Duration: 10/01/2017 to 09/30/2022

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

NEEDS. The long-term goal of this collaborative project is to develop strategies to prevent and control enteric diseases of cattle, swine, and poultry, ultimately to decrease the incidence of enteric diseases in food animals, and decrease zoonotic food and water-borne illnesses in the USA.  Illnesses caused by enteric food-borne pathogens continue to remain a prominent public health challenge in the USA (https://www.cdc.gov/ncezid/dfwed/edeb/).  Despite many concerted efforts to control enteric pathogens as well as zoonotic pathogens in food animals at both on-farm (pre-harvest) and food processing (post-harvest) environments, the incidence of diseases caused by enteric pathogens, and food and water-borne pathogens remains high and some diseases are increasing.  Nevertheless, a broad range of educational and scientifically-driven and practically-oriented control efforts have succeeded in decreasing the incidence of five key food-borne pathogens.  NC1202 originally started as NC62 in the 1960’s and has been an important contributor to research on the enteric diseases of swine. Over the last 15 years, our enteric diseases group has contributed significantly to an evolved and expanded effort to find interventions to prevent enteric diseases in livestock and prevent food-borne diseases. In this renewal proposal we remain committed to the prevention and control of animal and human diseases caused by enteric pathogens in cattle, swine, and poultry. A primary avenue for control is decreasing carriage and disease due to enteric pathogens in food animals.  Our collaborative efforts also harmonize with the new government-wide initiative to better understand, characterize and mitigate antimicrobial resistance (AMR) across the food chain. The proactive and collaborative efforts from our enteric diseases group will also enable us to contribute to meeting the national goals to further decrease the burden of microbial diarrheal illness by the year 2020 (https://www.healthypeople.gov/).

Enteric diseases account for multi-million dollar annual economic losses to the food animal industry due to reduced weight gain, mortality of young animals and treatment costs. For example, neonatal diarrhea and post-weaning diarrhea are the most important swine diseases and are caused by pathogenic bacteria and viruses including Enterotoxigenic E. coli (ETEC), porcine epidemic diarrhea virus (PEDV) and rotavirus. Similarly, Brachyspira and Lawsonia species represent the most important causes of bacterial enteric diseases of grow/finish pigs in the US. L. intracellularis causes porcine proliferative enteropathy and B. hyodysenteriae/ B. hampsonii are the etiologic agents of swine dysentery. Therefore, effective prevention and control of enteric diseases is critical to maintain production efficiency, produce wholesome pork, enhance food security and safety, and animal well-being.

Foodborne illness is a major public health concern in the USA.  The CDC estimates that each year about 1 in 6 Americans (~ 48 million people) get sick, 128,000 are hospitalized, and 3,000 die of foodborne diseases.  In 2015, the USDA ERS estimated that foodborne illnesses in the USA impose over $15.5 billion in economic burden on the economy each year. Of this, ~$14 billion/year is attributed to medical costs, productivity losses, and costs associated with premature deaths due to diseases caused by five leading foodborne pathogens. Norovirus caused the most illnesses whereas non-typhoidal Salmonella spp., norovirus, Campylobacter spp., and T. gondii caused the most hospitalizations; and non-typhoidal Salmonella spp., T. gondii, L. monocytogenes, and norovirus caused the most deaths. Scarce data precluded estimates for other known infectious and non-infectious agents, such as chemicals.

The Foodborne Diseases Active Surveillance Network (Food-Net) was established in 1995 and is a collaborative program among CDC, 10 state health departments, USDA FSIS, and FDA. Food-Net conducts surveillance for bacterial (Campylobacter, Listeria, Salmonella, enterohemorrhagic E. coli (EHEC) O157 and non-O157, Shigella, Vibrio, and Yersinia) and parasitic (Cryptosporidium, Cyclospora) infections diagnosed by laboratory testing of patient samples. It is not surprising that the US President, Congress, and USDA have made national food safety a high priority mission. Statistics from Food-Net 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 food-borne 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 animal pathogens 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.

While most food-borne 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 complicating brain damage, paralysis and miscarriage, inflammatory diseases, among other diseases. The incidences of autoimmune disorders are rapidly increasing and a number of these syndromes are triggered by enteric pathogens. For example, C. jejuni is a leading cause of bacterial food-borne gastroenteritis that can trigger serious autoimmune diseases. Acute neuropathies, such as Guillain Barré Syndrome (GBS), Miller Fisher Syndrome (MFS), Inflammatory Bowel Disease (IBD) and Reiter’s Arthritis (RA), have all been associated with Campylobacter infection. It has now been demonstrated that antibiotic resistance gene carriage by enteric pathogens can exacerbate these autoimmune manifestations when antibiotics are used.

Unique dynamic interactions between the enteric pathogens, animals and humans, their gut microbiota (microbiome), sharing the same environment, is considered within the “One Health” approach. In the upcoming project, we will attempt to more fully understand the role of the gut microbiome in contributing to or preventing enteric diseases and will perform studies to determine how the productive functions of the gut microbiome can be manipulated without the need for antibiotics. This new NC1202 project will develop and employ inter-disciplinary systems approaches to address critical areas that will enhance animal health, food safety and food security by maintaining efficient pork, beef and chicken production and reducing reliance on antibiotic use through development of alternative approaches for sustainable food animal agriculture. The list that follows are the major and significant enteric animal, zoonotic and AMR pathogens on which the NC1202 group will continue to work  to seek novel prevention and control measures.

• Enterohemorrhagic E. coli (EHEC). Cattle are important reservoirs of EHEC and other Shiga toxin-producing  E. coli (STEC), a leading cause of U.S. outbreaks of foodborne illness. Colonization of the colon in cattle results in shedding of the organism in feces. Control of fecal shedding of EHEC such as serotype 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. These organisms are still highly prevalent in cattle and pre-harvest interventions are needed to reduce carriage levels.


• Enterotoxigenic E. coli (ETEC). ETEC causes diarrhea in neonatal animals (piglets, calves and lambs) by adhering to the intestinal epithelial cells and producing enterotoxins. ETEC cause death of 10.8% of all pre-weaning pigs and 1.5-2% of all weaned pigs. The incidence of neonatal diarrhea has been reduced substantially using vaccines, but post-weaning ETEC diarrhea remains an economically significant disease for the swine industry.


• Salmonella (non-typhoidal species). Each year, >1 million people in the United States become ill from a non-typhoidal foodborne Salmonella infection (11 percent of foodborne illnesses). Salmonella ranks 1^st among the 15 pathogens included in the 2015 USDA ERS report in terms of economic burden. The food animals that we study (cattle, swine, poultry) are common reservoirs of Salmonella.  Salmonella enterica is a common cause of diarrheal and systemic 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, particularly multidrug resistant Salmonella, have recently emerged.


• Campylobacter (all species). Campylobacter spp. are common causes of diarrheal illness acquired in the United States, and one of the most common causes of food-borne illness. Poultry, ruminant and swine are primary animal reservoirs. Campylobacter ranks fifth in terms of economic burden among the 15 pathogens based on the recent USDA ERS report.  C. jejuni infection has been causally linked to many autoimmune disorders including GBS, MFS, IBD, RA and now Irritable Bowel Syndrome (IBS). 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. CDC has listed antibiotic resistant Campylobacter as a serious antibiotic threat.


• Caliciviruses. Based on CDC estimates, enteric caliciviruses (Noroviruses, Sapoviruses) cause over 9 million cases of foodborne illnesses yearly (58% of foodborne illnesses in the U.S.), making them the most common cause of acute foodborne gastroenteritis in the US. 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.


• Coronavirus. Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea, vomiting, dehydration and high mortality in seronegative nursing piglets. PEDV outbreaks in the US have led to significant economic impacts as a result of the high mortality among seronegative nursing piglets and decreased pork production. PED is the most devastating in nursing piglets (under 3-weeks-old), causing 100% morbidity and 50-100% mortality. The mechanisms by which PEDV infection induces greater disease severity and deaths of nursing piglets versus weaned pigs are not clearly defined.


• Lawsonia. Proliferative enteropathy (ileitis) is a common enteric disease of weaned pigs and other animals caused by an intracellular bacterium, L. intracellularis. Infections are common and estimates of annual economic losses are ~$100 million for the US swine industry. Clinical signs include diarrhea, weight loss, and melena. Little is known of the pathogenesis and sensitive and specific methods for diagnosis are not universally available. Lawsonia infection is also known to be a risk factor for carriage of Salmonella by pigs.


