Duration: 10/01/2022 to 09/30/2027

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 livestock and poultry, ultimately to decrease incidence of enteric diseases in food animals, and zoonotic gastroenteritis in humans.  Illnesses caused by enteric pathogens of animal origin (foodborne or zoonotic 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 enteric diseases of animals and food and water-borne illnesses of humans remains high, and some are increasing. Nevertheless, a broad range of education and research-driven and practice-oriented control efforts have succeeded in controlling the incidence of key food-borne pathogens at low levels.  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 20 years, our enteric diseases group has contributed significantly to an evolved and expanded effort to find evidence-based interventions to prevent enteric diseases in food animals and food-borne diseases in humans. In this renewal proposal, we remain committed to the prevention and control of animal and human diseases caused by enteric pathogens. A primary avenue for control is decreasing carriage and disease due to enteric pathogens in food animals. Our collaborative efforts harmonize with the recent government-wide initiative to better understand, characterize, and mitigate antimicrobial resistance (AMR) across the food chain. Our proactive and collaborative efforts will also contribute to national goals to further decrease the burden of foodborne illness by the year 2030 (https://www.healthypeople.gov/).

Diarrheal diseases account for multi-billion-dollar annual economic losses to the food animal industry due to reduced weight gain, mortality of young animals and treatment costs. Diarrheal infections in neonates, post-weaning period and adult animals are very important diseases in swine and other livestock and are caused by pathogenic bacteria and viruses including enterotoxigenic E. coli (ETEC), Clostridium perfringens, Salmonella, porcine epidemic diarrhea virus (PEDV), rotavirus (RV), calicivirus and emerging coronaviruses [porcine deltacoronavirus (PDCoV)]. 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 are critical to maintain production efficiency, produce wholesome meat, enhance food security and safety, and animal well-being.

Foodborne illness is a major public health concern in the US. The CDC estimates that about 1 in 6 Americans get sick, 128,000 are hospitalized, and 3,000 die of foodborne diseases each year. Norovirus caused the most illnesses (5.5 million) whereas non-typhoidal Salmonella spp caused the most economic losses ($4.1 billion) followed by Toxoplasma gondii ($3.7 billion), Listeria monocytogenes ($3.2 billion), norovirus ($2.6 billion), and Campylobacter ($2.2 billion).

The Foodborne Diseases Active Surveillance Network (FoodNet) was established in 1995 and is a collaborative program among CDC, 10 state health departments, the USDA FSIS, and the FDA. FoodNet 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. Data from 2019 FoodNet report highlight three main points: 1) The number of infections diagnosed by culture-independent diagnostic tests (CIDTs) increased 32% compared with the previous 3 years; 2) Progress in controlling major foodborne pathogens in the United States has stalled; and 3) Incidence of some pathogen infections are decreasing, whereas others are increasing. These facts can be partially explained by the increased testing of samples and the increased use of more sensitive CIDTs in addition to the culture-based diagnostic tests compared with previous years. Importantly, 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. For example, chickens are an important source of Salmonella and Campylobacter infections and S. Typhimurium infections in humans declined after widespread vaccination of chickens against this Salmonella serotype. In addition, several of these agents are also animal pathogens or have close relatives that are animal pathogens. Thus, investigating the host-microbe relationship in animal models or animal populations could solve these problems in humans.

Although 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 incidence of autoimmune disorders is 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. The 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 specific antibiotic resistance carriage by enteric pathogens can further exacerbate these autoimmune manifestations when antibiotics are used.

Unique dynamic interactions between enteric pathogens, animals, humans, and their gut microbiota (microbiome), sharing the same environment, are considered within the “One Health” concept. This new NC1202 project will develop and employ interdisciplinary approaches to address critical areas that will enhance animal health, food safety and food security by maintaining efficient livestock and poultry production and reducing reliance on antibiotic use through the development of alternative approaches for sustainable food animal agriculture.

IMPORTANCE & CONSEQUENCES.  The USDA Economic Research Service (ERS) estimated that the total cost of foodborne illness for the 15 leading foodborne pathogens in the USA in 2018 imposed over $17.6 billion in the economic burden on economy, due to medical costs, productivity losses, and costs associated with premature deaths due to diseases. 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-billion-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 among the most important swine diseases and are caused by pathogenic bacteria and viruses including ETEC, Lawsonia, Brachyspira, PEDV, PDCoV and RV. Foodborne illness incidence can be reduced with new knowledge and new detection procedures that have been and will be developed through this collaborative research. Production systems for food animals have evolved toward large size and complexity. At the same time, there is a new initiative to withdraw the use of growth promoting and prophylactic antibiotics. Due to the recent ban on use of antibiotics for growth promotion in food animals, there is a new emphasis 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 incidence and costs accompanied by burgeoning chronic disease rates.

FEASIBILITY. Based on recent data by FoodNet, foodborne illness is still common and costly, yet a preventable-public health problem. The significant foodborne pathogens include but are not limited to norovirus, Salmonella, Campylobacter, STEC, Listeria, Clostridium perfringens, and 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, such as the widespread vaccination of chickens against Salmonella Typhimurium was strongly associated with the decreased incidence of the related foodborne illness in humans due to this pathogen. CDC believes this success demonstrates the feasibility of preventing foodborne illnesses; research, collaboration and dissemination of successful innovations will be essential to continue this trend.

