibiotic resistance and virulence genes that were frequently expressed and likely carried on mobile genetic elements(29). Moreover, house flies have been reported as mechanical vectors of nosocomial infections of multidrug-resistant bacteria in clinical settings(22). We also demonstrated that house flies commonly carry E. coli O157:H7 in feedlots(3, 48) and are capable of transmitting this pathogen to the cattle digestive tract(1). Consequently, house flies play an important role in the ecology of this pathogen in the cattle-production environment. However, the majority of these STEC studies focused only on the O157 serotype. Other STEC serotypes (O104:H4) are of great public health importance based on the 2011 European outbreak. Thus, there is a need to evaluate the effect of insect pest management in feedlots on STEC prevalence in cattle.

VIRUSES:

Norovirus:

Norovirus and other emerging viruses (Saif Lab).

Human noroviruses (HuNoVs) are emerging foodborne pathogens that cause an estimated 23 million cases of foodborne illness annually in the US (33, 50). HuNoVs are highly contagious; as few as 100 infectious viral particles can cause infection. However, HuNoV-contaminated foods can meet bacteriologic standards and still be distributed to the market. Because HuNoVs are uncultivable, surrogates are needed to evaluate disinfection/decontamination methods. The ideal surrogate virus should share resistance to disinfectants comparable to that of HuNoVs to avoid over estimation of disinfectant effects. Porcine sapovirus (SaV) Cowden strain is an enteropathogenic calicivirus like HuNoVs, but unlike HuNoV and human SaV, it is cultivable in vitro. It belongs to the genus Sapovirus within the Caliciviridae, and like HuNoVs, it replicates in the small intestine and causes gastroenteritis. Most importantly, our lab has successfully adapted this strain to cell culture in readily available pig kidney cell lines (8, 37). Like HuNoVs, TC-Po/SaV is resistant to the following: low and high pH (pH 3.0 to 8.0); high (56ºC) temperatures; and standard chlorine treatment. Therefore, TC-Po/SaV is an improved surrogate for HuNoVs compared to the current surrogates, feline calicivirus and murine norovirus, to study its persistence on food surfaces and disinfection procedures. Animal models are also critical for evaluation of NoV and SaV vaccines and therapeutics (antivirals). Our finding that pigs and calves express gut histoblood group antigens related to those in humans, which influence genetic susceptibility to HuNoV (10), was a breakthrough to establish the first and only animal model susceptible to HuNoV infection. Porcine NoVs belong to genogroup II (GII) and comprise 3 genotypes (GII.11, GII.18 and GII.19). They are genetically and antigenically closely related to human NoVs (58). Moreover HuNoV RNA has been detected from pigs (32, 34), raising public health concerns of possible interspecies transmission between pigs and humans. To assess the risk to humans, it is important to know the prevalence of NoVs circulating in swine. We screened 1,874 fecal samples from clinically healthy finisher pigs in nine North Carolina swine farms during 2009 by RT-PCR-coupled hybridization assays using primers and probes specific for porcine NoVs. The porcine NoV-negative samples were tested further by RT-PCR assays with GII (including human) NoV-specific primers and calicivirus universal primers, respectively. The representative RT-PCR products were sequenced for identification. The overall prevalence of porcine NoVs was 18.9%, and no human NoVs were detected. Although studies from Europe, Asia and New Zealand have reported lower porcine NoV prevalence rates (<1% to 15%), other North American studies report higher rates (20% to 25%). Additional studies of porcine NoVs and SaVs from NC member states are needed to assess their geographic and age prevalence in pigs and to confirm their relatedness to human strains.

New porcine caliciviruses, St-Valerien-like viruses, were detected from Canadian swine in 2009. Genomic characterization suggests that they represent a new genus within Caliciviridae. However, no sensitive diagnostic assays to detect these caliciviruses or prevalence data were available. We detected a similar virus, NC-WGP93C strain, from US pig fecal samples (57), characterized its genome, designed a real-time RT-PCR assay and performed a prevalence study of healthy finisher pigs. The NC-WGP93C strain is genetically similar to the Canadian strains (89.3-89.7% nucleotide and 95.9-96.3% amino acid identity, respectively) for the predicted capsid protein VP1. The new caliciviruses were detected in all nine North Carolina farms tested (prevalence of 23.8%; range 2.6- 80%), indicating that St-Valerien-like viruses are prevalent in some US swine farms. Further study is needed to learn if they cause disease in pigs or humans or are food safety hazards.

Rotavirus:

Epidemiology in animals (Saif Lab).

Despite available vaccines, rotaviruses (RVs) continue to be one of the most common causes of diarrhea in calves, piglets and infants (25, 46). A notable advance is the recent development of RT-PCR assays to monitor the epidemiology of group A and nongroup A RVs by NC-1041 collaborators. An important finding is the emergence of group C RV as a major cause of diarrhea in neonatal piglets. Lack of cell adapted group C RVs and a corresponding vaccine, necessitates additional research in this area to control these emerging RVs. Surveys of the dominant strains of RVs circulating in animals of different ages, their diversity and relatedness to human strains and their shedding by RV vaccinated animals is critical to evaluate vaccine efficacy and aid in the design of new vaccines to prevent RV disease across animal species and humans. Ongoing research focuses on new oral adjuvants (vitamins, probiotics) and vaccine approaches to improve RV vaccines using the neonatal gnotobiotic (Gn) piglet model (9, 63). Gn pigs resemble infants physiologically, immunologically and in their colonization by related microflora and susceptibility to human rotavirus (HRV) diarrhea. This model provides controlled conditions to constrain confounding variables in ways not possible in infants or conventional pigs naturally infected with diverse RV field strains.

Prevention of Rotavirus (Kuhlenschmidt Lab).