• 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. SD has devastating economic impacts on pig production, but changes in management designed to eliminate SD have 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.


• Cryptosporidium. Cryptosporidium is the most common enteric pathogen of calves and is responsible for significant economic losses to the dairy and cattle industries. Cryptosporidium is also one of the most common causes of waterborne illness in the United States. It is also a significant threat to water resources and human health. There are still no effective treatments for humans or livestock with cryptosporidiosis.


• Antimicrobial resistance (AMR). The widespread use of antimicrobials in both food animals and humans has heightened concerns about the emergence of AMR, which impacts animal health, public health, food safety and environmental exposure. The role of the microbiome in transmission of AMR is a new avenue for understanding the extent and emergence of this problem. The NC1202 group has expertise and extensive research experience in AMR with focus on epidemiology, emergence, transmission, molecular mechanisms as well as development of innovative and sustainable approaches to mitigate AMR. The AMR mitigation strategies include but are not limited to development of non-antibiotic alternatives to antibiotics, manipulation of the gut microbiota to improve gut health, boostering innate defense to enhance disease resistance, and vaccine development. Historically, the AMR has been one of important research areas of NC1202 multistate project and will continue to be an significant topic of enteric diseases of food animals.  Notably, the AMR component of this new NC1202 project does not duplicate another new project with focus on AMR (NC_temp1206).  
   

A search of CRIS database (http://cris.csrees.usda.gov/search.html) showed that no other multistate research projects focused on enteric diseases of food animals. Therefore, NC1202 will continue to be a leader and important contributor to research on the enteric diseases of swine, cattle, and poultry in the US. The CRIS search did reveal several approved individual research projects that have some degree of overlap with the multistate project proposed here; however, those individual Project Directors are active participants in the NC1202 multistate research project.

IMPORTANCE & CONSEQUENCES.  In 2015, the USDA ERS estimated that foodborne illnesses in the US impose over $15.5 billion in economic burden on the public each year. Approximately $14 billion/year are for medical costs, productivity losses, and costs of premature deaths for diseases caused by the top five leading 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. Diarrheal diseases account for multi-million dollar annual economic losses to the food animal industry due to reduced weight gain, mortality of young animals and treatment costs. Neonatal diarrhea and post-weaning diarrhea and diarrhea in grow/finish pigs are the most important clinical conditions of swine and are caused by pathogenic bacteria and viruses including ETEC, Lawsonia, Brachyspira, PEDV and RV. 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 EHEC O157:H7 to the beef industry from 1993-2003 was estimated at $2.671 billion. 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. FoodNet indicates significant progress in control of illness caused by Campylobacter, Salmonella, E. coli, Listeria, Cryptosporidium and Yersinia. The decline in incidence has resulted from the 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 food-borne agents approaches 2010 targets, rates can be further reduced with new knowledge and new detection procedures developed through this collaborative research. Production systems for food animals have evolved toward larger size and complexity. At the same time, there is a new initiative to withdraw the use of growth promoting antibiotics. Due to the recent ban on use of antibiotics for growth promotion in food animals, there is a new push towards natural, grass-fed, and organic production systems. These new animal systems pose new challenges associated with food-borne pathogens as animals encounter diversely contaminated water and soil. 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. The consequences of inaction are increases in disease and costs accompanied by burgeoning chronic disease rates.

FEASIBILITY. Based on the recent improved 2011 estimate by CDC, foodborne illness is still a common, costly, yet preventable, public health problem. The CDC estimates that each year roughly 1 in 6 Americans (or 48 million people) get sick, 128,000 are hospitalized, and 3,000 die of foodborne diseases.  The significant foodborne pathogens include but are not limited to norovirus, EHEC O157, Listeria, Campylobacter, Salmonella, Clostridium perfringens, Staphylococcus aureus. Progress has been made in preventing and controlling foodborne illness; the incidence of several high impact pathogens has declined based on targeting them for control and prevention. The 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 diverse areas of expertise are needed to devise scientific strategies for pathogen control, to educate agricultural experts and producers, and to apply new 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 NC1202 group has bacteriologists, virologists, molecular biologists, epidemiologists, pathologists, systems biologists and immunologists with a history of successful collaboration and productivity in developing innovative strategies.  The tangible research benefits of the multistate committee collaboration are highlighted in following specific “Objective 4. Group interaction” section of this proposal.

 

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 and 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. We also expect to develop effective and sustainable approaches to mitigate AMR.  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. A major expected outcome will be increased understanding of the 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 NC1202 project addresses critical, timely, cross-cutting research areas and objectives (e.g. antimicrobial resistance, intestinal microbiome) that will enhance food safety while maintaining efficient pork, beef and poultry production.

 

Related, Current and Previous Work

Our NC1202 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.  During the last 5 years, we have published over 300 peer-reviewed scientific manuscripts, held annual NC1202 meetings in conjunction with the Conference of Research Workers in Animal Diseases (CRWAD), held two animal health symposia and trained people at all levels of sophistication to the prevention and control of animal and human diseases caused by enteric pathogens.  To enhance national collaborations for solving challenging agriculture issues, NC1202 became the first multistate committee to include faculty from 1890 institutions in membership in 2016. This section shows a small selection of projects to demonstrate our productivity and show our follow-up strategies.

Enterohemorrhagic E. coli (EHEC) and other Shiga toxin (Stx)-producing E. coli (STEC) in cattle (Moxley Lab, Nebraska). EHEC are important foodborne pathogens, both nationally (Scallan, et al., 2011) and globally (Majowicz et al., 2014). These organisms cause severe illness in humans, including hemorrhagic colitis and hemolytic uremic syndrome (HUS), with raw and undercooked beef constituting important sources (Kaper and O’Brien, 2014). The U.S. Department of Agriculture, Food Safety and Inspection Service (USDA, FSIS) has declared seven serogroups of EHEC (STEC positive for the colonization factor, intimin) as adulterants in raw, non-intact beef (USDA, FSIS, 2012). These seven serogroups are O26, O45, O103, O111, O121, O145 and O157. Collectively, the first six serogroups are called non-O157 EHEC, which are estimated to cause approximately 71% of the illnesses attributable to EHEC in the United States (Brooks et al., 2005). A major problem with prevention of non-O157 EHEC illnesses is their reliable detection in pre- and post-harvest samples, which is needed to identify infected cattle and contaminated beef. Progress has been made, in part due to our efforts through NC-1202 and other USDA funding sources (Moxley et al., 2015; Stromberg et al., 2015a, 2015b, 2015c, 2016a, 2016b, 2016c; Woods et al., 2016). However, a major problem, both with culture and molecular methods for different reasons, has been the difficulty in distinguishing these organisms from background microbiota, especially from other E. coli and Enterobacteriaceae (Moxley & Acuff, 2014).

Pathophysiology of Enterotoxigenic E. coli (ETEC) in swine (Moxley Lab, Nebraska). ETEC remain a significant problem to the swine industry, although significant advances in knowledge have been made regarding the pathophysiology of these organisms, accomplishments due in part to past NC-1202 funded research (Wijemanne et al., 2014, 2015). However, a major problem is that an effective post-weaning vaccine is still not available, and consequently an effective way to prevent infection of weaned pigs is still lacking to the swine industry.

Porcine epidemic diarrhea virus (PEDV) (Saif/Jung Lab, Ohio). The mechanisms by which PEDV infection induces greater disease severity and deaths in nursing versus weaned pigs, have been defined in our previous studies (Annamalai et al., 2015; Jung et al., 2015; Jung and Saif, 2015). Goblet cells are critical to maintain intestinal homeostasis and integrity of intestinal epithelium. Based on our studies, we speculate that during early stages of PEDV infection, goblet cell mucins in the small intestine may be reduced, possibly leading to an impaired mucus layer and increased susceptibility to secondary enteric bacterial or viral infection. These hypotheses will be tested in our future studies.

