Enteric Diseases of Food Animals: Enhanced Prevention, Control and Food Safety

<p>NON-TECHNICAL SUMMARY: We will survey and characterize emerging and new enteric viruses in swine and cattle or wildlife reservoirs and assess their environmental transmission in food or water. Their zoonotic potential will be evaluated based on their similarity to the corresponding human strains and in epidemiologic studies. Vaccines or other preventive measures (probiotics) will be developed to reduce their incidence and prevalence in swine and cattle and consequently in food and water. </p>
<p>APPROACH: We will use molecular approaches including monoclonal antibodies (ELISA), RT-PCR and real time PCR and sequence analysis to diagnose, characterize and differentiate field and wildlife strains of coronaviruses (TGEV, PRCV, bovine CoV, etc), rotavirus (serotypes and serogroups), enteric caliciviruses and newly recognized enteric viruses. Cell culture systems will be developed and optimized to quantitate enteric viruses. We will determine the relatedness between the animal viruses including similar viruses from wildlife, and human strains to assess zoonotic potential. We will also examine their food and water-borne transmission to assay environmental reservoirs. Pathogenicity, virulence and impact of host genetic factors, gut microflora, probiotics and antibiotics on enteric or respiratory coronaviruses, rotaviruses, enteric caliciviruses and newly identified enteric viruses will be studied in gnotobiotic or conventional pigs. Susceptibility of pigs and calves to human or wildlife counterpart viruses will also be examined to investigate interspecies viral transmission. We will use the ELISA, ELISPOT (antibodies and cytokines) and flow cytometry to assess innate, antibody, cellular and cytokine responses to evaluate mucosal and systemic immune responses to coronaviruses, rotaviruses, enteric caliciviruses and novel subunit (VLP) vaccines derived from these viruses. We will determine possible immune correlates of passive and active protective against these viruses in pigs in order to develop efficacious vaccines and adjuvants. </p>
<p>PROGRESS: 2013/01 TO 2013/09
Target Audience: Veterinarians, Veterinary Diagnosticians, Swine and Poultry Practitioners, Producers, Researchers, and Veterinary Virologists and Immunologists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Graduate students and postdoctoral fellows acquired research skills in epidemiology, vaccine development, pre- and postharvest control strategies, as well as in basic research relating to mechanisms of disease pathogenesis. This training resulted in the completion of Graduate students’ dissertations and enhanced their skills to conduct independent research. Similarly researchers were trained in extension education such that they can effectively communicate with different stakeholders. How have the results been disseminated to communities of interest? Research findings were presented at several local, national and international meetings. Written and electronic media was distributed to stakeholders and research findings were published in peer reviewed journals. What do you plan to do during the next reporting period to accomplish the goals? 1) Caliciviruses: Pathogenesis of GIII.2 bovine norovirus, CV186-OH/00/US strain in gnotobiotic calves. Elucidate the role of innate and adaptive immunity in the clearance of NoV from the infected intestine and the prolonged virus shedding and diarrhea. An immunologic study is underway to investigate if prolonged BoNoV RNA fecal shedding by infected Gn calves correlates with increases in anti-inflammatory immune responses that might reduce clearance of the virus in the intestine. Prevalence and Molecular Characterization of SaVs and NoVs in pigs. We will study the prevalence and diversity of enteric caliciviruses in swine in OH, NC and collaborating NC1202 states and in different ages of pigs using molecular assays or newly developed assays based on the newly identified viruses. These studies are crucial since NoVs most closely related to HuNoVs have been detected in swine by our lab and others, raising the issue of the zoonotic potential of swine NoVs. We will also investigate if selected porcine NoVs, SaVs or new caliciviruses cause disease in Gn pigs to study their pathogenesis and to develop methods for their prevention and control. 2) Coronaviruses: Pathogenesis of an OH strain of PEDV in Gn pigs and isolation in cell culture. The long-term goal of our research is to understand the disease mechanisms, pathology and innate immune responses in pigs of varying ages to understand age-dependent susceptibility to PEDV and to develop PEDV vaccines to enhance immunity in nursing pigs. We plan to investigate how innate immunity develops after PEDV infection, how it impacts viral shedding and virulence and how it contributes to enhanced recovery as related to pig age (nursing vs. weaned). Our findings will contribute to a comprehensive understanding of the pathogenesis and innate immunity to US PEDV strains to promote potential new innovative treatments for control and prevention of US PEDV infections. We will serially passage PEDVstrains in Vero cells to generate an attenuated PEDV candidate vaccine strain. We plan to test the attenuation of the cell passaged PEDV isolates in specific pathogen-free nursing pigs that are the major target of fatal PEDV infections in the field. Cell culture-based VN assays will be developed for the detection of antibodies from field pigs and for the evaluation of vaccine efficacy in the future. 