We have identified a porcine intestinal GM3 ganglioside receptor that is required for sialic acid-dependent rotavirus recognition of host cells. In addition, we previously demonstrated exogenously added GM3 can competitively inhibit porcine rotavirus binding and infectivity of host cells in vitro. Sialyllactose, the carbohydrate moiety of GM3, is approximately 3 orders of magnitude less effective than GM3 at inhibiting rotavirus binding to cells. Furthermore, production of therapeutic quantities of GM3 ganglioside for use as an oral carbomimetic in swine is cost prohibitive. In an effort to circumvent these problems, a sialyllactose-containing neoglycolipid was synthesized and evaluated for its ability to inhibit rotavirus binding and infectivity of host cells. Sialyllactose was coupled to dipalmitoylphosphatidylethanolamine (PE) by reductive amination and the product (SLPE) purified by HPLC. Characterization of the product showed a single primulin (lipid) and resorcinol (sialic acid) positive band by thin layer chromatography and quantification of phosphate and sialic acid yielded a 1:1 molar ratio. Mass spectroscopy confirmed a molecular weight coinciding with SLPE. Concentration-dependent binding of rotavirus to SLPE was demonstrated using a thin-layer overlay assay. Using concentrations comparable to GM3, SLPE was also shown to inhibit rotavirus binding to host cells by 80%. Furthermore, SLPE was shown to decrease rotavirus infection of host cells by over 90%. Finally, preliminary results of in vivo animal challenge studies using newborn piglets in their natural environment, demonstrated SLPE afforded complete protection from rotavirus disease. The efficacy of SLPE in inhibiting rotavirus binding and infection in vitro and in vivo, coupled with its relatively low-cost, large-scale production capabilities make SLPE a promising candidate for further exploration as a possible prophylactic or therapeutic nutriceutical for combating rotavirus disease in animals. Most importantly, the results presented here provide proof of concept that the nutriceutical approach of providing natural or synthetic dietary receptor mimetics for protection against gastrointestinal virus infectious disease in all species is plausible.

Prevention using probiotics (Saif Lab).

Probiotics mediate their effects in a species specific, but largely undefined manner(40, 52). The probiotic, L. rhamnosus reduced HRV diarrhea and shedding in children by unknown mechanisms(17, 30). L. acidophilus enhanced IgA antibody and Th1 responses to live attenuated (Att) AttRV in the Gn pig model(63). We plan to test probiotic strains to aid in recovery from RV diarrhea or to function as RV vaccine adjuvants. This is based on our prior Gn pig data(62-64) and ongoing microarray analysis detailing strain-specific anti-or pro-inflammatory properties. Further, soluble components in unpasteurized colostrum (col)/milk (antibodies, Th2/Th3 cytokines, sCD14), by influencing the microenvironment of the intestine, promote probiotic bacterial colonization, persistence and influence the regions colonized. Both breast milk [containing IgA inductive IL-4 and TGF²(35)] and probiotics synergistically influence development of the mucosal immune system of neonates. Colonization with probiotic bacteria will drive subsequent maturation to more balanced Th1/Th2 profiles and stabilize immunoregulatory T cell (Treg cell) responses. Thus, col/milk and defined probiotic bacteria administered to neonates should synergize to enhance development of humoral immunity, DC maturation, cytokine secretion, and T cell responses, thereby moderating infection by enteric pathogens (RV) and reducing diarrhea. Our studies of probiotics will comprehensively address gaps in knowledge of maturation of neonatal immunity, establishment of gut homeostasis and interactions between microflora and enteropathogenic viruses or oral vaccines. Our findings will improve neonatal oral vaccines and provide innovative, inexpensive treatments for viral diarrheas. We will elucidate changes in the naïve immune system of milk-fed neonatal Gn pigs colonized with selected probiotic strains. Findings will provide new insights on the impact of microflora on modulating infection of neonatal Gn pigs by virulent RV, a leading cause of neonatal diarrhea, or on efficacy of AttRV oral vaccines.

Other Enteric Viruses (Poultry):

Enteric viral diseases of poultry (Zsak Lab, Sellers Lab).

Enteric disease and associated production loss is an ongoing economic problem for the poultry industry in the US and abroad. The etiologies of the recognized enteric disease syndromes,Poult Enteritis Complex (PEC) and Poult Enteritis Mortality Syndrome (PEMS) in young turkeys, and Runting-Stunting Syndrome (RSS) in broiler chickens,are largely unknown, although many intestinal viruses have been implicated(11-15, 53, 54). Despite decades of research targeting a number of these viruses, a definitive cause of the poultry enteric disease syndromes has not been identified, and many times the suspect viruses are also detected in otherwise healthy flocks, suggesting an unknown virus or combination of viruses may be involved(12-15, 53, 54). There is a pressing need to identify the novel viruses present in the poultry gut,an important first step in determining their roles in the enteric disease syndromes and flock performance problems. The next generation of nucleic acid sequencing technologies has allowed researchers to determine the full genomic profile of organisms in complex environmental samples. Our use of these technologies to investigate the poultry gut environment has allowed us to discover new enteric viruses (12, 15, 65). Further, there are no commercial vaccines that target any of the viruses suspected of causing the signs of enteric disease(11-15, 53, 54). The proposed research will expand the existing poultry gut viral sequence database and allow us to develop novel, up-to-date molecular diagnostic assays(55, 66) and to develop and improve intervention strategies through design of recombinant vaccines targeting enteric viruses.

PARASITES:

Cryptosporidium parvum (Kuhlenschmidt Lab).

We have discovered and characterized a subset of long-chain polyunsaturated fatty acids (L-PUFA), originally isolated from bovine colostrum, which block in vitro C. parvum and Toxoplasma gondii host cell infectivity as well as both T. gondii and Plasmodium gallinaceum infectivity in vivo. Current results suggest L-PUFA are inhibiting parasite-host cell invasion by inhibiting sporozoite microneme secretion and gliding motility, processes that are required for host cell infectivity. We are collaborating with Dr. David Sibley to evaluate a variety of synthetic calcium-dependent protein kinase (CDPK) inhibitors for their ability to block Cryptosporidium infectivity in vitro. This is part of our long-term goal to develop small molecule inhibitors of the Apicomplexa-conserved, parasite-specific microneme secretion pathway that is required for host cell invasion. Proof of concept experiments demonstrating the ability of LPUFA to block in vivo infectivity of T. gondii and P. gallinaceum in mice and chickens, respectively, are currently underway. Preliminary results of these experiments indicate certain LPUFA are able to prevent disease when given at the time of parasite challenge

Objectives

1. Focus on emerging diseases: We will identify, characterize and develop improved detection and prevention methods related to newly recognized, novel or emerging causes of zoonotic enteric disease and enteric pathogens of food animals.
   
2. Focus on preventions and interventions: We will develop and improve preventative measures and interventions to reduce the incidence and prevalence of infections of food animals with enteric and foodborne and waterborne pathogens.
   
3. Focus on disseminating knowledge: We will provide training or continuing education to disseminate new information to students, producers, veterinarians, diagnostic labs and others to implement interventions and preventative measures.
   
4. Group interaction: The group will interact in a variety of ways to facilitate progress including direct collaborations with joint publications, sharing of resources (pathogen strains, gene sequences, statistical analysis, bioinformatics information/expertise), and friendly feedback and facilitation for all research efforts at annual meetings.
   