Immune response to porcine epidemic diarrhea virus (PEDV) (Yoo Lab, Illinois).  Porcine epidemic diarrhea virus (PEDV) is an emerging and highly contagious virus in the US (Stevenson et al. 2013). PEDV caused major economic losses to the pork industry (Lee 2014). The immune response, especially the early innate response, is essential to protect host cells from invading viruses. Accumulating evidence indicates that type I IFN also has pleiotropic effects on immune cells and promote adaptive responses (Kasper and Reder 2014; Piconese et al., 2015). The role of type III in the intestinal epithelium is critical for antiviral host defense and cannot be replaced with type I IFNs (Pott et al., 2011; Mahlakoiv et al., 2015). Therefore, such principle cytokines are ideal targets of invading viruses to disarm the host immune surveillance.  Therefore, an understanding of viral antagonism against host innate immunity is essential for the rational design of prophylactic and control strategies as well as for advancement of our knowledge on virus-host interaction and pathogenesis. PEDV is an RNA virus that belongs to the Alphacoronavirus genus in the family Coronaviridae (Marthaler D. et al., 2013). It has been shown that the rapid production of type I IFNs by plasmacytoid DCs upon exposure to coronaviruses efficiently inhibits virus replication (Totura and Baric 2012).  Overall, type I IFNs seem to restrict various coronaviruses, and many coronaviruses code for IFN antagonizing proteins. Limited information is available for PEDV as to the importance of host innate immunity and the virus-cell interactions for viral modulation of type I IFN production (Ding et al, 2014). There is a need to conduct such studies to understand the pathogenesis of PEDV, which will in turn provide new insights into the design of a new vaccine candidate for control of the disease.

Pathogenesis and emerging antibiotic resistance in Campylobacter (Zhang Lab, Iowa) Campylobacter jejuni clone SA is a zoonotic foodborne pathogen and is responsible for the majority of sheep abortion in the United States. We analyzed its prevalence in feedlot dairy cattle and found it accounted for approximately 7% of the C. jejuni population from ruminants. We also developed a “directed genome evolution” strategy to understand why this clone is hypervirulent.  This strategy successfully identified SNPs in porA, encoding the major outer membrane protein (MOMP).  MOMP is responsible for its ability to induce systemic infection and abortion (1). Additionally, we found that fluoroquinolone-resistant Campylobacter is increasingly prevalent in ruminants and a multi-drug resistant gene cfr(C) has emerged in florfenicol-resistant Campylobacter. These findings provide key information for prevention and control of foodborne transmission of Campylobacter in animal reservoirs and to humans via the food chain.

Evolution of virulence and the role of Campylobacter in Autoimmunity (Mansfield Lab, Michigan). We have made significant progress in pathogenesis studies of  Campylobacter jejuni by developing several mouse models that permit the study of strains with selected gene knockouts. We developed mouse models of autoimmune diseases arising after C. jejuni infection including Inflammatory Bowel Disease and Guillain Barré Syndrome.  We know that the evolution of C. jejuni in the host can lead to changes in its surface coat that trigger autoimmune disease in genetically susceptible hosts and that antimicrobial resistance can enhance such disease.

Lawsonia intracellularis enhances shedding of Salmonella enterica (Isaacson Lab, Minnesota). Our data has shown that pigs co-infected with S. enterica serovar Typhimurium and L. intracellularis shed higher levels of S. enterica and for a longer period of time (Borewicz, et al, 2015 and Patterson, et al, 2016). Our hypothesis is that an L. intracellularis infection of pigs increases the risk of salmonellosis in humans. The goals of this project are to determine the duration and quantity of S. enterica shed by pigs co-infected with L. intracellularis and to determine if vaccination with an L. intracellularis specific vaccine mitigates S. enterica shedding. A third goal is to quantify microbiome changes in the intestinal tract in response to infections with these two pathogens to identify new targets that could be exploited to reduce shedding of S. enterica.

Organoids for study of Lawsonia intracellularis pathogenesis (Gebhart Lab, Minnesota). Lawsonia intracellularis is the etiological agent of proliferative enteropathy (PE) which is responsible for high economic losses in pig production worldwide (Vannucci and Gebhart, 2014). The absence of an in vitro model to study the pathogenesis of proliferation caused by L.intracellularis in the small intestine has limited the understanding of PE pathogenesis and therefore, constrains the development of methods for control and prevention of the disease. Intestinal organoids are a promising in vitro model for PE because they resemble the primary tissue architecture (Fatehulla, et al, 2016, Zhang, et al 2014). In addition, the establishment of an intestinal organoid model for L.intracellularis infection will broaden the interest in the veterinary research community for the use of organoids as an improved in vitro technique that can be an alternative to decrease the use of animals as in vivo models.

Comparative genomic analyses of Brachyspira hyodysenteriae and B. hampsonii (Gebhart Lab, Minnesota). Anecdotal information and data from experimental trials have indicated that different strains of B. hyodysenteriae can cause varying levels of clinical severity in the host. The completion of the first genome of B. hyodysenteriae in 2009 identified many additional putative virulence factors including some chemotaxis-related, adhesion and/or surface proteins, proteases, peptidases, anykyrin-like proteins, phages and a plasmid (Bellgard et al, 2009), few of which had been studied before. Although experimental trials have demonstrated that both clades of B. hampsonii show clinical severity comparable to virulent B. hyodysenteriae strains (Vannucci et al, 2013), no efforts have been made to understand the genetic factors responsible for this virulence. The limited knowledge of the genetic factors associated with virulence of B. hyodysenteriae and/or B. hampsonii continue to impede the development of specific diagnostic assays and vaccines. The proposed research is the first attempt to identify the genotypic and phenotypic characteristics of B. hyodysenteriae and B. hampsonii using novel genomic, molecular and microbiological approaches.

Emerging and re-emerging enteric food- and water-borne agents: pathogenesis, epidemiology and antimicrobial resistance (Shah/Sischo Lab, Washington). The WSU Food- and Water-borne Disease Research Group conducts research on the agents responsible for diverse food- and water-borne zoonotic diseases that affect both food animals and people. The primary research goals include investigations on identifying and characterizing emerging and re-emerging enteric food- and water-borne agents (Salmonella enterica, Campylobacter, Escherichia coli, Listeria monocytogenes, Vibrio parahaemolyticus, etc.) including factors underlying their prevalence, distribution and antibiotic resistance (Afema et al., 2015, 2016; Shah et al., 2016; Subbiah et al., 2011, 2016; Besser et al., 2014; Davis et al., 2015; Campioni et al., 2013).  Using molecular and genetic approaches, we are identifying genes and genetic markers of food- and water- borne pathogens for the development of new or improved molecular detection tools, immunoprophylactics (eg., antibodies, vaccines) and therapeutics (eg., probiotics) (Al-Adwani et al., 2013; Paul et al., 2013; Crespo et al., 2013; Shah et al., 2012; Short et al., 2016). The unit also collaborates with other Washington State agencies, including the Public Health Laboratory and the Department of Ecology, in surveillance for emerging infectious diseases. The unit plans to continue to focus on emerging infectious disease detection and prevention.

Campylobacter jejuni physiology, epidemiology, and control (Rajashekara Lab, Ohio). We characterized the contributions of transducer like proteins (Tlps), polyphosphate kinase, respiratory proteins, and the twin-arginine translocations system to the pathobiology of C. jejuni (Chandrashekhar et al., 2015 a,b; Pina-Mimbela et al., 2015; Drozd et al., 2014; Malde et al., 2014; Gangaiah et al., 2009 and 201; Kassem et al., 2011, 2012, 2013, 2014). We showed that formate, a small chain fatty acid and a byproduct of food breakdown in the gut, might affect both energy metabolism and microaerobic survival in C. jejuni (Kassem and Rajashekara 2014).  We characterized the epidemiology and antimicrobial resistance of Campylobacter in different food animals, including cattle and turkeys (Sanad et al., 2011, 2013, 2014; Kashoma et al., 2014, 2015, and 2016; Kassem et al., 2016). We investigated the efficacy of on-farm practices to control litter as a source of Campylobacter infection within and between flocks (Kassem et al., 2010). Using high-throughput chemical screens, we identified small compounds for control of Campylobacter (Kumar et al., 2016) and Salmonella.  Our recent results suggest that EcN impedes C. jejuni invasion and intracellular survival by affecting HT-29 cells barrier function and tight junction integrity, which would help us develop non-antibiotic approach to control Campylobacter.