MULTISTATE EFFORTS. The magnitude of this problem dictates a team-based approach to devising and implementing preventative strategies. Individuals with markedly varied areas of expertise are needed to devise scientific strategies for pathogen control, educate agricultural experts and producers, and 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, parasitologists, molecular biologists, epidemiologists, pathologists, systems biologists and immunologists with a long history of successful 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 of enteric and foodborne pathogen infections in food animals. 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. 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 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 multistate committee focuses on bacteria, viruses, and parasites that cause enteric diseases in food animals. Many of these pathogens are also zoonotic and cause foodborne illnesses in humans. When we search CRIS and NIMSS, we find no multistate projects that target enteric diseases of food animals except NC1202. Many research projects of NC1202 members have been funded by USDA. NC1202 facilitates multistate collaboration and interdisciplinary research by bridging faculty members in different fields to solve problems comprehensively. In the last 5 years, we have published over 130 peer-reviewed research manuscripts, held annual NC1202 meetings in collaboration with the Conference of Research Workers in Animal Diseases (CRWAD), and trained people at all levels of sophistication for 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 1890 land-grant college and university faculty in membership in 2016. This section presents representative projects to demonstrate our productivity and show our follow-up strategies.

Escherichia coli (Hardwidge/Nagaraja/Renter Labs, Kansas State; Moxley Lab, Nebraska; Zhang Lab, Illinois; Liu Lab, California; Rajashekara Lab, Ohio)

Cattle are important reservoirs of enterohemorrhagic E. coli (EHEC) and other Shiga toxin-producing E. coli (STEC), the leading causes of foodborne illness worldwide. We developed and validated multiplex PCR assays that can identify 137 serogroups of STEC, evaluated and improved the performance of broth and chromogenic agar media for the detection and isolation of USDA-regulated O157 and non-O157 STEC in pre- and post-harvest samples from cattle. We quantified the frequency, distribution, and variability of fecal shedding and super-shedding of STEC in feedlot cattle over time, determined the prevalence, concentration, and molecular characterization of USDA-regulated O157 and non-O157 STEC in feces or rectal swabs, and on hides and carcasses of beef and dairy cattle at slaughter, and determined the effects of dietary fiber from distillers grains on the prevalence of USDA-regulated O157 and non-O157 STEC in rectal swabs of feedlot cattle. We identified intra-bacterial activities for type III secretion system effector proteins. For disease control, we determined the efficacy of a humanized monoclonal antibody specific for Shiga toxin-2 to protect against post-diarrheal brain infarction in gnotobiotic piglets infected with E. coli O157:H7.

Enterotoxigenic E. coli (ETEC) is a significant cause of diarrhea in neonatal animals (piglets, calves, and lambs). By mapping small peptides (epitopes) for virulence and immunity, we signified virulence domains among ETEC toxins and adhesins and identified protective epitopes against ETEC diarrheal disease (Lu et al., 2019a; Lu et al., 2019b).  By combining epitope vaccinology and structural vaccinology concepts, we developed a novel vaccinology platform called multiepitope-fusion-antigen (MEFA) for development of cross protective multivalent vaccines against heterogeneous pathogen strains or different pathogens (Seo et al., 2020).  By applying this MEFA vaccinology platform, we developed polyvalent antigens and broadly protective vaccines against ETEC diarrhea (Lu et al., 2020; Seo et al., 2021). We investigated the impact of several non-nutrient additives on ETEC infection in newly weaned pigs and found that dietary supplementation of B. subtilis, algae-derived β-glucan or botanical extract could enhance disease resistance and performance of the pigs by strengthening intestinal barrier function and mucosal immunity.

Colibacillosis caused by avian pathogenic E. coli (APEC) is an economically important disease of poultry worldwide. We identified small molecules as growth and quorum sensing inhibitors with efficacy comparable with or better than sulfadimethoxine (currently used antibiotic) against APEC infection in chickens (Kathayat et al., 2020).  We also identified Lactobacillus probiotic and its derived small peptides effective in reducing APEC colonization in chickens as well as the potential antibacterial target of these peptides.

Escherichia albertii (Lin Lab, TN; Moxley Lab, Nebraska; Sahin Lab, Iowa)

Escherichia albertii is an emerging foodborne human enteric pathogen. However, the prevalence and major animal reservoirs of this significant pathogen are still not clear. We performed comprehensive microbiological, molecular, comparative genomics and animal studies to understand the status and features of E. albertii in the US domestic and food animals. For the first time, we found and characterized E. albertii in poultry at the pre-harvest level (Hinenoya et al. 2021).

Salmonella (non-typhoidal species) (Moxley Lab, Nebraska; Shah Lab, Washington)

Salmonella ranked first among the top 5 pathogens causing the most foodborne illness costs in the 2018 USDA ERS report.  Food animals (cattle, swine, poultry) are common reservoirs of Salmonella spp.  Salmonella spp. is a common cause of diarrheal and systemic disease in livestock with economic losses estimated to be $12 billion. Many strains of Salmonella, particularly multidrug-resistant (MDR) Salmonella, have recently emerged in both animals and humans. We developed an infrared radiation (IR) spectroscopy rapid detection method for MDR S. enterica. We studied the prevalence of Salmonella in backyard poultry in WA State. We completed antibiograms and whole genome sequence assembly and annotation, followed by antimicrobial resistance gene analysis of a set of bovine S. enterica isolates, performed the global transcriptomic analysis of tyramine and D-glucuronic acid metabolic pathways in Salmonella, reported the genomic organization and role of Salmonella pathogenicity island 13 in nutritional fitness of Salmonella, and identified common highly expressed genes of S. Enteritidis.