Methods

OBJECTIVE 1- FOCUS ON EMERGING DISEASES:

AIM 1- NEW DIAGNOSTICS FOR EMERGING BACTERIAL PATHOGENS:

Foodborne pathogen nanodetection (Lin Lab).

We established a partnership with Nano Detection Technology (NDT) to develop rapid, sensitive, in-the-field foodborne pathogen detection for E. coli O157:H7. We will identify new antigens for other bacteria of interest (non-O157 E. coli, Campylobacter, Salmonella, Listeria) and generate immune reagents for NDT detection. We will perform a literature search, bioinformatic analysis, and genome and proteome searches for unique highly specific targets. Novel antigen-antibody pairs will be designed and developed using established molecular and immunological approaches. The antigen-antibody pairs are expected to enhance specificity and sensitivity, allowing for lower detection limits than currently available methods. Then, we will evaluate sensitivity, specificity and detection of pathogens from different food matrices (milk, meat) using spiking with foodborne pathogens from NC members. Rapid diagnostic for ruminant C. jejuni clone SA (Zhang Lab).

We will 1)develop a PCR-based method for rapid detection and identification of C. jejuni clone SA in ruminants and use this diagnostic to monitor the emergence and transmission of this highly pathogenic clone and 2)examine prevalence of the C. jejuni clone SA on cattle farms and identify management practices to control prevalence. We have sequenced this clone and, using comparative genomics, we have identified several candidate gene sequences that appear to be unique to C. jejuni clone SA as a basis for the specificity of the assay. We will evaluate the feasibility of these targets by using different C. jejuni isolates and then assess the specificity and sensitivity of the PCR assay using experimental materials and clinical samples. To continue to monitor the emergence of this pathogenic clone, we will collect sheep abortion isolates from diagnostic laboratories in different NC states and analyze them with PFGE and MLST. To understand the epidemiology of C. jejuni clone SA, we will examine prevalence in cattle feces and milk and identify risk factors associated with prevalence on farms. Novel strongly hemolytic (NSH) Brachyspira and swine dysentery emergence (Gebhart Lab).

Increases in US incidence of Brachyspira and its impact on pig production globally, indicate urgent need for better surveillance and diagnostics including antimicrobial susceptibility testing methods and molecular strain typing. We will 1)develop/evaluate enhanced diagnostics for B. hyodysenteriae, 2)establish standard antimicrobial minimum inhibitory concentration testing for clinically important Brachyspira spp, and 3)develop molecular methods for epidemiological tracking of individual strains of B. hyodysenteriae. We will further develop molecular subtyping methods to trace B. hyodysenteriae isolates within and between outbreaks and determine sources of outbreaks to understand the epidemiological significance of re-emerging B. hyodysenteriae. Recent studies show that 70% of Brachyspira-positive cultures from diseased swine are non-typable. Many are both strongly hemolytic on blood agar media and associated with bloody diarrhea and colitis consistent with virulent Brachyspira infection. We will 1)perform further genotypic and phenotypic analyses of over 20 isolates of NSH Brachyspira to characterize them to species and determine significance, 2)determine the epidemiological significance of NSH Brachyspira by showing its association with colitis in pigs from multiple locations while demonstrating the absence of other intestinal pathogens in affected pigs, and 3)assess pathogenesis of NSH Brachyspira for pigs in challenge studies to fulfill Koch's postulates. AIM 2-DISCOVER EMERGING VIRAL PATHOGENS AND INTERVENTIONS:

Screens for emerging enteropathogenic viruses; prevalence/diversity of enteric caliciviruses (Saif Lab).

We will continue screening for new coronaviruses and other enteropathogenic viruses (including RVs and caliciviruses) in wild and domestic animals (swine and cattle). Mechanisms of interspecies/ zoonotic transmission of animal enteropathogenic viruses and their genetic relatedness to the corresponding human viruses are unknown. Such information is critical to understand evolution, diversity, re-emergence and control of animal enteropathogenic and zoonotic viruses. Prior studies involved collaborators from MN, WA, WI and other NC states to identify new CoV strains from mink (67) and wild ruminants and collaborations with J Craig Ventor Institute to sequence the full CoV genomes (5, 25). Also, we will study the prevalence and diversity of enteric caliciviruses in swine in OH and other states and in different ages of pigs using molecular assays (68, 69). These studies are crucial since NoVs most closely related to HuNoVs have been detected in swine, raising issues of zoonotic potential of swine NoVs. We will also investigate if selected porcine NoVs or new caliciviruses cause disease in the Gn pig model to study their pathogenesis and methods for control. Specificity of human NoVs to plant surfaces (Saif Lab).

We will investigate attachment, uptake and systemic dissemination of HuNoVs and SaVs within unprocessed leafy vegetables by using HuNoVs, HuNoV virus-like-particles (VLPs) and TC-Po/SaV. The binding to the plant cell wall extracts of HuNoV VLPs and TC-Po/SaV will be quantified by ELISA. The binding specificity of human NoVs to plant surfaces will be studied using HuNoVs and HuNoV VLPs. Finally, the specific molecules to which HuNoV VLPs bind will be assessed by blocking attachment with specific antibodies, various plant lectins, etc to design inhibitor molecules to block attachment, in conjunction with NC colleagues (IL). Enteric poultry virus discovery, diagnostics and vaccines (Zsak Lab, Sellers Lab).

Unidentified viruses or viral communities may play specific roles in enteric disease syndromes and could act as predictors of enteric disease. The Zsak Lab will discover and characterize novel poultry enteric RNA and DNA viruses and viral communities, and associate these etiologic agents with disease syndromes by sequencing the metagenome of the enteric virus microbiome of poultry from various US regions. We will investigate in vivo and in vitro isolation and propagation techniques for novel enteric viruses and perform pathogenesis studies with isolates in turkeys and chickens. We will also develop molecular diagnostic tests to determine prevalence of novel poultry enteric viruses on farms and their geographic distribution. Based on genome sequence of novel poultry enteric viruses found and characterized, conserved sequences will be identified as targets for RT-PCR, PCR or real-time RT-PCR diagnostics. We will also develop molecular platforms for rationale vaccine design to control enteric diseases of poultry, including mass delivery capability and companion diagnostics to distinguish naturally infected from vaccinated birds. The goal is selection of safe, live vaccine vector candidates that will ideally 1) be enterotropic, 2) induce an initial rapid gut mucosal immune response, and 3) induce a systemic cellular/humoral immune response in the bird. Live vaccine vector candidates will be sought because inactivated or non-replicating DNA vaccines do not produce good protective immunity during the first 14 days post-hatch.