Porcine epidemic diarrhea virus (Wang Lab, Ohio). Our research team aims to generate safe and effective, live attenuated vaccines for PEDV. Dr. Wang’s laboratory is currently studying which genes and mutations contribute to PEDV virulence and immunogenicity in pigs (Lin et al., 2015a; Lin et al., 2015b; Liu et al, 2016). Virus stocks of the highly virulent PEDV and its infectious dose have been determined as a standardized challenge pool to evaluate PEDV vaccine efficacy in the future (Liu et al., 2015). Our studies will generate live attenuated PEDV vaccine candidates, and expand knowledge of immunology, pathology and molecular biology of PEDV infection in pigs. We will also investigate whether there are natural reservoirs for PEDV, such as wild pigs.

Porcine deltacoronavirus (Saif/Jung Lab, Ohio). Our previous study verified the enteropathogenicity of porcine deltacoronavirus (PDCoV) in young pigs (Jung et al., 2015). Cultivable, cell culture-adapted PDCoV (TC-PDCoV) strains were also isolated in our previous study (Hui et al., 2015). The TC-PDCoV strain OH-FD22 has been serially passaged in LLC-PK cells (Jung et al., 2016). Our findings suggest that evaluation of a higher cell-culture passaged TC-PDCoV OH-FD22 strain is needed to verify attenuation and vaccine potential.

Caliciviruses (Wang Lab, Ohio). Salad crops and fruits are increasingly recognized as vehicles for human norovirus (HuNoV) transmission. We found that H-type HBGA-like carbohydrates exist in lettuce tissues and that GII.4 HuNoV VLPs can bind the exposed fucose moiety, possibly in the hemicellulose component of the cell wall (Esseili et al., 2012; Gao et al., 2016). These results suggest that specifically bound HuNoVs cannot be removed by simple washing, which may allow viral transmission to consumers. We also attempted to grow HuNoVs in vitro (Takanashi et al., 2014) and performed molecular epidemiological studies of porcine caliciviruses (Oka et al., 2016).

RV epidemiology in animals (Saif/Vlasova Lab, Ohio). A notable advance is the recent development of RT-PCR assays to monitor the epidemiology of group A,B and C RVs by NC1202 collaborators (Amimo et al., 2013a; Amimo et al., 2013b). An important finding is the emergence of new genotype of group C RV as a major cause of diarrhea in neonatal piglets (Amimo et al., 2013b (78)). Comprehensive epidemiology study of RVs is critical to evaluate vaccine efficacy and aid in the design of new vaccines to prevent RV disease across animal species and humans.

Prevention using probiotics (Saif/Vlasova/Rajashekara Lab, Ohio). We have used mono- and dual-probiotic associated Gn pig models demonstrated that species-specific effects of different probiotics were associated with immune maturation, immune stimulation and regulation of the immune homeostasis depending on the microenvironment (Chattha et al., 2013; Vlasova et al., 2013; Kandasamy et al., 2014; Kandasamy et al., 2016; Vlasova et al., 2016a; Vlasova et al., 2016b).  Recently, we also have completed extensive studies on the role of probiotics treatment on host immunity, physiology, and viral infections.

Pathogenesis mechanisms of ETEC and vaccine development (Zhang Lab, Kansas; Moxley Lab, Nebraska). ETEC strains are the predominant cause of diarrhea in neonatal and post-weaning diarrhea (PWD) in pigs.  Diarrhea is also the main reason for using antibiotics in herds of swine.  Vaccination is the most economical and likely effective approach to control PWD and to reduce antibiotic use. Unfortunately, there are still no efficacious vaccines currently available against PWD caused by ETEC. This laboratory focuses on the elucidation of a better understanding of pathogenesis of ETEC diarrhea and the development of vaccines against ETEC diarrhea. Over the past years, we have developed and applied toxoids and toxoid fusions for safe and effective immunogens for ETEC vaccine development, and have invented MEFA (multiepitope fusion antigen) technology for multivalent and broadly protective vaccines against ETEC diarrhea (Duan et al., 2012, 2013; Fekete et al., 2013; Harshish et al., 2013; Nandre et al., 2016; Rausch et al., 2016;cRuan et al., 2012, 2013, 2014, 2015; Santiago-Mateo et al., 2012; Zhang et al., 2012, 2013, 2014, 2015)

Prevention and control of enteric bacteria and viruses (Chang/Hardwidge/Nagaraja/Renter Labs, Kansas). We use interdisciplinary research and outreach teams to generate and disseminate important research results relevant to prevention and control of important pathogens such as E. coli O157:H7, other Shiga toxin-producing E. coli, Salmonella, Campylobacter, Norovirus, and antimicrobial resistance of enteric bacteria, primarily in beef and pork production systems. Through our focus on disseminating new knowledge to stakeholders, as well as our direct collaboration and interaction with beef and pork producers, these research outcomes had an immediate impact on human and animal health risks thereby providing benefits for public health and food animal production systems (Agga et al., 2015; Amachawadi, 2015; Cernicchiaro, 2014, 2016; Chen, 2016; Chopyk, 2016; Cull, 2015; DeMars, 2016; Dewsbury, 2015; Feuerbacher, 2014; Ison, 2015; Kankanamalage, 2015; Kim, 2015, 2016; Kumar, 2015; Noll, 2016; Ruter, 2014; Shivanna, 2014, 2015; Shridhar, 2016; Smith, 2016; Stromberg, 2015; Zhou, 2015)

High-affinity iron acquisition in C. jejuni (Lin Lab, Tennessee). The high affinity enterobactin (Ent)-mediated iron scavenging is tightly linked to Campylobacter pathogenesis.  Our molecular studies also have revealed novel features of FeEnt acquisition in Campylobacter and led to a new model for FeEnt acquisition (Xu et al., 2010, 2015; Zeng et al., 2009, 2013a, 2013b; Zeng & Lin, 2014, 2016).  By targeting FeEnt receptor CfrA, different vaccination strategies have been developed and evaluated for Campylobacter control in poultry (Sahin et al, 2015; Liu et al., 2016). Together, the FeEnt iron acquisition system in C. jejuni offers promising targets for intervention against Campylobacter infection. 

Antimicrobial resistance (AMR) in C. jejuni (Lin Lab, Tennessee). Campylobacter has become increasingly resistant to various antibiotics and we have examined emergence, transmission and molecular mechanisms of AMR for developing effective mitigation strategies. Specifically, we have made significant discoveries for antimicrobial peptide resistance (Hoang et al., 2011a, 2011b, 2012), beta-lactam resistance (Zeng, 2013c, 2014a, 2015a), conjugative gene transfer (Zeng, 2015b), and chicken gut resistome (Zhou, 2012).

Alternatives to antibiotic growth promoters (AGPs) (Lin Lab, Tennessee).   Developing effective alternatives to AGPs is urgently required in order to maintain current animal production levels without threatening public health.  Our recent functional microbiota research in chicken strongly suggested that intestinal bile salt hydrolase (BSH) is a key mechanistic microbiome target for developing novel alternatives to AGPs; we have identified a unique BSH enzyme from a chicken Lactobacillus salivarius strain, developed an efficient high-throughput screening system to discover BSH inhibitors, and performed a series of functional, structural, and broiler studies to develop innovative alternatives to AGPs (Geng & Lin, 2016; Lin et al., 2013, 2014; Lin 2014; Smith et al., 2014; Wang et al., 2012; Xu et al., 2016).

Surveillance of antimicrobial resistance (Bisha Lab, Wyoming).  The incidental exposure of wildlife to antibiotics at anthropogenic foci of antimicrobial use (e.g., livestock operations and urban areas), coupled with the ecological propensities of particular wildlife species (e.g., movement capabilities and AMR carriage across agricultural landscapes and the subsequent deposition of AMR through fecal shedding), is recognized as a potential mechanism to promote the dissemination of AMR throughout the food production continuum (Radhouani et al., 2014; Carroll et al., 2015; Greig et al., 2015). To evaluate the wildlife-associated AMR problem, we couple ecosystem-level understanding of the wildlife-agricultural interface with advanced laboratory evaluation for AMR surveillance and the elucidation of specific AMR phenotypes and mechanisms.

Diagnostics of foodborne pathogens (Scaria Lab, South Dakota). The South Dakota Animal Disease Research and Diagnostic Laboratory (SD-ADRDL) receives large number of samples from swine, cattle and poultry operations in the Midwest. We isolate  foodborne pathogens such as Salmonella enterica, Escherichia coli and various species of the genus Clostridium from these samples for routine diagnostics. Typing and characterization of these isolates for their potential to cause outbreaks will be beneficial in controlling these pathogens in the food supply system. We will use Next Generation Sequencing (NGS) methods to type and track these pathogens isolated by SD-ADRDL. Data from the NGS will be deposited in NCBI for public access.