Campylobacter spp. (Zhang/Sahin Lab, Iowa; Rajashekara Lab, Ohio; Mansfield Lab, Michigan)

Campylobacter ranks fifth in terms of economic burden among the top foodborne pathogens based on the 2018 USDA ERS report. Poultry, ruminants and swine are primary animal reservoirs and Campylobacter can cause important diseases in animals. Besides gastroenteritis, Campylobacter infection has been causally linked to many autoimmune disorders including Guillain-Barré syndrome (GBS) and Miller Fisher syndrome (MFS). Another growing problem is the increasing prevalence of antibiotic resistant Campylobacter. The CDC has listed antibiotic resistant Campylobacter as a serious public health threat. We conducted a longitudinal study on multiple commercial broilers for up to 10 consecutive production cycles for Campylobacter presence and revealed important epidemiological information. We investigated the effect of different fluoroquinolone treatments (enrofloxacin and danofloxacin) on the development of fluoroquinolone resistance in Campylobacter jejuni in calves. To identify factors important for systemic infections by a hypervirulent C. jejuni clone SA, we used a novel pathogenomic strategy, which revealed that single nucleotide polymorphisms (SNPs) in porA (encoding the major outer membrane protein) were responsible for the hypervirulence of clone SA in systemic infection and abortion induction. We developed an experimental pregnant sheep model and determined the efficacy of commercial and experimental bacterins (Wu et al., 2020) and antibiotic treatment (Yaeger et al., 2020) against clone SA induced abortion. We evaluated the potential of a novel approach to combat fluoroquinolone resistant Campylobacter by inhibition of expression of the MDR efflux gene cmeABC via antisense peptide nucleic acid (PNA) and identified two novel narrow spectrum Campylobacter specific small molecule growth inhibitors, which reduced C. jejuni burden in broiler chicken’s ceca and had minimal impact on the cecal microbiota. We showed that experimental evolution of Campylobacter jejuni leads to loss of motility, rpoN (s54) deletion, and genome reduction.

Lawsonia (Gebhart Lab, Saqui-Salces Lab, Minnesota)

Lawsonia intracellularis is an obligate intracellular bacterium that causes proliferative enteropathy in many animal species, most notably pigs. We evaluated the permissibility of macrophages to L. intracellularis infection in vitro and showed interaction, survival and propagation of L. intracellularis in macrophages. We developed a novel diagnostic platform to investigate swine emerging pathogens and new variants of endemic viruses via next-generation sequencing (NGS) coupled with in situ hybridization (ISH). To study the pathogenesis of Lawsonia intracellularis and the intestinal epithelium responses, we have developed swine intestinal organoids (enteroids), a culture system that reproduces all the cells and dynamics of the intestine in vitro. We have successfully infected the enteroids with L. intracellularis, thus providing a swine-specific in vitro model for the study of proliferative enteropathy.

Antimicrobial resistance (AMR, Lin lab, Tennessee; Moxley Lab, Nebraska; Zhang/Sahin Lab, Iowa; Bisha Lab, Wyoming)

Polymyxins (e.g. colistin) are the drugs of last resort to treat MDR bacterial infections in humans. We have performed mechanistic studies to examine the emergence, transmission, and regulatory mechanisms of colistin resistance. We performed an in-depth review, identified and summarized several factors that potentially influence in vivo horizontal gene transfer (HGT) efficiency in the intestine. We also identified and characterized a restriction-modification enzyme reducing conjugation efficiency in Campylobacter jejuni. We identified a novel MDR (phenicols, lincosamides, oxazolidinones, and pleuromutilins) mechanism conferred by a variant cfr gene (cfrC) in Campylobacter coli from cattle (Tang et al. 2017). We investigated the role of wildlife in the acquisition of associated AMR bacteria and genes, and subsequent dispersal across the landscape following interactions with livestock. We evaluated targeted control of invasive European starlings on feedlots and found that decreased population of European starlings was not associated with corresponding reductions in bovine fecal prevalence of ciprofloxacin-resistant E. coli (Carlson et al. 2020). We evaluated the use of high throughput detection and characterization of enterococci from wildlife by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) following culture-based isolation.

Immune and other interventions (Lin Lab, Tennessee; Rajashekara Lab, Ohio; Sun Lab, Arkansas; Farnell Lab, Texas)

We have successfully produced enterobactin (Ent) conjugate vaccine candidates that are promising to control enteric pathogens, such as E. coli, C. jejuni, and S. enterica, in food animals (Wang et al., 2019; Zeng et al., 2021). We have performed multiple chicken trials to test three bacterial bile salt hydrolase (BSH, a promising antimicrobial target) inhibitors (Geng et al., 2020; Poudel, 2021) and investigated the biological basis of the antibacterial phenomenon using state-of-the-art metabolomics, metagenomics, and computational approaches. Encapsulation technology was also developed to improve bioavailability of BSH inhibitors.  We successfully developed innovative and cost-effective encapsulation technologies to improve viability and stability of powdered probiotics for controlling enteric disease. We evaluated Campylobacter colonization in chickens vaccinated with several recombinant attenuated Salmonella Vaccines expressing Campylobacter surface antigens and found highly promising results. Our studies have identified small molecule inhibitors, probiotic bacteria and their derived peptides having a promising effect in reducing Salmonella load in chickens. In addition, our studies demonstrated the immune modulating properties of probiotic E. coli Nissle 1917 (EcN) in human intestinal cells and showed EcN effect on Campylobacter in vitro and in chickens where EcN reduced C. jejuni load in the cecum. Also, we developed mRNA and adenoviral vaccine candidates and monoclonal antibodies against C. jejuni and Clostridium perfringens infections.

Microbiota-host interactions (Sun Lab, Arkansas; Zhang Lab, Oklahoma; Mansfield Lab, Michigan)

We investigated intestinal bile acids, bile acid-metabolizing bacteria, and inflammatory responses on influencing C. jejuni colonization and induction of colitis, and on influencing Clostridium perfringens and coccidia-induced intestinal inflammation using chicken and mouse models. We profiled the ileal microbiomes of chickens following an experimental induction of necrotic enteritis (NE) and correlated them with disease severity. We have identified a microbiota signature associated with NE severity. We are developing tractable mouse models transplanted with human microbiota to study the mechanisms by which horizontal transfer of antibiotic resistance occurs in the context of the gut microbiota. We determined the influence of host gut microbiome in 3-month-old infants on susceptibility to allergic outcomes up to 3 years of age and showed that three-month-old infants harboring a fecal microbiome with increased abundance of Escherichia coli-Shigella, and Bifidobacterium had higher risk for eczema at 1–3 years but were protected at 0–1 year.