The Sellers lab studies Runting & Stunting Syndrome (RSS) a widespread enteric disease of poultry that causes severe weight suppression, lack of flock uniformity, diarrhea, and a significant increase in feed conversion ratio. The etiologic agent(s) of RSS is not identified, but disease is reliably reproduced using filtered intestinal homogenates from RSS affected broilers implying a viral etiology. Although several novel viruses were isolated, the clinical disease has not been reproduced by experimental infection. Metagenomic studies in our lab identified viral sequences only in RSS+ birds. A novel chicken astrovirus was detected and adapted to grow in a cell line. We will: 1)determine full length genome sequences for both astroviruses (gut content and cell culture adapted) and 2)perform RT-PCR amplification and cloning of the capsid of gut derived astrovirus into a baculovirus expression system. Then, the baculovirus expressed capsid protein will be used to generate diagnostic tools: capsid specific antisera in rabbits, indirect ELISA to measure capsid specific antibodies, and a riboprobe for in situ hybridization. We are currently evaluating experimental vaccines in broiler breeders and assessing protection against RSS in the progeny. OBJECTIVE 2-FOCUS ON PREVENTIONS AND INTERVENTIONS:

AIM 3-DETERMINE BACTERIAL COLONIZATION AND VIRULENCE MECHANSIMS:

Role of TAT System and PPK1 and PPK2 in C. jejuni pathophysiology (Rajashekara Lab).

We will investigate how the TAT system contributes to C. jejuni pathophysiology using genetic, molecular and biochemical approaches. We plan to identify TAT dependent proteins using proteomics coupled with mass spectrometry by comparing proteomes of different cellular fractions of wild type and a tatC deletion mutant using 2D-DIGE. To determine molecular mechanisms by which PPK1 and PPK2 mediate diverse functions in C. jejuni, we plan to use deep sequencing to identify genes that are regulated by poly P. We will identify C. jejuni genes regulated by the two poly P associated enzymes by comparing the transcriptome of the ppk1 and ppk2 knockout mutants with the wild type when grown in rich and minimal media. Future studies are directed at understanding mechanisms underlying contributions of poly P to diverse phenotypes and to identify candidate genes for control of C. jejuni in humans and animal reservoirs. Putative toxin in non-invasive C. jejuni poultry strains (Joens Lab).

We have demonstrated in the past that 78% of C. jejuni poultry strains are non-invasive or invade epithelial cells at a very low frequency when compared to human clinical or bovine isolates. Non-invasive poultry isolates produce a high amount of intestinal fluid in the inoculated piglet model. Three proteins with very high homology to the C. jejuni genome have been identified in the intestinal fluid of pigs infected with the bacterium. We will mutate the genes encoding these proteins and examine their role in fluid production in the piglet model. Highthroughput approach to study C. jejuni and Salmonella Enteritidis pathogenesis (Shah lab).

We will directly compare pathogenic and non-pathogenic strains of Salmonella and Campylobacter using comparative genomics and proteomic approaches to understand molecular pathogenesis. We then apply a suite of tools, such as signature tagged mutagenesis, in vivo induced antigen technology, suppression subtractive hybridization, custom designed microarrays and next generation sequencing such as RNA and DNA-seq to characterize the differences between organisms under the broad hypothesis that the differential pathogenesis is controlled at the genetic, transcriptional, and protein level. Direct host-pathogen interaction often contributes, thus we will investigate host immune responses. The long-term goal is to identify diagnostic markers and improved vaccines, probiotics or immunotherapeutics for control of Salmonella and Campylobacter in poultry. Determining how C. jejuni initiates autoimmunity (Mansfield lab).

C. jejuni infection has been shown to trigger autoimmune diseases. LOS is a major antigen on the outer surface of C. jejuni that interacts with host epithelium. We will determine if autoimmune sequelae vary with differences in C. jejuni LOS profiles and differences in LOS region. Using 45 C. jejuni strains proven to cause GBS neuropathy in humans, we will determine the ganglioside profiles using thin layer chromatography (50) and determine the LOS class by examining loci involved in ganglioside synthesis using PCR of class-specific ORFs (43). More than 5 major C. jejuni LOS classes have been identified; most but not all genes within these intervals are characterized (22, 43). LOS structure of an isolate is determined by the genes present and by various mutations, thus C. jejuni strains with the same LOS class will not necessarily express the same LOS structure. We will determine allelic composition of these strains at the 3 GBS-associated galactosyl-transferase loci (23). Finally, testing of strains in GBS mouse models will validate which antigenic determinants are associated with initiation of autoimmunity. AIM 4-EPIDEMIOLOGY OF BACTERIAL PATHOGENS IN FOOD ANIMAL POPULATIONS:

Epidemiology in beef cattle (Rajashekara Lab).

We plan to expand our findings to include more samples from NC collaborators to provide a better spatial representation of C. jejuni clones in US beef cattle. We will also study how wildlife (Starlings) contribute to epidemiology of Campylobacter on dairy farms and their implications for public health. Comparison of genes in C. jejuni isolates showing different fitness traits in beef cattle (Joens Lab).

In conducting a longitudinal cohort study for the presence of C. jejuni in calves from range through the feedlot to processing, we identified different C. jejuni isolates in the same calf at a different time point during their stay in the feedlot. In one instance, we identified three different isolates by PFGE in the same calf. We will sequence the genomes of these unique strains and make comparisons. AIM 5-DEVELOP PREVENTION AND CONTROL METHODS FOR BACTERIAL PATHOGENS AND ANTIBIOTIC RESISTANCE:

Evaluate cost-effective vaccines against Campylobacter (Lin and Joens labs).

The Lin lab has compelling evidence that CmeC (an OMP of CmeABC multidrug efflux system) and CfrA (FeEnt receptor) are promising Campylobacter vaccine candidates(34, 73, 75, 76). We will evaluate, 1)in ovo DNA vaccination; 2)an oral live Salmonella-vectored vaccine; and 3)intranasal immunization with an encapsulated subunit vaccine.