PEDV diagnostics and vaccine (Ramamoorthy Lab, North Dakota). Despite improved technology, the lag time in developing effective vaccines and diagnostic tests typically takes several months to years, depending on the complexity of the pathogen. Therefore, the two major current focus areas of this station are to develop rapid response diagnostics and vaccines for newly emerging pathogens, using PEDV as a model (Ssemadaali, 2016; Song, 2016; Singh, 2016)

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 pathogens of livestock 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

Detection of non-O157 EHEC and other STEC (Moxley Lab, Nebraska & Nagaraja Lab, Kansas). This collaborative project will develop and validate  new and improved culture and molecular detection methods for non-O157 EHEC and other STEC. This work will be a continuation of previous efforts as described in cited publications above. Studies to improve culture methods will target better control of background microbiota and a search for unique biochemical markers for the specific identification of non-O157 EHEC. Studies to improve molecular detection will focus on validation and application of new methodologies and reagents, e.g., waveguide-based optical biosensor, multiplex oligonucleotide-ligated (MOL)-PCR, and continued development and implementation of monoclonal antibodies for non-O157 EHEC.

Field surveillance and molecular epidemiology of food- and water- borne pathogens and antimicrobial resistance (Shah/Sischo Lab, Washington). The unit is conducting field surveillance studies for food- and water- borne pathogens from food animals in WA state using traditional microbiological methods and newer molecular methods (e.g., PCR). Antimicrobial resistance profiling is conducted following CLSI guidelines. For molecular characterization, the unit is using PFGE and MLVA. Next generation sequencing tools (e.g., illumina, ION) are used for sequencing whole genome and transcriptomes of pathogens. Comparative genomics studies are conducted using commercial and open source bioinformatics software (e.g., Geneious Pro, CLC Bio, etc.). For identifying genes and genetic markers, we are utilizing high throughput mutagenesis, transcriptomics and proteomic approaches.    

Develop rapid response serological assay against PEDV (Ramamoorthy lab, North Dakota). A combination of bioinformatic and wet lab methods will be used. Immunogenic targets for B cells will be predicted using immune-informatic tools. Diagnostic antigen targets will be rapidly synthesized using invitro transcription and translation. The developed assay was both sensitive and specific in detecting antibody responses to PEDV.

Epidemiology of RV in animals (Saif and Vlasova Lab, Ohio). We will determine the epidemiology of RV in pig populations by RT-PCR and sequencing.

Rapid and specific identification of antibiotic resistant bacteria (Bisha lab, Wyoming). We will develop methods using Matrix-Assisted Laser Desorption Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS) and other mass spectrometry-based methods for rapid and specific identification and characterization of antibiotic resistant (AMR) bacteria from food, food animals, wildlife and environment. We are devising inexpensive paper-based analytical devices (μPADs) amenable to field-based detection of bacterial pathogens and indicator bacteria via a simple coupling of chromogenic substrates processed by enzymes indicative of target microorganisms, and have partnered with the company Access Sensor Technologies to facilitate the process. We will employ a ‘multi-omics’ approach for determination and characterization of AMR, including phenotypic (culture-based, biochemical testing, and MALDI-TOF MS) and genotypic (whole genome sequencing and metagenomics). We will focus our examinations to AMR derived from wildlife, food production animals, and associated environment. By combining the data outputs of these methods, the acquired information will provide needed insight into the magnitude and causation of the wildlife-associated AMR problem, and help determine what actions should be taken to limit this risk.

New approaches for diagnostics of foodborne pathogens (Scaria, South Dakota). To develop NGS based tracking methods, we will sequence whole genomes of at least 500 isolates of foodborne pathogens with the final goal of using the whole genome data as the references for tracking any outbreaks or infections originating from animal or food sources. Sequencing reactions will be carried out using Illumina 2x250 cycles paired end sequencing chemistry (Amachawadi RG et. Al 2016). Mapping of the virulence genes from each sample will be carried out by sequence homology searches against virulence factor database (VFDB) data. We have installed a local copy of VFDB and this will allow us to complete these searches in automated mode.

 

OBJECTIVE 2. FOCUS ON PREVENTION AND INTERVENTION

Pathobiology of ETEC and vaccine development (Zhang Lab, Kansas). Equipped with the safe and immunogenic toxoid and toxoid fusion antigens and the MEFA technology, we can finally develop a broadly protective oral vaccine against ETEC diarrhea.  We will identify neutralizing epitopes from each individual ETEC adhesin and enterotoxin, integrate individual epitopes into a backbone immunogen, express this multivalent immunogen in a vaccine vector strain for an oral vaccine, and confirm vaccine efficacy in pig challenge studies.  This technology also provides us a platform to develop multivalent vaccines against other enteric diseases. We will also continue our efforts in understanding disease mechanism and virulence prevalence, including identification of novel virulence factors or vaccine antigens and examination effect of vaccination at pig but health.  That further improves our effort in developing broadly effective ETEC vaccines.

Microbiomes and enteric pathogens (Hardwidge, Nagaraja, Renter Labs, Kansas). We are developing and using techniques to study the role of the intestinal microbiota in the control of enteric pathogens in both livestock and in the mouse pathogen Citrobacter rodentium, which is used as a model organism for the study of attaching/effacing pathogens.

Manipulating the pathogenesis Campylobacter jejuni and enterohemorrhagic E. coli (EHEC) with the microbiome (Mansfield Lab, Michigan). We are studying the role of the microbiome in acute enteritis due to Campylobacter jejuni (invasive) and enterohemorrhagic E. coli (EHEC; adherent). We expect to determine which cellular signals and pathways are differentially regulated upon adherence, invasion or translocation of humanized microbiota mice by C. jejuni or EHEC in absence/ presence of innate cells or particular microbiota.

Evaluation of ETEC vaccine (Moxley Lab, Nebraska). We will test the protective efficacy of novel vaccines against post-weaning enterotoxigenic Escherichia coli of swine developed by the Kansas station (Zhang). These studies will involve the development of a novel live fimbria-toxoid multiepitope fusion antigen (MEFA) vaccine that will be tested in post-weaned conventional pigs.

Campylobacter and Salmonella studies (Rajashekara Lab, Ohio). We are deploying traditional and molecular microbiological techniques in our research on Campylobacter and Salmonella. These include: 1) high throughput screening of small molecules, 2) metagenomics (16S rRNA gene sequencing) to characterize gut microbiome, 3) mutagenesis; 4) physiological assays for respiration, 5) quantitative real time PCR to quantify gene expression and gene copy numbers in matrices, 6) standard isolation and enumeration culture techniques, 7) broth microdilution assay to test antimicrobial resistance, 8) PCR arrays to test innate and adaptive immunity in response to probiotic. 

Campylobacter (Zhang Lab, Iowa). We will utilize animal models and molecular techniques to understand how the sequence polymorphisms in the major outer membrane protein affect the virulence of C. jejuni clone SA. Recombinant DNA technologies will be used to evaluate a subunit vaccine against sheep abortion. Whole genome sequence analysis and animal models will be used to detect emerging antibiotic resistance and the persistence of antibiotic-resistant Campylobacter. Molecular and genetic tools will be used to analyze antibiotic resistance mechanisms and the factors influencing the development of antibiotic resistance in bacterial pathogens.

Lawsonia intracellularis enhances shedding of Salmonella enterica (Isaacson Lab, Minnesota). To determine the duration and quantity of S. enterica shed by pigs infected with S. enterica or S. enterica and L. intracellularis groups of pigs will be orally challenged with one or both microbes and S. enteric shedding monitored using a most probable number analysis tool. These data also will be compared to non-challenged pigs. A comparison of singly or co-challenged pigs that were vaccinated with a commercial vaccine against L. intracellularis will be performed and shedding S. enterica will be determined. Finally, DNA will be extracted from the collected fecal samples, subjected to amplification of the 16S rRNA gene, and sequenced to determine the composition of the microbiomes in these pigs.