Rapid Microbial Diagnostics (Bisha Lab, Wyoming)

We developed transparency-based electrochemical and paper-based colorimetric analytic detection platforms to detect indicators of fecal contamination E. coli and Enterococcus species in a single assay. We developed an LC-MALDI-TOF-MS protocol in conjunction with supervised principal components analysis, to detect a protein biomarker which correlated to β-lactam resistance in AMR E. coli. A rapid PCR method was developed and validated for the detection of colistin-resistant E. coli containing mcr-1 in the feces of feral swine. Antimicrobial sensitive/resistant phenotypes and host sources of E. coli were also predicted based on differential fatty acid abundance.

Porcine Epidemic Diarrhea Virus (PEDV) (Saif/Wang Lab, Ohio; Yoo Lab, Illinois; Ramamoorthy Lab, North Dakota; Chang Lab, Kansas)

PEDV is the most important enteric viral pathogen in the swine industry worldwide. We established a reverse genetics system for PEDV, studied viral virulence determinants (Hou et al., 2019) and viral modulation of innate immune response in cells and hosts. We studied the effects of vitamin A and gestational stage on regulation of the gut-mammary immune axis and neonatal piglet protection (Langel et al., 2019a; 2019b; 2020). Additionally, our studies established that PEDV vaccination of sows in the second trimester optimally stimulated the gut-MG-sIgA axis resulting in greatly improved lactogenic immune protection in suckling piglets. We studied the molecular epidemiology of PEDV, the pathogenesis of PEDV variants in pigs, and the genetic evolution of animal coronaviruses, including PEDV. We will continue to study the pathogenesis of PEDV and develop safe and effective PEDV vaccines. In addition, we reported the structure-guided optimization of a series of inhibitors of the coronavirus 3C-like protease.

Porcine deltacoronavirus (PDCoV) (Kenney/Saif /Vlasova/Wang Lab, Ohio)

PDCoV causes gastroenteritis in pigs and a recent study showed that it is a zoonotic pathogen. We showed the susceptibility and transmissibility of food animals by experimental infection of chickens and turkeys (Boley et al., 2020), and gnotobiotic calves with PDCoV. We identified the host receptor for PDCoV and showed the cross-species potential of PDCoV through infection of multiple cell lines (Li et al., 2018). To better prevent disease spread, we studied the origin, host ranges and evolution of PDCoV. Wild birds represent a natural reservoir for porcine deltacoronavirus. We studied the prevalence of deltacoronaviruses in wild birds in the Mississippi and Atlantic flyways (Paim et al. 2019). We established a reverse genetics system for PDCoV and investigated whether PDCoV evolved directly from sparrow deltacoronavirus (Niu et al., 2021). We will use this infectious clone to study the virulence genes of PDCoV and develop PDCoV vaccines. We will continue to investigate whether swine, other mammals and avian deltacoronaviruses are co-evolving, the mechanisms of its broad host range, and develop diagnostic, preventive, and therapeutic methods to control PDCoV infection in pigs and zoonosis.

Rotavirus (Saif/Vlasova Lab, OH) 

Rotavirus is an important enteric viral pathogen of pigs and cattle and a zoonotic pathogen. We investigated rotavirus C (RVC) prevalence, genetic characteristics, and pathogenesis. The inability of RVCs to efficiently replicate in cell culture leads to the lack of vaccines and tools for serodiagnosis. Using our newly developed virus-like particle (VLP)-based ELISA, we demonstrated that gilts (first parity animals) with diarrheic litters had significantly lower RVC antibody titers in milk (but not in serum) compared with those of sows with healthy litters. Additionally, our comparative pathogenesis studies demonstrated that current vs. historic RVC strains can cause more severe clinical disease and higher virus replication (Chepngeno et al., 2020). These findings necessitate development of control and prevention tools and necessitate further studies.

We discovered novel probiotics for the swine industry to combat rotavirus diarrhea. We have demonstrated that host glycans (histo-blood group antigens and sialic acids) play contrasting roles in infection with human and pig rotavirus A strains (Guo et al., 2021). Similarly, probiotic and commensal bacteria express similar glycans and can regulate glycan availability on the host epithelium, altering the ability of rotaviruses to attach and infect. These tri-directional interactions need to be considered to formulate optimal probiotics for the swine industry. We also demonstrated that protein-calorie malnutrition induced an immune-deficiency state in gnotobiotic pigs aggravating rotavirus disease and reducing rotavirus vaccine efficacy (Fischer et al., 2017; Miyazaki et al., 2018; Michael et al., 2021a; 2021b). We have demonstrated that Escherichia coli Nissle 1917 modulates rotavirus immunity and it can be successfully formulated to increase its stability and simplify its administration. We will continue to develop diagnostic, prevention, and therapeutic methods to control rotavirus infection.

Ostertagia ostertagi in cattle (Xiao Lab, Maryland).

O. ostertagi is a gastrointestinal parasite of cattle. We found that bovine neutrophils form extracellular traps and produce IL-10 in response to O. ostertagi. Bovine CD4+ T cells was synergistically activated by neutrophils and IL-12.