The Joens lab will continue to develop an efficacious vaccine using an attenuated Salmonella vector to express C. jejuni proteins shown to be involved in colonization of broilers. They identified and extracted 3 proteins involved in C. jejuni biofilm production. Two of these genes have been cloned and mutated by insertion of a chloramphenicol-resistance cassette into each ORF. The vector containing the CAT gene and the flanking bases of each gene was then introduced by electroporation into C. jejuni and the mutation transferred into the genome via a double crossover. Both mutant strains have demonstrated a significant reduction in colonization of inoculated birds when compared to wild type. These genes will be cloned into attenuated Salmonella vectors and examined for immunogenicity in broilers. Small molecules to prevent C. jejuni (Rajashekara Lab).

We will use high-throughput chemical screens to identify small compounds for control of Campylobacter. High throughput assays enable screening of large libraries of chemical compounds that alter pathogen virulence thereby interfering with growth and survival of pathogenic bacteria. Small molecules confer important advantages as drug candidates, they lack toxicity, their small size allows diffusion into targets cells and they are suitable for mass application. Examine molecular basis of antimicrobial peptide (AMP) resistance in Campylobacter (Lin Lab).

Endogenous AMPs are efficient components of host defense and limit bacterial infections at the GI mucosal surface. AMPs are also increasingly recognized as novel peptide antibiotics to combat antibiotic resistance. C. jejuni may have the means to evade killing by potential peptide antibiotics and by innate immunity. However, AMP resistance mechanisms are still largely unknown in C. jejuni. Availability of this information will not only provide insights into host-pathogen interactions and reveal novel intervention targets to control Campylobacter infections in humans and animal reservoirs, but also help us to develop more sustainable peptide antibiotics. We have initiated AMP resistance studies (26, 27, 35), which ideally positions us for future in-depth characterization of identified molecular targets. Antibiotic growth promoter mechanisms and antibiotic resistant pathogen control (Lin Lab).

Worldwide, there is a trend to limit AGP use in food animals to protect food safety and public health; however, this limit poses challenges for the animal feed and feed additive industries. The lack of information about microbial function, diversity and dynamics in the animal intestine in response to AGP treatment has hampered development of effective alternative strategies to improve animal production without use of AGPs. We will use systemic and targeted approaches to study the mode of action of AGP and develop effective AGP alternatives to improve animal health as well as sustainability of animal production. STEC in insects in confined beef cattle (Zurek Lab).

We will 1)assess prevalence of four STEC serotypes (O104, O111, O26, O157) in house flies in confined beef cattle environments and evaluate their potential to transmit STEC to steam-flaked (SF) corn. Fresh SFcorn is attractive to house flies and is a hotspot for bacterial contamination from flies (Fig. 1). We will 2)apply and evaluate fly screens to prevent fly-bacterial corn contamination. Because isolation of E. coli O157:H7 from flies is possible without enrichment (3, 57), we will 3)assess the use of house flies as indicators of genotypic diversity of STEC in the confined beef cattle environment. House flies and SFcorn will be sampled from cattle feedlots bi-weekly during fly season. SFcorn alone will be sampled monthly in winter when house flies are not active outside. Adult flies and corn samples will be collected and flies surface sterilized(82), individually homogenized and plated directly on selective and differential STEC media(49) for culturing STEC (O111, O26, O157). STEC O104 will also be detected and confirmed (65). Corn will be mixed in buffer and plated on STEC agar. CFUs will be counted and total STEC calculated per fly or gram of corn. We will confirm O26 and O111 by serotyping, non-O157 STEC by PCR (48) and 6 major STEC virulence genes by multiplex PCR(6). This is a 2 yr study at 3 sites (KS, TX, OK), with and w/out fly screens. Data from winter x summer months and from sites with and w/out fly screens will be analyzed and compared. Genotypic diversity of representative strains for each serotype from each source (flies and corn) will be assessed by PFGE and compared to that of STEC isolates from cattle feces(4). Transcriptional profiling of a homologous attenuated pathogenic L. intracellularis isolate (Gebhart Lab).

L. intracellularis causes proliferative enteropathy; little is known of the genetic basis of virulence, pathogenesis or physiology of this obligate intracellular bacterium. We will identify and compare differentially-expressed genes in pathogenic and non-pathogenic L. intracellularis during in vitro infection of cell culture and characterize potential virulence factor-encoding genes expressed in experimentally-infected pigs. Intestinal piglet epithelial cells (IPEC-J2) will be infected with both pathogenic and non-pathogenic cultures of the homologous isolate of L. intracellularis. Total RNA will be harvested from infected cell monolayers during the exponential phase of growth. For the in vivo study, total RNA will be extracted from infected intestinal mucosa. In both the in vitro and in vivo experiments, bacterial mRNA will be purified by subtractive hybridization and sequenced using next generation sequencing technology. Transcription readings will be mapped onto a reference genome to characterize and compare the bacterial transcriptome. This will provide information about virulence factor-encoding genes, especially those lost during attenuation in cell culture, as well as elucidate their specific ability to induce proliferative lesions. AIM 6-DEVELOP PREVENTION AND CONTROL METHODS FOR VIRAL AND PARASITIC INFECTIONS:

Rotavirus prevention (Kuhlenschmidt Lab).

Our goal is to further develop sialyllactose dipalmitoyl-phosphatidyl-ethanolamine (SLPE) as a feed additive to protect weanling piglets from rotavirus disease. We showed that neonatal piglets are completely protected from rotavirus disease by feeding SLPE every 12 hours. This study will examine dosage and delivery methods to determine 1) if SLPE will also protect weanling pigs from disease and 2) if SLPE can be administered as a nutriceutical in feed rather directly per os and 3) to evaluate the safety of SLPE as a porcine nutriceutical. The propagation, purification of Group A porcine rotavirus (OSU strain (P9(7)G5), measurement of in vitro and in vivo infectivity in the presence and absence of SLPE, are determined(8, 33, 52, 53). The (Saif Lab) will determine the genotypes of group A RVs and the dominant non-group A RVs from calves and pigs with diarrhea using RT-PCR-or real time PCR. Sequence analysis of representative strains will reveal new strains, their genetic diversity, relatedness to human strains and identify new interspecies reassortant strains. Data on the dominant RV strains in the field is needed to determine the effectiveness of existing vaccines and to design new vaccines. Enteric viral vaccines and therapeutics (Saif Lab).