Organoids for study of Lawsonia intracellularis pathogenesis (Gebhart Lab, Minnesota).  The general objective of this research is to evaluate the use of intestinal organoids as an in vitro model for the L. intracellularis infection. The specific aims are to establish the survival and growing rate of intestinal organoids in microaerophilic conditions, to determine a protocol for L. intracellularis inoculation in the organoids, to establish the best time point to inoculate L. intracellularis in the organoids, and to verify the optimum duration of L. intracellularis growth within the organoids. High passage L .intracellularis does not lead to enterocyte proliferation in vivo. Using organoids as an in vitro model, the same methodology described above will be used to verify differences between high and low passage L.intracellularis isolates regarding morpho- and histological changes in organoids. This will allow further investigations regarding mechanisms involved in enterocyte proliferation.

Comparative genomic analyses of Brachyspira hyodysenteriae and B . hampsonii (Gebhart Lab, Minnesota). B. hyodysenteriae strains “B204” and “field” will be grown anaerobically and the DNA will be extracted and purified and sent to University of Minnesota Genomics Center (UMGC) for whole genome sequencing. The resultant sequence reads will be assembled and mapped to a previously closed and annotated reference genome (strain “WA1”) using CLC Genomics workbench® .The aligned genomes will be visualized using MAUVE v2.3.1 to identify regions of difference in high-virulence and low-virulence strains, and a list of such genes will be recorded. Any newly identified genetic components of interest that are not annotated in the reference genome will be queried against the Conserved Domain Database (CDD) using a BLAST search in order to annotate such genes and predict their potential functions. A virulence gene presence/absence map will be created for all four strains and the association of the genotype with the phenotype will be tested using PLINK v1.07 and MATLAB.

Enterobactin (Ent)-mediated iron acquisition for Campylobacter infection (Lin Lab, Tennessee). We hypothesize that Campylobacter can utilize various catecholate siderophores and Ent specific antibodies function as an effective bacteriostatic agent against Campylobacter infection.  To test this, we plan to 1) determine utilization of various catecholate siderophores by Campylobacter and examine inhibitory effect of Ent antibodies on catecholates-dependent growth of Campylobacter; and 2) evaluate in vivo efficacy of Ent antibodies-based immune intervention strategies in a chicken model of C. jejuni infection. This project will not only improve our understanding of Ent-mediated high affinity iron acquisition in Campylobacter but also result in major conceptual advances in the development of new vaccine and therapeutics against C. jejuni and other Gram-negative pathogens.   

Salmonella and Campylobacter studies (Shah/Sischo lab, WSU). We are employing traditional and molecular biological tools in our research on Salmonella and Campylobacter. These include (i) high throughput screening of mutant libraries of these pathogens for pathogenicity, and persistence in the environment (ii) characterization of individual genes of these pathogens for their contribution to infectivity in food animals such as poultry to identify suitable vaccine candidates (iii) identification of non-antibiotic alternatives (e.g., CpG islands) as immunopotentiating agents to enhance innate immunity against Salmonella and (i) characterize microbiomes of dairy animals to identify suitable probiotic agents to reduce calf mortality and prevent infection with food borne pathogens.

Regulatory mechanisms of beta-lactamase expression in C. jejuni (Lin Lab, Tennessee). We have obtained compelling evidence showing that a lytic transglycosylase (LT), which cleaves peptidoglycan and generates muropeptide signal, plays a critical role in β-lactam resistance by inducing β-lactamase production in C. jejuni.  We hypothesize that the LT-mediated cell wall metabolism regulates the expression of β-lactamase in C. jejuni via multiple key molecular components and the LT is a promising target for the discovery of new antimicrobials for mitigation of β-lactam resistance.  We will identify and characterize the key players involved in the LT-mediated β-lactamase induction in C. jejuni using a panel of biochemical and molecular tools (e.g. RNAseq, in vivo pull down, and random transposon mutagenesis).  In addition, we will determine the LT crystal structure, perform structure-based inhibitor discovery, and evaluate LT inhibitors using both in vitro and in vivo systems.

Development of novel alternative to antibiotic growth promoters (AGP) (Lin Lab, Tennessee). We have previously identified bacterial enzymes called Bile Salt Hydrolases (BSH) which modify bile acids in the host and influence energy metabolism. Compelling evidence shows that inhibition of BSH activity would cause weight gain.  On this basis, we will develop a world-leading research program that will develop BSH inhibitors showing promise as non-antibiotic animal growth promoters in this project.  We also will test these novel growth promoters in a chicken husbandry model and will investigate the biological basis of the phenomenon using state-of-the-art metabolomics, metagenomics and computational approaches.

Develop non-antibiotic alternatives (Scaria Lab, South Dakota). To develop non-antibiotic alternatives for the control of antibiotic resistant bacteria, we will utilize the colonization resistance of the healthy gut microbiota. We will isolate bacteria from healthy microbiota that provide colonization resistance by identifying species that produce beneficial metabolites such as butyrate and propionate. Strains will be isolated using strict anaerobic culture methods and will be identified using MALDI-TOF. Inhibitory of these strains against drug resistant bacteria will be then identified using co-culture assays.

Campylobacter studies (Rajashekara Lab, Ohio). We are deploying traditional and molecular microbiological techniques in our research on Campylobacter and Salmonella. These include: 1) high throughput screening of small molecules, 2) metagenomics (16S rRNA gene sequencing) to characterize gut microbiome, 3) mutagenesis; 4) physiological assays for respiration, 5) quantitative real time PCR to quantify gene expression and gene copy numbers in matrices, 6) standard isolation and enumeration culture techniques, 7) broth microdilution assay to test antimicrobial resistance, 8) PCR arrays to test innate and adaptive immunity in response to probiotic. 

Immune response to porcine epidemic diarrhea virus (PEDV) (Yoo Lab, Illinois).  IFN antagonisms mediated by PEDV will be determined in cells expressing individual genes using reporter assays. Porcine reproductive and respiratory syndrome virus has been known as an IFNα suppressor and will be used as a control virus. All of PEDV genes will be cloned and expressed in cells. Cells expressing each gene of PEDV will be stimulated, and IFN modulation will be determined using the Dual luciferase assay system. The findings from the reporter assays will be validated by VSV bioassays. The dilution corresponding to 50% of cells exhibiting GFP expression will be determined as the end-point inhibition. These assays are available in my laboratory and no difficulty is anticipated. To determine the cellular target for IFN suppression by PEDV, IRF3 and NF-κB pathways will be examined for individual viral proteins using the IRF3- and NF-κB-specific reporters, and this study will identify whether the inhibition is through the IFR3 pathway or the NF-κB pathway. Cellular proteins interacting with PEDV proteins will be identified by yeast 2 hybrid assays and their specific interaction will be determined. PEDV has been shown to activate NF-κB during the late stage of infection (Cao et al., 2015a; Cao et al., 2015b). However, viral activation of NF-κB is complicated and often time-dependent. Viral infections may inhibit the NF-κB activity at early times for optimal survival, and then activate it at late times for inflammation. It is probable that PEDV regulates NF-κB for production of the pro-inflammatory and anti-inflammatory cytokines. Limited information is available as to how PEDV modulates NF-κB during infection. PEDV N protein suppresses the NF-κB-mediated IFN-β production. In the coming years, the expression profiles of cytokines and chemokines induced by PEDV will be determined. All viral 23 genes will be examined and screened to identify the cytokine and chemokine regulators. Quantitative RT-PCR will be performed with SYBR green mix for swine cytokine and chemokine genes for differential expression of different cytokines. TNF-α, IL-1b, IL-6, and IL-15 will be examined. Also examined will be IL-6, IL-8, IL-10, MCP-1, and RANTES regulated by different PEDV proteins upon stimulation with TNF-α in PEDV protein expressing cells. We have established a system to determine such activities, and the assay system is in possession.

Porcine epidemic diarrhea virus (Saif/Jung Lab, Ohio): We will try to identify pharmacological or biological mediators such as epidermal growth factor that promote stem cell regeneration or maturation to shorten the time for epithelial cell renewal. Combined use of preventive (vaccination) and therapeutic interventions would synergistically reduce PEDV or/and PDCoV death losses from dehydration and enhance recovery from the diseases caused by PEDV, PDCoV, or both viruses.

Porcine deltacoronavirus (Saif/Jung Lab, Ohio). We will use cell culture assays combined with Gn pig studies and histolgical/immunohistochemistry analysis to determine the pathogenicity of PDCoVand to develop and assess vaccine candidates.  