Cryptosporidium parvum (Witola Lab, Illinois)

C. parvum is a highly prevalent protozoan parasite that causes a serious diarrheal syndrome (cryptosporidiosis) in calves, but for which no effective drugs nor vaccine exist. C. parvum depends solely on glycolysis for metabolic energy production. We have shown that inhibitors for C. parvum’s unique bacterial-type lactate dehydrogenase (CpLDH) and plant-like pyruvate kinase (CpPyK) enzymes of the glycolytic pathway can stop the growth of this parasite and prevent disease development in infected mice models. In the next five years, we aim to derivatize optimized CpLDH and CpPyK inhibitors and test them for efficacy and safety in treating C. parvum infection in bovine calves.

Modeling pathogen infections using a livestock enteroid/organoid system (Vlasova lab, Ohio; Saqui-Salces and Gebhart Labs, Minnesota; Sang Lab, TN; Farnell lab, TX)

Animal models and cell culture in vitro are primarily used in virus and antiviral immune research.  However, the limitation of these models to recapitulate viral pathogenesis in humans has been made clear. It is also imperative to introduce more efficient systems to validate emerging pathogens in both domestic and wild animals. Organoids are three-dimensional structures derived from stem cells under differential conditions mimicking endogenous organogenetic niches to develop miniatures of organs (i.e., mini organs). They are used in validating emerging animal pathogens, particularly zoonotic pathogens (e.g., rotavirus and Lawsonia intracellularis), in domestic and wild animals.

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 and zoonotic enteric infections of humans with 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 grants and publications, sharing of resources (pathogen strains, gene sequences, statistical analysis, bioinformatics information/expertise), and relevant feedback and facilitation for all research efforts at annual meetings.
   

Methods

During our annual meetings, NC1202 members give presentations and discuss details of methods, such as animal models, new technology and new equipment. Then, protocols are often shared, and new collaborations are established to supplement each other’s research needs. Methods for specific projects are described below.

OBJECTIVE 1 – FOCUS ON EMERGING DISEASES

Antimicrobial resistance (Lin Lab, TN; Moxley Lab, Nebraska)

Using comprehensive molecular, genomics and lipid A analyses, we will perform in-depth characterization of new targets (RpoE and PmrR) that are essential for polymyxin resistance in E. coli.

We will characterize the prevalence and nature of mobile genetic elements in MDR Salmonella enterica from cattle using whole genomic sequencing and bioinformatics. We will use infrared radiation spectroscopy or alternate methods that enable rapid detection of MDR S. enterica and we will identify risk factors for MDR Salmonella enterica in cattle production system.

Rapid Microbial Diagnostics (Bisha Lab, Wyoming)

We will utilize methods including phenotypic (culture-based, MALDI-TOF MS), cytomic (flow cytometry, microscopy), and molecular (digital PCR, whole genome sequencing, metagenomics) to better understand and characterize microbial resistance, stress and survival in the environment, food, and animals.

Prevalence of deltacoronaviruses in wild birds (Vlasova/Wang/Saif Lab, OH)

We will continue utilizing RT-PCR to identify and using sequence analyses to characterize partial and complete genome sequences of avian deltacoronaviruses.

Cross-species transmission of coronaviruses (Kenney/Saif/Wang Lab, OH)

We will use PDCoV as a model to perform surveillance studies and animal challenge studies, and use reverse genetics, cell culture adaptation, bioinformatics and RNA sequencing to understand how PDCoV spreads among pigs, birds, and humans.

Rotavirus C prevalence, genetic characteristics, and pathogenesis (Vlasova/Saif Lab, OH) 

We will continue our studies using a combination of methods for molecular and serological diagnostics using our innovative tools, such as virus-like particles (VLPs), and experimental animal studies to identify, characterize and control the emerging viruses. The VLPs generated in our previous studies will be evaluated as a vaccine candidate in experimental and on-farm trials.

OBJECTIVE 2. FOCUS ON PREVENTION AND INTERVENTION

Enterobactin (Ent)-based immune intervention against Gram-negative pathogens (Lin Lab, TN)

We will continue to assess Ent antibody-based immune intervention strategies for controlling Gram-negative enteric pathogens, such as E. coli, Salmonella, and Campylobacter, in food animals. In addition, encapsulation technology will be developed to improve in vivo stability and bioavailability of Ent-specific egg yolk powder for passive immune therapy. 

Functional and translational characterizations of gut bile salt hydrolase (BSH) (Lin Lab, TN)

Using multidisciplinary approaches, we will further study structure-function relationship of BSH and discover new BSH inhibitors. With aid of the validated BSH inhibitors, we also plan to evaluate the effects of the BSH inhibitors on Clostridium difficile infection in a mouse model. We will comprehensively examine which and how specific intestinal bile acid signatures influence in vivo C. difficile germination, cell growth, and toxin production for disease development.

Vaccines against ETEC in pigs (Zhang Lab, Illinois; Moxley Lab, Nebraska)

We apply computational biology and structural biology techniques to optimize the vaccinology platform MEFA, to construct polyvalent antigen for cross protective immunity, and to develop broadly protective vaccines against ETEC and combination vaccines for ETEC and other enteric pathogens.  We also use animal models to human enteric disease and disease prevention.

Diet-based interventions against ETEC in pigs (Liu Lab, California)

We will continue focusing on diet-based approaches to prevent ETEC infection in weaned pigs using in vivo disease challenge models.

Coccidial infection in poultry (Liu Lab, California)

We will focus on coccidial infection on growth performance and intestinal mucosal immunity of broiler chickens. Different dietary approaches will be evaluated to prevent the negative impacts of coccidial infection in poultry health.

Antibiotic resistant Campylobacter (Zhang/Sahin Lab, Iowa State)

We use animal models (chicken, sheep and cattle) to determine the effect of antibiotic treatment on the development of antibiotic-resistant C. jejuni following oral inoculation. In addition, we use metagenomic approaches to study the effect of antibiotic treatment on gut microbiota and antimicrobial resistance development. Additionally, we will continue to evaluate antisense-based intervention (PNA) and antimicrobial alternatives to control antibiotic resistant Campylobacter.