Together with NC groups, we will develop new calicivirus and RV vaccines (VLPs, adjuvants, delivery systems) to reduce diarrhea in animals and environmental and food contamination. We will use monoclonal antibodies to porcine and bovine Igs provided by IA and NE to define systemic and mucosal antibody responses to various group A rotavirus serotypes from swine and cattle. Comparisons of innate and adaptive mucosal immunity will be done using monoclonal antibodies in ELISA and ELISPOT assays to measure B and T cell responses. We will compare immunologic reagents and assays to quantitate innate, humoral and cellular immune responses in swine. We will define the role of host immune responses in disease pathogenesis and evaluate existing and novel enteric viral vaccines. We will also determine how the gut microflora modulates intestinal homeostasis and its impact on pathogenesis of enteric pathogens. We will study how different probiotic bacteria colonize different parts of intestine and modulate host cellular pathways. We will use high throughput microarray analysis to determine the host-gut mucosal transcriptome responses temporally in the Gn pig model to identify different cellular pathways regulated by certain lactic acid bacteria, and we will study the interaction of different probiotic bacteria with enteric pathogens such as RV to understand how they modulate intestinal infections and neonatal immunity. Cryptosporidium parvum prevention (Kuhlenschmidt Lab).

Our goal is to evaluate selected LPUFA and CDPK inhibitors, alone or in combination, as novel therapeutic and prophylactic agents to protect and treat apicomplexan parasitic diseases, such as toxoplasmosis, malaria, and cryptosporidiosis in animals and people. Several LPUFA and CDPK inhibitors will be screened using in vitro infectivity and qPCR assays and selected compounds will be tested in vivo using animal models (mice and calves). Progress will be measured by demonstrating dose-dependent inhibition of parasite infectivity, protection from disease and inhibition of microneme secretion in vitro. Propagation, purification of C. parvum oocysts, measurement of infectivity in the presence and absence of small molecular weight LPUFA or CDPK inhibitors, and measurement of parasite motility and microneme secretion activity will be performed as previously described (29, 60, 71, 72). AIM 7. FOSTER COLLABORATIONS AMONG MEMBER STATES. Sharing and collaborations are strongly encouraged and promoted by the committee. Additional information on collaborations is described in an attached document.

Measurement of Progress and Results

Outputs

• AIM 1<p>A rapid, sensitive, in-the-field Nano Detection Technology foodborne pathogen detection system is functioning.<p>A PCR assay is available and functioning for rapid detection and identification of the C. jejuni clone SA<p>Prevalence of the C. jejuni clone SA in ruminants is known and potential interventions are identified<p>Enhanced diagnostics for Brachyspira. hyodysenteriae<p>Standard operating procedure for antimicrobial minimum inhibitory concentration testing is established for clinically important Brachyspira spp,<p>Molecular methods for epidemiological tracking of individual strains of Brachyspira hyodysenteriae are available<p>Understanding of whether novel hemolytic Brachyspira is a new cause of swine dysentery in the USA<p>Understanding of the epidemiology of novel hemolytic Brachyspira so that control and prevention can be initiated in the USA<p>Understanding of whether novel hemolytic Brachyspira are a primary pathogen of swine
• AIM 2<p>Porcine and bovine rotavirus strains and rotavirus-like particles with known P and G types are available for reference diagnostic strains and for potential vaccine applications<p>Immunologic reagents and assays to quantitate innate, humoral and cellular immunity in swine are available<p>Understand the prevalence of novel enteropathogenic viruses in swine and poultry<p>Understand attachment, uptake and systemic dissemination of HuNoVs and SaVs within unprocessed leafy vegetables<p>Genome sequences for astroviruses of poultry<p>Diagnostic tools for poultry astroviruses<p>Understand diversity of swine caliciviruses
• AIM 3<p>Understand the genetic basis for fluid production by pathogenic C. jejuni in piglets<p>Understand mechanisms underlying contributions of TAT and polyP to diverse phenotypes in C. jejuni and use this information to identify candidate genes for control of C. jejuni<p>Understand differences between pathogenic and nonpathogenic strains of C. jejuni and Salmonella Enteritidis in order to make new vaccines<p>Understand how to avoid induction of Guillain Barré Syndrome neuropathy when giving C. jejuni vaccines to animals and humans<p>Understand the mechanisms by which bacterial pathogens induce autoimmune disease in susceptible animals and humans
• AIM 4<p><p>Understand fitness traits of different strains of C. jejuni in calves on range to the feedlot<p>Understand prevalence, distribution, and molecular epidemiology of C. jejuni strains in US beef cattle<p>Understand potential risk associated with human consumption of bacterial pathogen contaminated cattle products
• AIM 5<p>Understand the ability of several C. jejuni gene products to act as the basis for a poultry vaccine<p>Understand the best formulation for a C. jejuni poultry vaccine 1) in ovo DNA vaccination; 2) an oral live Salmonella-vectored vaccine; and 3) intranasal immunization with an encapsulated subunit vaccine<p>Identify several small molecules that could act as drug candidate inhibitors for C. jejuni<p>Understand antimicrobial peptide targets and resistance in C. jejuni<p>Understand the mode of action of antibiotic growth promoters and develop effective alternatives<p>Understand the prevalence of four STEC serotypes (O104, O111, O26, O157) in house flies in confined beef cattle environments and evaluate their potential to transmit STEC to steam-flaked corn<p>Understand whether house flies can act as indicators of genotypic diversity of STEC in the confined beef cattle environment<p>Understand whether fly screens can prevent fly-bacterial corn contamination<p>Understand differentially-expressed genes in pathogenic and non-pathogenic Lawsonia intracellularis to identify important virulence factors inducing proliferative lesions
• AIM 6<p>Develop SLPE feed additives to protect piglets from rotavirus <p>Understand safety, dosage and delivery methods for SLPE<p>Develop new Calicivirus and Rotavirus vaccines<p>Understand how probiotic bacteria colonize the intestine, modulate host cellular pathways, affect

Outcomes or Projected Impacts

• Enhanced health and well being of food animals and humans
• Increased profitablility of food animal production systems including poultry, swine and cattle
• New diagnostics that will provide enhancements to profitability for diagnostic labs, universities and diagnostic test manufacturers
• New vaccines that will provide enhancements to profitability for universities and vaccine manufacturers
• Increased profitability from production of novel small molecule pathogen preventatives
• A wide range of basic science information(genome, immunological, agricultural)that will foster future novel products and procedures

Milestones

(2013): <p>Targets for pathogen detection have been identified<p> Molecular testing is initiated to understand gene targets for diagnostics and vaccines<p> Pathogen/commensal gene sequencing work is initiated<p> Genome sequencing for emerging viral pathogens in initiated<p> IRB documents are written and submitted for approval for epidemiological studies<p> Basic bacteriology studies are initiated to identify relevant genes for vaccines and diagnostics<p> Genes composing LOS/LPS of bacteria are examined for presence/absence and allelic variation<p> Basic studies for small molecule inhibitors are initiated<p> Initiate probiotic studies<p> Conduct NC annual meeting<p> Initiate new outreach efforts<p> Student mentoring and awards<p>