Porcine epidemic diarrhea virus (Wang Lab, Ohio). We will combine traditional cell culture adaptation method and the state-of-the-art technology, reverse genetics, to rationally design safe attenuated PEDV vaccine candidates. We will perform both in vivo pig and in vitro cell culture experiments to study the phenotype (growth kinetics, fidelity, etc.) of PEDV. We also perform molecular epidemiology of enteric coronaviruses in wild pigs.

Caliciviruses (Wang Lab, Ohio). We will use virological and biochemical methods to perform studies on Caliciviruses.

Prevention using probiotics (Saif/Vlasova/Rajashekara Lab, Ohio).  Ongoing research focuses on new oral adjuvants (vitamins, probiotics) and vaccine approaches to improve RV vaccines using the neonatal gnotobiotic (Gn) piglet model transplanted with human infant fecal microbiota (HIFM). 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. We will use omics approaches (metagenomics, metabolomics and metatranscriptomics) to understand the impact of malnutrition on host metabolome/transcriptome, microbiome composition, gut immunity, HRV pathogenesis and HRV vaccine efficacy.

PEDV vaccine development (Ramamoorthy Lab, North Dakota).  The rapid-response PEDV vaccines will be developed by first-generation technology, using proprietary methods. Optimization of the process has been completed and the vaccine candidates are currently under test in a swine challenge model.

 

OBJECTIVE 3. FOCUS ON DISSEMINATING KNOWLEDGE

All personnel involved in this project 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.  Specific approaches are detailed in Outreach Plan.

 

OBJECTIVE 4. GROUP INTERACTION

NC-1202 members and their students will present their work in various national and international meetings. We will hold annual NC1202 meetings in conjunction with annual CRWAD meeting and continue to sponsor student awards for best oral and poster presentations at the CRWAD meeting.  NC1202 will continue to organize symposia focused on significant and timely animal health issues. 

The complexity and range of 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 NC1202 group has bacteriologists, virologists, molecular biologists, epidemiologists, pathologists, and immunologists with a history of successful collaboration and productivity in developing innovative strategies, partly reflected by joint grant/publications.  For example, following are some active integrated food safety projects in which our NC1202 members are project directors and involve several NC1202 members at the participating institutions as co-project directors: 1) Moxley, R. (PD, Univ. of Nebraska) and T.G. Nagaraja and David Renter (Co-PDs, Kansas State Univ.)  USDA NIFA Coordinated Agricultural Program Award, Shiga-toxigenic Escherichia coli (STEC) in the Beef Chain: Assessing and Mitigating the Risk by Translational Science, Education and Outreach; 2) Zhang, Q. (PD, Iowa State Univ.) and other Co-PDs who are NC1202 members: Jun Lin (Univ. of Tennessee), Gireesh Rajashekara (Ohio State Univ).  USDA NIFA Food Safety Challenge Grant, Novel Approaches for Mitigation of Campylobacter in Poultry; 3) Law, B. (PD, Univ. of Arizona) and other Co-PIs who are NC1202 members: Roy Curtiss III is a scientific advisor, and Ken Roland from the Curtiss lab is the Co-PI. USDA NIFA Food Safety Challenge Grant, The Development of an Efficacious Vaccine to Reduce Campylobacter in Chickens.  In the next five years, we will continue to keep active group interactions and develop competitive, multi-disciplinary, multi-institutional, and sustainable research to prevent enteric diseases in livestock and prevent zoonotic food and water-borne illnesses.

Measurement of Progress and Results

Outputs

• Development of an oral ETEC vaccine candidate
• Characterization of ETEC diarrhea disease mechanism
• Development of new and improved detection methods for EHEC
• Understand the modulatory mechanisms of PEDV for innate immunity of host.
• Identify PEDV components responsible for innate immune antagonism for type I and type III IFNs
• Successful development of the Human infant fecal microflora transplanted Gn pig provides a highly relevant model to comprehensively evaluate the interactions among enteric pathogens, diet and host factors and to test potential interventions
• Establishment of porcine small intestinal enteroids (IE) as an ex vivo preclinical platform will allow studies on interaction between human and porcine enteric pathogens, commensal and probiotic bacteria and macro-/micronutrients.
• The shedding of S. enterica in pigs infected with L. intracellularis will be measured and whether vaccination against L. intracellularis reduces shedding of S. enterica determined.
• We will evaluate the use of intestinal organoids as an in vitro model for L.intracellularis infection
• We will identify potential virulence-associated genes of Brachyspira hyodysenteriae and B. hampsonii to better understand the pathogenesis of swine dysentery and develop new vaccines and diagnostic tests.
• Dissemination of data to stakeholders through meetings and conferences, and publications in peer reviewed journals to share with the scientific community
• We have completed genetic characterization and antibiotic resistance profiling of several field strains of Salmonella isolated from cattle and poultry and STEC isolates from cattle in WA state
• We have identified several unique genes of Salmonella that contribute to pathogenicity in poultry
• Publications: refereed journal articles
• Products: reagents, detection methods, vaccines
• Presentations: scientific meetings, national and international
• New insights will be generated into the pathogenic mechanisms involved in systemic infection by C. jejuni clone SA.
• Significant new knowledge will be gained on the emergence, development and persistence of antibiotic-resistant Campylobacter.
• Successful development of practical and effective non-antibiotic alternatives to antibiotic growth promoters, such as BSH inhibitor, for enhanced animal health.
• Understand mechanisms of beta-lactamase induction and discover lytic transglycosylase inhibitors for mitigation of beta-lactam resistance in Campylobacter and other enteric pathogens.
• Develop novel iron-dependent immune interventions that effectively control the infections caused by Campylobacter and other Gram-negative pathogens.

Outcomes or Projected Impacts

• Improved detection of non-O157 EHEC
• Improved control of non-O157 EHEC
• Improved prevention of post-weaning ETEC infection of swine
• A broadly protective ETEC vaccine saves hundreds of millions dollars for the US swine producers.
• An effective ETEC vaccine reduces or eliminates antibiotic use in US swine farms
• Novel approaches to developing effective vaccines for intestinal pathogens in pigs
• A better understanding of disease mechanism improves efficiency in ETEC vaccine development.
• The WSU unit has a repository of >25,000 isolates of food- and water- borne pathogens.
• The WSU unit has trained 5 doctoral students, 3 Masters students and 3 post-doctoral fellows in the last 5 years.
• S. enterica is an important food borne pathogen and efforts to reduce shedding is important for public health. If vaccination against L. intracellularis does reduce shedding, it will be become an important tool to mitigate against food borne contamination with S. enterica.
• For the comparative genomic analyses of Brachyspira hyodysenteriae and B. hampsonii, using the virulence-associated genes identified as targets, it is expected that urgently needed diagnostic assays will be developed to detect infection or exposure to these pathogens. Additionally, it is expected that novel vaccine candidates targeting newly identified virulence-associated genes may be developed, which will dramatically improve the prevention of swine dysentery.
• This in vitro infection model for L.intracellularis infection will allow further investigations regarding the mechanisms of proliferation and help identify methods for control and prevention of proliferative enteropathy
• Understanding pathogenic mechanism will lead to novel strategies (such as vaccines) to control zoonotic transmission of Campylobacter.
• New intervention targets for antibiotic-resistant Campylobacter will be identified.
• Development of innovative strategies to control Campylobacter infection in humans and in animal reservoir would reduce the occurrence of foodborne illness in humans
• Our antimicrobial resistance studies may open new avenues for treatment and prevention of resistant foodborne pathogens important in animal health and food safety
• Research on the development of alternatives to antibiotic growth promoters will lead to novel ‘One Health’ measures for enhanced animal production, food safety, and human health.
• Development of new methods to control multistate outbreaks of foodborne pathogens such as Salmonella and E. coli
• Development of new direct-fed microbials that could be used non-antibiotic alternatives in livestock

Milestones

(2018):Sequence whole genome and transcriptomes of pathogens. New insights will be generated into the pathogenic mechanisms involved in systemic infection by C. jejuni clone SA. Identify PEDV components responsible for innate immune antagonism for type I and type III IFNs Rationally design safe attenuated PEDV vaccine candidates High throughput screening of small molecules against Campylobacter and Salmonella Discovery of innovative alternatives to antibiotics Identification and characterization of new drugs against enteric bacterial pathogens using high-throughput screening and virtual screening