3) Rotaviruses: Probiotics: effects on neonatal innate immune responses, immune homeostasis, RV infections and vaccine efficacy. In our future studies, we will assess whether lactobacilli and bifidobacteria will retain their beneficial effects as probiotics in Gn pigs colonized with commensals or defined microflora. We will also evaluate how selected Gram+, Gram- commensal bacteria (individually and combined) and a defined commensal cocktail modulates intestinal homeostasis and immune responses to AttHRV vaccine or VirHRV infection in neonatal Gn pigs. To improve the comprehensive knowledge of probiotic actions, we will delineate mechanisms governing their beneficial effects using metatranscriptomic and metabolomic approaches. 4) Campylobacter: Association of Polyphosphate (poly P) Kinases with C. jejuni Invasion and Survival in Human Epithelial Cells. Two kinases in C. jejuni have been associated with virulence:polyphosphate kinase 1 (PPK1) and poly phosphate kinase 2 (PPK2). PPK1 is responsible for polyP generation and PPK2 for GTP biosynthesis from poly P. GTP is a molecule involved in signaling processes as well as protein and capsule synthesis. We plan to determine the contribution of component(s) present in the outer material (OM) from the wild-type, ?ppk1 and ?ppk2 to C. jejuni invasion and intracellular survival in human epithelial cells in vitro. Chemotaxis transducers in C. jejuni with a role in sensing essential nutrients. Chemotaxis mediated motility enables Campylobacter jejuni to navigate through various environmental gradients and allow for its establishment in diverse niches. Here we plan to elucidate the mechanisms underlying the ability of some Tlps to sense specific nutrients such as iron and phosphate and these findings will better define pathways of iron and phosphate regulation in this important pathogen. Chemical treatments to reduce C. jejuni in chicken litter microcosms. C. jejuni persists in the infected chickens and is heavily shed in feces; therefore, chicken litter is a significant source of infection for new flocks. Here, we plan to investigate the impact of aluminum sulfate, sodium bisulfate, and magnesium sulfate on C. jejuni’s survival in litter microcosms. Our findings have the potential of providing a relatively non-cumbersome and efficient approach for reducing C. jejuni in chicken litter; there by reducing the pathogen in on-farm environments. The impact of formate on C. jejuni survival phenotypes. Formate, a byproduct of fermentation in the hosts’ gut, is a primary energy source for C. jejuni. C. jejuni also possesses two terminal oxidases, and formate, in other bacteria, can inhibit these enzymes. Consequently, formate utilization may represent a conflict between energy production and microaerobic survival in this bacterium. To investigate this dichotomy, we will assess the impact of formate on C. jejuni’s survival traits. Our findings will reveal how this bacterium may use formate to sense its environment and potentially adapt to the conditions that prevail in the gut. </p>
<p>IMPACT: 2013/01 TO 2013/09
What was accomplished under these goals? Focus on emerging, zoonotic and foodborne diseases PEDV. Porcine epidemic diarrhea virus (PEDV) is a devastating and deadly, newly emerged enteric viral infection of swine with high mortality in baby pigs. We studied the pathogenicity of Ohio PEDV strain PC21A in germfree pigs and developed new assays (real-time RT-PCR; immunofluorescent antibody, IFA) for PEDV detection. Germfree pigs (10-35-days-old) were inoculated with the PEDV fecal filtrate confirmed to be bacteriologically sterile and free of other major swine viral pathogens. Clinical signs and virus shedding were monitored until necropsy. Pigs were euthanized for pathology at acute, mid-stage and later-stages of infection. Pigs exhibited acute severe diarrhea/vomiting post-inoculation at 24-48 hr, followed by dehydration and collapse. Pathologic lesions, limited to small and large intestines, included severe atrophic enteritis with vacuolation of superficial epithelial cells in cecum and colon. Consistent with histopathology, PEDV antigens were observed in the epithelium of atrophied villi of the small and large intestines, as determined by IFA. By real-time RT-PCR, all infected pigs had viral RNA titers in feces and transiently in serum. Our data suggest that the PC21A strain of PEDV is highly enteropathogenic and acutely affects the entire intestine, leading to severe atrophic enteritis and death (Jung, et al. 2013. Pathogenesis of a US strain of porcine epidemic diarrhea virus (PEDV) in gnotobiotic pigs. Submitted). Studies of the adaptation of Ohio strains of PEDV to cell culture are in progress. The data from our preliminary studies contributes to a comprehensive understanding of the pathogenesis of newly emerged US PEDV strains. These findings and cell culture adaptation of PEDV will lead to new measures for control and prevention of PEDV infections in US swine. Campylobacter: Our objectives were to determine the prevalence, antimicrobial susceptibility, and genetic relatedness of Campylobacter spp. recovered from commercial turkey Farms in Northwest, Ohio. Overall Campylobacter prevalence was 55.8%. The flaA-RFLP subtyping detected 62 types for C. coli and 8 types for C. jejuni isolates, with most (80%) of flaA-types constituting Farm homogenous groups. MLST of 99 systematically selected Campylobacter strains resulted in 23 sequence types (STs), consisting of 8 STs for C. jejuni and 15 STs for C. coli isolates. Six novel STs; four for C. jejuni and two STs for C. coli, were detected. C. coli isolates displayed greater resistance than C. jejuni to most antimicrobials. This study highlights the high prevalence and genotypic diversity of antimicrobial-resistant Campylobacter spp. recovered from commercial turkey production systems.Transducer Like Proteins (Tlps) of C. jejuni mediate substrate specific chemotaxis and virulence. We investigated the role of C. jejuni Tlps in sensing specific nutrients and their role in its pathobiology. Chemotaxis assays revealed novel ligands for some of the Tlps and more than one Tlp was responsible for sensing a nutrient in C. jejuni. Our results highlight the contribution of Tlps to C. jejuni adaptation to micro environments both inside and outside their host.We also investigated the pathogenesis of C. jejuni induced abortion in pregnant ewes. Typical abortion and stillbirth occurred in pregnant ewes inoculated with ovine or bovine abortion strains. Pathological lesions including suppurative necrotizing placentitis, or endometritis coincided with 1) increased apoptotic cell death, 2) increased expression of the host genes related to cellular necrosis and pro-inflammatory responses, and 3) decreased expression of the genes that account for embryonic development. Since the abortion associated C. jejuni isolates share genotypic similarities with clones present in the human population, these strains might represent a potential public health risk. 2) Focus on preventions and treatments:Rotaviruses. Rotaviruses (RVs) are a leading cause of diarrhea in children and young animals. Because current oral vaccines are only partially effective in animals and in infants in impoverished countries, alternative affordable vaccines or treatments are needed. Probiotics can alleviate diarrhea in children and enhance specific immune responses. Neonatal Gn pigs are the only animal model susceptible to human (HRV) diarrhea. We demonstrated that probiotics modify neonatal innate and T cell immune responses and immune homeostasis to reduce rotavirus disease severity and increase vaccine efficacy in piglets. Effects of co-colonization with Lactobacillus rhamnosus GG (LGG) and Bifidobacterium lactis Bb12 (Bb12) on attenuated (Att) HRV vaccination and virulent HRV (VirHRV) challenge were assessed in Gn pigs. These 2 probiotics were selected to mimic gut commensals in breastfed infants. Diarrhea and HRV shedding were significantly reduced in colonized pigs following VirHRV challenge. LGG+Bb12 colonization promoted immunomaturation as reflected by increased frequencies of CD4, SWC3a, CD11R1, MHCII expressing immune cells in intestinal tissues and blood post-challenge. Colonization modulated frequencies of toll-like receptors (TLR) suggesting that probiotics likely exert anti-inflammatory (TLR2 and 4 downregulation) and antiviral (TLR3 upregulation by HRV dsRNA) actions via TLR signaling. Probiotic colonization differentially modulated frequencies of intestinal and systemic apoptotic and proliferating mononuclear cells suggesting that probiotics regulate immunotolerance and control excessive lymphoproliferative reactions upon VirHRV challenge. Thus the selected probiotics exerted beneficial effects by promoting immunomaturation, regulating immune homeostasis and moderating HRV diarrhea. We further elucidated the T cell and cytokine responses to AttHRV and VirHRV in the Gn pigs co-colonized with LGG+Bb12. Higher protection from VirHRV challenge in the colonized vs non-colonized pigs coincided with higher ileal T regulatory cells (Tregs) before and after challenge, and higher serum TGF-β and lower serum and biliary proinflammatory cytokines postchallenge. Probiotic colonization induced systemic Th1 immunostimulatory effects with the oral AttHRV vaccine that coincided with lower diarrhea severity and reduced virus shedding postchallenge compared with the noncolonized vaccinated pigs. By enhancing Th1 responses to oral AttHRV vaccine, while inducing Treg responses to VirHRV, the selected probiotics were beneficial in improving responses to an enteric vaccine and alleviating the severity of an enteric infection. Recombinant monovalent llama-derived nanoantibody fragments (VHH) as a passive treatment for rotavirus-induced diarrhea. Recombinant VHH nanoantibodies (VHH nanoAb) were effective passive antibody treatments for RV diarrhea in piglets. We determined the neutralizing activity of RV VP6-specific llama-derived single domain VHH nanoAbs against different RV strains in vitro and then evaluated their passive protection against VirHRV in Gn piglets. Supplementation of the milk diet with baculovirus-derived 3B2 VHH clone nanoAbs for 9 days conferred full protection against RV diarrhea and significantly reduced virus shedding. Administration of comparable levels of porcine IgG Abs protected 4 of 6 piglets from HRV diarrhea, but significantly reduced virus shedding in all piglets. G6P[1]-VP6 rotavirus-specific IgY Abs purified from eggs of hyperimmunized hens failed to protect piglets against HRV diarrhea or virus shedding when administered in similar quantities. Oral administration of VHH nanoAb did not interfere with host immunoglobulin isotype Ab responses to HRV and did not induce host serum or gut Ab responses to the treatment. Thus passive oral administration of rotavirus VP6-VHH nanoAb is a broadly reactive and effective treatment against RV-induced diarrhea in neonatal pigs. </p>