(2014): <p>Testing of targets for pathogen detection has been completed<p> Testing of gene/gene products as vaccine candidates is initiated<p> Comparative genomics is underway using genome sequences<p> Epidemiology studies are initiated<p> Gene targets for vaccines and diagnostics are tested<p> Emerging disease information is published<p> Bacterial outer membrane molecules are tested for eliciting autoimmunity in animal models<p> Small molecule inhibitors are identified and in vito testing is initiated<p> Evaluate effects of probiotics<p> Conduct NC annual meeting<p> Initiate new outreach efforts<p> Student mentoring and awards<p>

(2015): <p>Diagnostic assays have been assembled<p> NC collaborators share bacterial strain collections for test validation<p> Epidemiology sample collection in various geographic areas is underway<p> Bacterial sample collections from epidemiologic studies are analyzed for allelic variants<p> Gene targets for vaccines and diagnostics are tested<p> Emerging disease information is published<p> LOS variants are tested in mouse models for initiation of autoimmunity<p> Small molecule inhibitors are tested in animal models<p> Evaluate effects of probiotics<p> Conduct NC annual meeting<p> Initiate new outreach efforts<p> Student mentoring and awards<p>

(2016): <p>Validation of diagnostic assays is initiated<p> Epidemiologic data is analyzed<p> Vaccines are formulated and tested<p> Emerging disease information is published<p> LOS variants are tested in mouse models for initiation of autoimmunity<p> Small molecule inhibitors are tested in food animals<p> Publish probiotic basic science information<p> Conduct NC annual meeting<p> Initiate new outreach efforts<p> Student mentoring and awards<p>

(2017): <p>Field and laboratory based tests are complete and publications and patent applications are submitted<p> Epidemiology studies are written up and published<p> Small molecule inhibitor licensing is initiated for those compounds showing efficacy and safety<p> Vaccines are evaluated for safety and field tested<p> Emerging disease information is published<p> Genome sequence of emerging pathogens is uploaded to databases<p> Majority of work has been published<p>Conduct NC annual meeting<p> Hold next Rushmore national meeting<p> Student mentoring and awards<p>

Projected Participation

View Appendix E: Participation

Outreach Plan

GENERAL APPROACH. Over the next 5 year period our NC1041 group will continue to provide information to veterinarians, researchers, animal scientists, diagnostic lab personnel, medical professionals, scientific extension personnel, cattle, swine and poultry producers, industry organizations, students and the general public using oral, written and web-based formats. We expect that continuing education will be an important component of our outreach in a wide variety of venues. Our group has demonstrated repeated successes in outreach efforts. We all collaborate on a regular basis to enhance these efforts. The discussion below gives more detail in our proposed methods for outreach and some specific examples of effective ongoing strategies.

SCIENTIFIC PEER-REVIEWED JOURNAL ARTICLES. A primary form of dissemination of research results and description of important outcomes and impacts will be through scientific peer-reviewed journal articles. We have found that this science based approach leads to greater dissemination and acceptance of emerging disease information as well as basic science findings and new diagnostic and preventative technologies. It also helps for speeding translational approaches to market. Because our work in NC1041 ranges from highly basic to highly applied, we target a wide scope of scientific journals for publication. As ever, the ultimate goal is to choose the BEST FIT journal to define the most interested audience for the highest impact and greatest dissemination. Scientific citation indices help all of us to understand which journal fits our needs for each specific dataset. The following are examples of the diverse journals that published our work in the last 5 year period including Emerging Infectious Diseases, PLoS One, Infection and Immunity, BMC Microbiology , Microbial Pathogenesis, Foodborne Pathogens and Disease, Biomacromolecules, Applied Environmental Microbiology, Journal of Antimicrobial Chemotherapy, Microbiology, Antimicrobial Agents and Chemotherapy, Journal of General Virology, Journal of Virology, Viral Immunology, Experimental Biology, Journal of Parasitology, Journal of Nutritional Biochemistry, IEEE Sensors Journal, Journal of Food Protection, Veterinary Microbiology, Veterinary Immunology and Immunopathology, Veterinary Pathology, Journal of Poultry Science, Journal of Veterinary Diagnostic Investigation, Journal of the American Veterinary Medical Association, American Journal of Veterinary Research, Canadian Journal of Veterinary Research, Journal of Microbiological Methods, Journal of Clinical Microbiology, Clinical Vaccines, and Immunology among others. Our members are a distinguished and highly productive group who published over 281 scientific manuscripts in the last 5 years. We expect an equivalent productivity in the next 5 years if this multistate project is approved.

SCIENTIFIC MEETINGS. Scientific meetings will continue to be the major venue for sharing our new findings with presentations (talks, posters) at international, national, regional and local scientific conferences. Each year, the new data will be analyzed and presented at the NC1041 annual meeting in conjunction with the Conference of Research Workers on Animal Diseases (CRWAD) meeting. At this event, the NC1041 group will host a two day meeting where all units will present their progress and findings to the group and provide a written AES unit report for posting at each state's AES website. Furthermore, we have organized the Rushmore meetings on a continuing basis to provide a national venue for sharing important information on enteric diseases. This meeting has always emphasized sharing of information on both human and animal diseases and provides an important forum for imparting of information on zoonotic diseases. We will host another Rushmore meeting at a time during the next 5 year period when enough novel work is available to draw interest from a wide audience. In addition to these enteric disciplines focused meetings, NC1041 investigators are expected to present at a variety of forums yearly. Examples of meetings that our members attend regularly are the American Society for Microbiology, the International Conference on Emerging Infectious Diseases, FACEB, the Conference on Helicobacter, Campylobacter and Related Organisms, the Plant Animal & Genome XVIII International Conference Meeting, the American Dairy Science Association annual meeting, the International Society for Veterinary Epidemiology and Economics, Keystone Meetings, the American Society for Virology annual meeting, and the International Conference on Caliciviruses among others. Presentations will be given to producer groups and veterinary specialty groups including the AVMA annual meeting, the Beef Industry Food Safety Council (BIFSCO), the American Dairy Science Association annual meeting, the Ontario Cattlemens Association, the American Association of Bovine Practitioners, the American College of Veterinary Pathologists, the American Association of Avian Pathologists, SCAD II, the Leman Swine Conference, the International Pig Veterinary Society, the American Association of Equine Practitioners, the American College of Veterinary Internal Medicine, the American Association of Swine Veterinarians and the Passion for Pigs Symposium among others. Within these scientific meeting venues we will take advantage of opportunities to advocate for better methods of control of enteric diseases in food animals based on our research.