Campylobacter-associated sheep abortion (Zhang/Sahin Lab, Iowa)

We use directed genome evolution (DGE), a positive selection method, to identify the specific mutation responsible for the hypervirulence of abortifacient C. jejuni clone SA.

Epidemiology and control of Campylobacter in poultry (Zhang/Sahin Lab, Iowa)

We will use metagenomic approaches and several probiotic screening methods along with in vivo chicken studies to develop microbiota-based interventions for Campylobacter in poultry.

Interventions for Campylobacter (Rajashekara Lab, Ohio)

We will continue to investigate several approaches to control Campylobacter in poultry production, including small molecules screening, testing probiotic EcN, and mixed RASV vaccines.

Role of Campylobacter in EED and stunting in children in LMIC (Rajashekara Lab, Ohio) We will continue our studies, in collaboration with University of Florida and Haramaya University, Ethiopia, to conduct a longitudinal study to identify the prevalence, abundance, and diversity of Campylobacter spp. in livestock and in the environment surrounding young children.

Antibiotic Independent Approaches to Control Salmonella in Poultry (Rajashekara Lab, Ohio)

We plan to optimize the oral delivery of small molecule inhibitors, probiotics and peptides and test them in chickens under field simulated conditions to uncover the antimicrobial potential of these approaches for industrial use.

Novel Antibiotic independent approaches to control colibacillosis in poultry (Rajashekara Lab, Ohio)

Our future studies will evaluate the small molecules, peptides, and probiotic bacteria for oral delivery in water or feed to reduce APEC infection and assess their impact on performance parameters. Further, we will elucidate their mechanisms of action using proteomics and biochemical approaches.  

Interventions for Campylobacter jejuni and Clostridium perfringens infections (Sun Lab, Arkansas)

We will perform chicken and mouse infection experiments with mRNA, protein, and histopathology evaluation using intestinal tissues; targeted metabolomics of bile acids on intestinal bile acid changes; in vitro cell culture on bacterial infection, host immune response and cell death; proteomics of bacteria, microbiota, and host cells/tissues to evaluate bacterial infection; shotgun library and Phage Display to develop recombinant mRNA and adenoviral bacterial vaccines, and monoclonal antibodies; in ovo vaccination.

Lawsonia (Gebhart and Saqui-Salces Labs, Minnesota)

We will use the swine organoid model to identify the specific cell type(s) invaded by L. intracellularis and to define the cell proliferation-invasion cycle of L. intracellularis in intestinal epithelial cells.

Microbiota-Host interactions (Mansfield Lab, Michigan State)

We will use fecal transplantation in various mouse models with different genetic/immunological/microbiota background along with metagenomics approaches to study allergic diseases in depth.

Porcine enteric coronaviruses (Saif/Wang Lab, Ohio; Yoo Lab, Illinois). We use reverse genetics to study viral gene functions and rationally design a new generation of safe and efficient live attenuated vaccine (LAV) candidates that do not reverse to virulent wild type virus or recombine with circulating field strains. We will perform both in vivo pig studies and in vitro experiments to study the phenotype of these candidates.

Effects of vitamin A and gestational stage on regulation of the gut-mammary immune axis and neonatal piglet protection (Saif/Vlasova lab, OH). These studies involve dietary manipulations and PEDV vaccination-challenge or RV challenge protocols of pregnant sows and their suckling piglets.

Coronavirus inhibitors (Chang Lab, Kansas)

We will use structure-guided optimization of a series of inhibitors of the coronavirus 3C-like protease for antiviral therapy.

Rotavirus (Saif/Vlasova Lab, Ohio)

• Novel probiotics for swine industry to combat rotavirus diarrhea. These studies are conducted using three models: porcine small intestinal enteroids, gnotobiotic and conventional pigs, in which bi- (virus-bacteria and bacteria-epithelial cell) or tri-directional interactions are studied using a broad range of methods
• Effects of protein-calorie malnutrition on rotavirus disease and immunity. We will continue evaluating interactions between the diet, immune system, and microbiome in gnotobiotic piglets as well as the outcomes of such interaction on rotavirus disease and vaccine efficacy.
• Escherichia coli Nissle 1917 modulation of rotavirus immunity. Evaluation of the probiotic EcN effects on rotavirus disease, vaccination and antibiotic-induced intestinal perturbations will continue utilizing our well-characterized gnotobiotic pig model of rotavirus disease.

Mucosal immune response to Ostertagia ostertagi in cattle (Xiao Lab, Maryland)

Immunostaining, flow cytometry and ELISA are used to examine the presence of O. ostertagi, phenotype of immune cells in tissues and antibodies in the serum from O. ostertagi-infected and non-infected cattle. We use confocal microscope to visualize bovine neutrophil responses to O. ostertagi. Chemiluminescent measurements are used extensively for the quantification of NETosis. Flow cytometry and microscopy were performed to determine the phenotype of immune cells from OO-infected and non-infected cattle. In additional to in vitro assay, calves were challenged with pathogen OO, and neutrophils in tissues from infected animals were examined for expression of MHC II and IL-10. Cell isolation, flow cytometry and cytokine array were performed to determine the activation status of bovine CD4+ T cells in response to different stimuli.

OBJECTIUVE 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

NC1202 members collaborate on a regular basis and many NC1202 members have established collaborations. Publications generated by multiple NC1202 members are listed in the Literature Cited. The list below summarizes grants lead by multiple members.