(2019):Identify genes and genetic markers utilizing high throughput mutagenesis, transcriptomics and proteomic approaches. Significant new knowledge will be gained on the emergence, development and persistence of antibiotic-resistant Campylobacter Development of an ETEC vaccine candidate Metagenomics to characterize gut microbiome Determine the pathogenicity of PDCoV to develop and assess vaccine candidates Perform both in vivo pig and in vitro cell culture experiments to study the phenotype (growth kinetics, fidelity, etc.) of PEDV

(2020):New and improved culture and molecular detection methods for non-O157 Shiga toxin-producing Escherichia coli will be developed and validated Novel strategies (such as vaccines) to control zoonotic transmission of Campylobacter. Assessment of vaccine efficacy in pig challenge studies

(2021):Novel approaches to developing effective vaccines for intestinal virus in pigs Assessment of ETEC vaccine in reduction of antibiotic uses, and at effect of gut health. Development of innovative siderphore-based immune intervention against enteric Gram-negative pathogens

(2022):Novel vaccines against post-weaning enterotoxigenic Escherichia coli of swine will be developed. New intervention targets for antibiotic-resistant Campylobacter will be identified New oral adjuvants (vitamins, probiotics) and vaccine approaches are developed to improve RV vaccines using the neonatal gnotobiotic (Gn) piglet model transplanted with human infant fecal microbiota (HIFM).

Projected Participation

View Appendix E: Participation

Outreach Plan

Over the next 5 year period our NC1202 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. Following are our major outreach plan 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. Our members in NC1202 group are a distinguished and highly productive group who published over 300 peer-reviewed 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 NC1202 members and their students will present their work in numerous national and international meetings. Each year NC1202 also will hold a two-day meeting in conjunction with the CRWAD meeting; all units will present their progress and findings to the group and provide a written AES unit report.  


• SEMINARS/SEMINAR SERIES. Each member of the NC1202 group will actively give seminars each year to impart research results and promote input and sharing of information at different levels.


• GRADUATE STUDENT TRAINING. For our group, graduate education is a continuing commitment in order to train the next generation of scientists in enteric diseases of food animals. In the next 5 years we will continue to provide excellent training and opportunities for graduate students.


• INTERNET OUTREACH. A major emphasis will be keeping the state agricultural experiment station websites up to date and accurate for annual productivity of the NC1202 group.

 

Organization/Governance

The recommended Standard Governance for multistate research activities include the election of a Chair and a Secretary (also the Chair-Elect).  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 Secretary is responsible for documenting the progress content of the meeting, communicating the results to the membership, and preparing NC1202 annual report for submission to NIMSS.

Furthermore, ad hoc committees will be formed to organize specific initiatives including research initiatives and formal and informal meetings to disseminate results.  For example, we have gained experience from recent organization of two animal health symposia.  In the next five years, specific symposium topic will be identified in annual NC1202 meeting and a special adhoc committee will be assembled to organize symposium. A chairperson along with the committee will be responsible for organizing the plenary presentations, abstract submissions and selection and the venue arrangements.

Notably, NC1202 is the first multistate committee to include faculty from 1890 institutions in membership in the state.  In December 2015, four faculty from 1890 institutions attended NC1202 annual meeting; subsequently, two faculty members (from Tuskegee University and North Carolina A&T State University, respectively) joined NC1202 membership in early 2016.  NC1202 group fully recognize inclusion of 1890 faculty (as well as 1862 faculty) for working together to solve challenging and important agricultural issues.  In the next five years, we will continue to invite new 1890 faculty or other minority-serving institution faculty to join NC1202 group.

 

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Kandasamy S, Vlasova AN, Fischer D, Kumar A, Chattha KS, Rauf A, Shao L, Langel SN, Rajashekara G, Saif LJ. Differential Effects of Escherichia coli Nissle and Lactobacillus rhamnosus Strain GG on Human Rotavirus Binding, Infection, and B Cell Immunity. J Immunol. 2016 Feb 15;196(4):1780-9. doi: 10.4049/jimmunol.1501705. PubMed PMID: 26800875; PubMed Central PMCID:PMC4744595.

Kankanamalage AM. Uy WP, Mandadapu SR, Alliston KR, Chang KO, Kim Y, Lovell S, Groutas WC. Structure-Based Design and Optimization of Dipeptidyl Inhibitors of Norovirus 3CL Protease. Structure-Activity Relationships and Biochemical, X-ray Crystallographic and Cell-Based Studies. 2015. J Med Chem. 58(7):3144-55.

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Kashoma IP, Kumar A, Sanad YM, Gebreyes W, Kazwala RR, Garabed R, Rajashekara G. Phenotypic and genotypic diversity of thermophilic Campylobacter spp. in commercial turkey flocks: a longitudinal study. Foodborne Pathog Dis. 2014 Nov;11(11):850-60

Kashoma IP, Kassem II, Kumar A, Kessy BM, Gebreyes W, Kazwala RR, Rajashekara G. Antimicrobial Resistance and Genotypic Diversity of Campylobacter Isolated from Pigs, Dairy, and Beef Cattle in Tanzania. Front Microbiol. 2015 Nov 12;6:1240. doi: 10.3389/fmicb.2015.01240.

Kashoma IP, Kassem II, John J, Kessy BM, Gebreyes W, Kazwala RR, Rajashekara G. Prevalence and Antimicrobial Resistance of Campylobacter Isolated from Dressed Beef Carcasses and Raw Milk in Tanzania. Microb Drug Resist. 2016 Jan;22(1):40-52. doi: 10.1089/mdr.2015.0079. PubMed PMID: 26153978

Kasper LH, Reder AT. 2014. Immunomodulatory activity of interferon-beta. Ann. Clin. Transl. Neurol. 8:622-631.

Kassem II, Sanad YM, Stonerock R, Rajashekara G. An evaluation of the effect of sodium bisulfate as a feed additive on Salmonella enterica serotype Enteritidis in experimentally infected broilers. Poult Sci. 2012 Apr;91(4):1032-7. doi: 10.3382/ps.2011-01935. PubMed PMID: 22399744.

Kassem II, Khatri M, Esseili MA, Sanad YM, Saif YM, Olson JW, Rajashekara G. Respiratory proteins contribute differentially to Campylobacter jejuni's survival and in vitro interaction with hosts' intestinal cells. BMC Microbiol. 2012 Nov 13;12:258. doi: 10.1186/1471-2180-12-258. PubMed PMID: 23148765; PubMed Central PMCID: PMC3541246.

Kassem II, Chandrashekhar K, Rajashekara G. Of energy and survival incognito: a relationship between viable but non-culturable cells formation and inorganic polyphosphate and formate metabolism in Campylobacter jejuni. Front Microbiol. 2013 Jul 9;4:183. doi: 10.3389/fmicb.2013.00183. PubMed PMID: 23847606; PubMed Central PMCID: PMC3705167.

Kassem II, Khatri M, Sanad YM, Wolboldt M, Saif YM, Olson JW, Rajashekara G. The impairment of methylmenaquinol:fumarate reductase affects hydrogen peroxide susceptibility and accumulation in Campylobacter jejuni. Microbiologyopen. 2014 Apr;3(2):168-81. doi: 10.1002/mbo3.158. PubMed PMID: 24515965; PubMed Central PMCID: PMC3996566.

Kassem II, Rajashekara G. Formate dehydrogenase localization and activity are dependent on an intact twin arginine translocation system (Tat) in Campylobacter jejuni 81-176. Foodborne Pathog Dis. 2014 Dec;11(12):917-9. doi: 10.1089/fpd.2014.1797. PubMed PMID: 25268895.

Kassem II, Kehinde O, Kumar A, Rajashekara G. Antimicrobial-Resistant Campylobacter in Organically and Conventionally Raised Layer Chickens. Foodborne Pathog Dis. 2016 Sep 22. PubMed PMID: 27768387.

Kim Y, Shivanna V, Hua DH, Groutas WC, and Chang KO. Broad-spectrum protease inhibitors against 3C-like proteases of feline coronaviruses and feline caliciviruses.2015.  J Virol. 89(9):4942-50.

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Land Grant Participating States/Institutions

AL, AR, CA, IL, KS, MD, MI, MN, ND, NE, NY, OH, OK, SD, TN, TX, WA, WY

Non Land Grant Participating States/Institutions

Iowa State University - College of Vet Med, NC A&T State University, Texas Tech University, USDA-ARS