SEMINARS/SEMINAR SERIES. It is expected that each member of the NC1041 group will actively give seminars each year to impart research results and promote input and sharing of information. We also expect to organize seminars or seminar series to enhance communication and information dissemination. For example, in 2010 Dr. Mansfield organized a seminar in Food and Waterborne Diseases for the faculty and students of Michigan State University. People attending came from the Agricultural, Veterinary Medicine, Human Medicine, Microbiology and Food Science, and Human Nutrition departments. The speakers included Shannon Manning, PhD, MPH (Shigatoxin producing E. coli) and Robert Britton, PhD (Probiotics for treating enteric diseases). Other presentations were made at scientific conferences held by the National Institutes of Health at Cambridge, Maryland on Food and Water borne Pathogens, the Norman E. Borlaug International Agricultural Science and Technology Fellowship Program mentor at Lublin, Poland, and the American Society for Microbiology.

COURSES AND EXCHANGE. In addition to scientific venues, we have always provided information directly to end users in a variety of formats that have been driven largely by feedback regarding which formats are most successful for getting the message out. This has included outreach to US farms/ranches on a one to one basis as well as educational tours to developing countries to share new strategies for improved agricultural efficiencies. In particular Dr. Margaret Khaitsa's group at North Dakota State University (NDSU) has developed an educational exchange program that crosses international boundaries. The Department of Veterinary and Microbiological Sciences at NDSU in conjunction with the Department of Veterinary Public Health and Preventive Medicine, Makerere University (Mak), Kampala Uganda, developed a 3-credit short term course (4 weeks) "International Animal Production Disease Surveillance and Public Health". The course was designed to facilitate diversity in student training and exposure in order to produce a broadly inclusive, open minded and globally engaged science workforce. The course provides: 1) shared opportunities for faculty and students through collaborative research and/or fieldwork in Uganda and the United States in areas of mutual interest, 2) career development opportunities and an international perspective to agricultural and biomedical training, 3) a foundation for tomorrow's global citizens. This course was offered for the first time in summer 2007 (June 17 to July 11, 2007). In summer 2008 the course was offered for the second time (June 12 to July 17, 2008) with an increased number of students (13) distributed as follows: 6 from NDSU and 7 students from four other institutions, 4 from The Ohio State University, 1 from Oklahoma State University, 1 from Kansas State University and 1 from University of Minnesota. Additionally, in conjunction with the Department of Veterinary Public Health and Preventive Medicine, Faculty of Veterinary Medicine, Makerere University, Kampala, Uganda, NDSU participated in conducting a 2-day symposium on Global Infectious Diseases, Bio-security and Agro-security at the Sheraton Hotel, Kampala, Uganda, June 30 to July 1, 2008. The symposium was attended among others, by NDSU administration (President Joseph Chapman, Dean David Wittrock (Dean Graduate & Interdisciplinary Studies), Kerri Spiering, Director, Office of International Programs, Marinus Otte, Professor Department of Biological Sciences and Douglas Freeman, Professor & Chair Department of Veterinary & Microbiological Sciences). This will continue with additional efforts by other members of the group to engage in a wide range of educational programs for getting new technologies out to producers to produce healthier animals and safer food products.

GRADUATE STUDENT TRAINING. For our group, graduate education is a continuing commitment in order to train the next generation of scientists expert in enteric diseases of food animals. NC1041 members have trained many experts in enteric disease, who have become important players in ensuring animal health and public health. Some of the trainees have joined the group after they achieved a permanent position. In the next 5 years, students at all levels of education will participate in these proposed projects including undergraduates, graduates, postdoctoral fellows, veterinary students and animal science students. Graduate programs range in a variety of topics including food and waterborne illness, microbiology, microbial genetics, diagnostic sciences, vaccinology, parasitology, pathology, virology, nutrition and antimicrobial chemotherapy. We participate in master's level and doctoral level training programs. A good example of such programs is the Sellers Lab. Data generated on Runting and Stunting Syndrome and other emerging diseases of poultry is shared with poultry veterinarians and DVMs in the Master's in Avian Medicine (MAM) program at the Poultry Diagnostic and Research Center (PDRC) of the University of Georgia. In this case, the research proposed by students is integrated with the evolving knowledge of poultry infectious diseases, nutrition, and genetics, benefiting the research community and the poultry industry. It is recognized that poultry enteric diseases are known collectively as "production diseases" or "management diseases" in the poultry industry, consequentially, disease onset and severity can be substantially influenced by subtle changes to management techniques on farms. This requires combined knowledge of the biology of enteric viruses with knowledge of common and ever-changing industry practices in order to provide meaningful scientific insight into the management and control of enteric disease. In fact, this is true for the range of food animals studied by our group and both basic and applied approaches must be integrated with the needs of the relevant industries.

INTERNET OUTREACH. Future outreach will involve a larger suite of methods that rely on the internet. A major emphasis will be keeping the state agricultural experiment station websites up to date and accurate for annual productivity of the NC-1041 group. Each NC1041 member will maintain a personal website where their published information is delivered in an understandable manner to whoever wishes to access the site. We will publish in online forums for veterinarians, animal scientists and producers and will participate, where appropriate in online blogs and listservs to share new and paradigm changing information arising from our work. For this media, we will work with computer and abstracting experts to ensure that the information is accessible and easily found by those needing it. In all cases, the work will be imparted using a science based, best practices methodology to ensure acceptance of improved methods to prevent and intervene in these important enteric diseases of poultry, swine and ruminants.

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 Adviser and a CSREES Representative. Chairs are required to organize and run the annual meeting during their term. The Chair-elect is responsible for documenting the progress content of the meeting and communicating the results to the membership.

Furthermore, adhoc committees will be formed to organize specific initiatives including research initiatives and formal and informal meetings to disseminate results. In particular a special adhoc committee will be assembled to organize the Rushmore meeting. A chairperson along with the committee will be responsible for organizing the plenary presentations, abstract submissions and selection and the venue arrangements.

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Land Grant Participating States/Institutions

AL, AZ, CA, FL, GA, IA, IL, KS, KY, MI, MN, ND, NE, OH, SD, TN, WA, WI, WY

Non Land Grant Participating States/Institutions

Iowa State University - College of Vet Med, NC A&T State University, Southeast Poultry Research Laboratory, Texas Tech University