1. Jun Lin (Tennessee) and Glenn Zhang (Oklahoma State University) have collaborated on development of novel non-antibiotic approaches for mitigation of antimicrobial resistance in poultry (USDA NIFA award 2018-68003-27462).
2. Zhang, W (Illinois) collaborates with Moxley, R. (Nebraska), Cernicchiaro, N. (Kansas) on USDA NIFA project (2017-67015-31471) entitled “A broadly protective vaccine against porcine post-weaning diarrhea (PWD)”. 2020-2025.
3. A. Moxley and J.D. Loy (University of Nebraska-Lincoln) collaborate on a project entitled “Genetic Analysis and Rapid Detection of Multidrug-Resistant Salmonella enterica Isolates from Cattle”. Nebraska Beef Council and National Cattlemen’s Beef Association. 10/1/2020-9/30/2021.
4. Sang (Tennessee) has collaborated with Drs. Wenjun Ma and Frank Blecha (Kansas) on the antiviral potency and functional novelty of porcine interferon-omega subtype (USDA NIFA 2018-67016-28313).
5. Zhang, Q and Sahin, O (Iowa) collaborate with J.F. Coetzee and Z. Lin (Kansas) on USDA NIFA integrated project (2017-68003-26499): Mitigation of fluoroquinolone-resistant Campylobacter in cattle. 2017-2022.
6. Zhang, Q and Sahin, O (Iowa) collaborate with G. Rajashekara and L. Medeiros (Ohio), J. Lin (Tennessee) on USDA NIFA integrated project (2012-68003-19679) entitled “Novel and practical approaches for mitigation of Campylobacter in poultry”. 2012-2018.
7. Curtiss (Florida) and G. Rajashekara (Ohio) have collaborated on the development of a food safety vaccine to control Salmonella Enteritidis and reduce Campylobacter in poultry (USDA NIFA 2017-67017-26179).
8. Kenney, Saif and Wang (Ohio) collaborate on NIFA, USDA 2020-67015-31618. Title: Functional genomics approach in livestock to delineate host factors critical for emerging virus replication.
9. Saif, Vlasova, and Wang (Ohio) collaborate on NIH-USDA Dual Purpose with Dual Benefit (NICHD R01HD095881): Research in Biomedicine and Agriculture. Title: The impact of vitamin A on the gut-mammary gland-secretory IgA axis during enteric viral infections.
10. Kenney and Wang (Ohio) collaborate on OSU Seeds grant entitled “Interspecies transmission mechanisms of deltacoronavirus from birds to pigs”.

Measurement of Progress and Results

Outputs

• Develop vaccines and vaccine candidates against enteric diseases in food animals.
• Discover new alternatives to antibiotics.
• Understand the pathogenesis and immunogenicity of enteric pathogens.
• Develop new diagnostic assays for better detection of enteric pathogens.
• Establish novel enteroid/organoid models for the study of enteric pathogens in vitro.

Outcomes or Projected Impacts

• Development of innovative strategies to control zoonotic viral and bacterial pathogens in humans and in animal reservoirs 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
• Safe and effective vaccines for food animals will decrease morbidity and mortality rates in animals and contribute to sustainable agriculture.
• Control and prevention of zoonotic pathogens in food animal reservoirs will promote public health.

Milestones

(1):All activities proposed in the four Objectives will be carried out simultaneously and completed in next five years (2022-2027). Note to NCRA reviewers from Christina Hamilton, NCRA Asst Director: Please be aware that our NC Guidelines state, “For NC projects, this section (Milestones) should be eliminated and replaced with a timeline of each objective common to most competitive grants," so this section is acceptable as written.

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.


• 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 NC-1202 members and their students will present their work in numerous national and international meetings. Each year NC1202 also will hold a meeting in conjunction with the Conference of Research Workers in Animal Diseases (CRWAD) meeting.  


• 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 to train the next generation of scientists in enteric diseases of food animals. Every year, NC1202 will continue to sponsor 3-4 Graduate Student awards at CRWAD for their research presentation on enteric diseases of food animals.


• Virtual meeting and seminars become popular during the COVID-19 pandemic. We will use these tools to enhance group member communication and collaboration. Also, we anticipate that this will attract more new members to join NC1202 because of its flexibility.

Organization/Governance

The recommended Standard Governance for multistate research activities includes 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 Advisor 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 reports for submission to NIMSS.

Furthermore, ad hoc committees will be formed to organize 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 5 years, a specific symposium topic will be identified at the annual NC1202 meeting and a special ad hoc committee will be assembled to organize the 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 1890 faculty members in its membership.  In December 2015, three 1890 faculty members (from Tuskegee University, North Carolina A&T State University, and Tennessee State University) attended NC1202 annual meeting; subsequently, all three 1890 faculty members joined the NC1202 membership in early 2016.  The NC1202 group fully recognizes 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 members or other minority-serving institution faculty members to join NC 1202 group.

Literature Cited

1. Boley et al. 2020. Porcine Deltacoronavirus Infection and Transmission in Poultry, United States. Emerg Infect Dis. 26:255-265.


2. Carlson et al. 2020. Bird-livestock interactions associated with increased cattle fecal shedding of ciprofloxacin-resistant Escherichia coli within feedlots in the United States. Sci Rep. 10:10174.


3. Chandler et al. 2020. The Role of European Starlings (Sturnus vulgaris) in the Dissemination of Multidrug-Resistant Escherichia coli among Concentrated Animal Feeding Operations. Sci Rep.10:8093.


4. Chepngeno et al. 2020. Comparative Sequence Analysis of Historic and Current Porcine Rotavirus C Strains and Their Pathogenesis in 3-Day-Old and 3-Week-Old Piglets. Front Microbiol. 11:780.


5. Fischer et al. Protein malnutrition alters tryptophan and angiotensin converting enzyme 2 homeostasis and adaptive immune responses in human rotavirus infected gnotobiotic pigs transplanted with human infant fecal microbiota. Clin Vaccine Immunol. 24:e00172-17.


6. Geng et al. 2020. Evaluation of in vivo efficacy of bile salt hydrolase inhibitors using chicken model system. Scientific Reports. 10:4941.


7. Guo et al. 2021. Infection of porcine small intestinal enteroids with human and pig rotavirus A strains reveals contrasting roles for histo-blood group antigens and terminal sialic acids. PLoS Pathog. 17:e1009237.


8. Hinenoya et al. 2021. Isolation and characterization of Escherichia albertii in poultry at the pre-harvest level. Zoonoses Public Health. 68:213-225.


9. Hou et al. 2019. Engineering a live attenuated PEDV vaccine candidate via inactivation of the viral 2'-O methyltransferase and the endocytosis signal of the spike protein. J Virol. 93:e00406-19.


10. Kathayat et al. Small Molecule Adjuvants Potentiate Colistin Activity and Attenuate Resistance Development in Escherichia coliby Affecting pmrAB System. Infect Drug Resist. 13:2205-2222.


11. Langel et al. Oral vitamin A supplementation of porcine epidemic diarrhea virus infected gilts enhances IgA and lactogenic immune protection of nursing piglets. Vet Res. 50:101.


12. Langel et al. Stage of Gestation at Porcine Epidemic Diarrhea Virus Infection of Pregnant Swine Impacts Maternal Immunity and Lactogenic Immune Protection of Neonatal Suckling Piglets. Front Immunol. 10:727.


13. Li W et al. 2018. Broad receptor engagement of an emerging global coronavirus may potentiate its diverse cross-species transmissibility. PNAS. 115:E5135-E5143.


14. Lu et al. Mapping the neutralizing epitopes of F18 fimbrial adhesin subunit FedF of enterotoxigenic Escherichia coli (ETEC). Vet. Microbiol. 230:171-177.


15. Lu et al. Mapping the neutralizing epitopes of enterotoxigenic Escherichia coli (ETEC) K88 (F4) fimbrial adhesin and major subunit FaeG. Appl. Environ. Microbiol. 85:e00329-19. Erratum: 85:e01946-19.


16. Lu et al. Application of a novel epitope- and structure-based vaccinology-assisted fimbria-toxin multiepitope fusion antigen of enterotoxigenic Escherichia coli for development of multivalent vaccines against porcine postweaning diarrhea. Appl. Environ. Microbiol. 86:e00274-20.


17. Michael et al. Escherichia coli Nissle 1917 Enhances Innate and Adaptive Immune Responses in a Ciprofloxacin-Treated Defined-Microbiota Piglet Model of Human Rotavirus Infection. mSphere. 6:e00074-21.


18. Michael et al. Escherichia coli Nissle 1917 administered as a dextranomar microsphere biofilm enhances immune responses against human rotavirus in a neonatal malnourished pig model colonized with human infant fecal microbiota. PLoS One.16:e0246193.


19. Miyazaki et al. 2018. Protein deficiency reduces efficacy of oral attenuated human rotavirus vaccine in a human infant fecal microbiota transplanted gnotobiotic pig model. Vaccine. 36:6270-6281.


20. Moxley et al. Efficacy of urtoxazumab (TMA-15 humanized monoclonal antibody specific for Shiga toxin-2) against post-diarrheal sequelae caused by Escherichia coli O157:H7 infection in the gnotobiotic piglet model. Toxins 9:49.


21. Moxley et al. Intimate attachment of Escherichia coli O157:H7 to urinary bladder epithelium in the gnotobiotic piglet model. Microorganisms 8:263.


22. Niu et al. 2021. Chimeric Porcine Deltacoronaviruses with Sparrow Coronavirus Spike Protein or the Receptor-Binding Domain Infect Pigs but Lose Virulence and Intestinal Tropism. Viruses 13:122.


23. Paim et al. 2019. Epidemiology of Deltacoronaviruses (δ-CoV) and Gammacoronaviruses (γ-CoV) in Wild Birds in the United States. Viruses. 11:897.


24. Poudel et al. 2021. Effects of riboflavin and Bacillus subtilis on internal organ development and intestinal health of Ross 708 male broilers with or without coccidial challenge. Poultry Science 100:100973.


25. Seo et al. Preclinical characterization of immunogenicity and efficacy against diarrhea from MecVax, a multivalent enterotoxigenic E. coli vaccine candidate. Infect Immun. 89:e0010621.


26. Tang et al. Emergence of a plasmid-borne multidrug resistance gene cfr(C) in foodborne pathogen Campylobacter. J Antimicrob Chemother. 72: 1581-1588.


27. Wang et al. 2019. Characterization of the enterobactin specific antibodies raised by a new enterobactin conjugate vaccine. Applied and Environmental Microbiology. 85:e00358-19.


28. Wu et al. A homologous bacterin protects sheep against abortion induced by a hyper-virulent Campylobacter jejuni clone. Vaccines 8:662.


29. Yaeger et al. Experimental evaluation of tulathromycin as a treatment for Campylobacter jejuni abortion in pregnant ewes. Am J Vet Res. 81:205-209.


30. Zeng et al. 2021. Evaluation of the immunogenic response of a novel enterobactin conjugate vaccine in chickens for the production of enterobactin-specific egg yolk antibodies. Frontiers in Immunology. 12:629480.

Attachments

Land Grant Participating States/Institutions

AR, CA, DE, IA, IL, KS, KY, MD, MI, MN, MS, NC, ND, NE, NY, OH, OK, SC, SD, TN, TX, WY

Non Land Grant Participating States/Institutions

Iowa State University - College of Vet Med, Long Island University, Merck Animal Health , National Animal Disease Center, NC A&T State University, Texas Tech University, USDA-ARS-Dairy Forage Research Center, Madison, WI, USDA-ARS/Maryland