Duration: 10/01/2002 to 09/30/2007

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

Even though most people in the U. S. feel that their food is safe, food borne illnesses are a serious problem that affects and concerns everyone. The Center for Disease Control (CDC) estimates that each year in the U. S. there are approximately 76 million cases of infectious and noninfectious food borne illness that result in 325,000 hospitalizations and 5,000 deaths. Of the 76 million cases, 14 million are caused by known bacterial, viral, and parasitic pathogens. Several important bacterial and protozoal causes of food borne illness of humans are also recognized as important enteric pathogens of cattle and swine. The CDC estimates that Norwalk-like viruses (calciviruses) are responsible for 9.2 million of the 14 million (66%) cases of food borne illness that are caused by known pathogens. In their white paper Food Safety for FY 2003 the Cooperative State Research, Education, and Extension Service (CSREES) states that little is known about the epidemiology or specific role of several animal viruses, such as calicivirus in swine, and identifies these viruses as an emerging food safety issue. It is not surprising that the current and immediate past presidential administrations, Congress, and the USDA have made food safety a high priority.

Diarrheal diseases are economically important causes of production losses to livestock producers. The National Animal Health Monitoring System (NAHMS) survey Swine 2000 lists scours as the first and second most common infectious cause of piglet death during the preweaning and nursery phases of production, respectively. The National Pork Producers Council recognizes enteric diseases as one of the most costly problems facing pork producers and has listed four infectious diarrheal diseases among their top research priorities. Enteric diseases are no less important to the beef and dairy industries as evidenced by the results of the NAHMS survey Cattle and Calves Death Loss 1995 that identified enteric diseases as the most common infectious cause of neonatal death in dairy calves and the second most common cause of neonatal death in beef calves. Without continued research, food safety and diarrheal diseases of livestock will likely continue at the present rate or increase.

Over the past few decades the average size of swine and cattle herds has markedly increased, and it is not uncommon for both cattle and hogs to be moved great distances and animals, especially cattle, from numerous sources mixed together. Large national and multinational companies playing an ever-increasing role in livestock production, processing, and distribution of animal-origin food products. At the same time, the scope of food safety and animal disease problems has increased proportionally. Problems that begin at a local level have the potential to rapidly become national and even worldwide problems. For these reasons it is becoming increasingly more critical that research involves people with a wide range of expertise from multiple institutions in different regions of the country working together to find solutions. Also, the wide range of bacteria, viruses, and protozoa that cause enteric disease in livestock and food borne disease in humans, necessitates the collaboration of scientists with different areas of expertise. Individual institutions do not have available teams of scientists with the expertise offered by NC-1007 Technical Committee. The NC-1007 Committee consists of bacteriologists, virologists, molecular biologists, pathologists, and immunologists who have a productive history. The NC-1007 technical committee expects to be able provide new strategies for the prevention, treatment, and control of enteric pathogens of swine and cattle, which would result in cost benefits of millions of dollars per year. This information also would help prevent thousands of cases of human illness and, consequentially, result in savings of millions of dollars in medical expenses and lost time at work.

This NC-1007 project addresses the crosscutting research areas and objectives as identified in the North Central Regional Association Multistate Prioritization Process. This project will enhance pork and beef production and food safety by: providing information on mechanisms used by enteric pathogens to cause disease in humans and livestock; identifying ways in which these agents are transmitted to humans; improving the diagnosis of enteric pathogens in livestock, their environment, and our food; providing epidemiological information on possible emerging animal and human enteric agents; develop new and improved vaccines and non-antibiotic products for prevention and treatment of enteric infections; reduce antibiotic usage and antibiotic resistance; and more effective dissemination of information from the committee to possible users.

NC-1007 Crosscutting Areas for Regional Research

The NCA-02 assigned the following percentages for the proposed NC-1007 project:

1. Agricultural production, processing and distribution
   
2. Genetic resources development and manipulation (genomics and germplasm)
   
3. Integrated pest management
   
4. Natural resources and the environment
   
5. Economic development and policy
   
6. Social Change and Development
   
7. Food and nutrition

Related, Current and Previous Work

The CDC estimates that caliciviruses (Norwalk-like viruses) cause over 9 million cases/ year of food borne illnesses in the U. S. each year, making them the most common cause of acute food borne gastroenteritis in the U. S.1 Recently, caliciviruses that are genetically more closely related to human caliciviruses than to other animal caliciviruses have been identified in fecal samples from pigs and cattle.2,3 To date the only enteric calicivirus that has been successfully cultivated is a porcine virus, related to human enteric caliciviruses, that was obtained as a part of this multistate project, and which causes diarrhea and viremia in gnotobiotic pigs.4,5 The inability to isolate caliciviruses and the lack of an animal model seriously impedes research on human caliciviruses. Because the pig isolate is the only enteric calicivirus cultivated to date, work on developing it as a model for human infection will continue. Two members of NC-1007 have developed reverse transcriptase polymerase chain reaction (RT-PCR) assays for enteric caliciviruses, and have identified caliciviruses in bovine and porcine fecal samples (unpublished data). Genomic sequence data indicates that the bovine strains are more closely related to bovine enteric and human enteric caliciviruses than other animal caliciviruses. The zoonotic potential of bovine and porcine enteric caliciviruses, as well as their role as pathogens of calves and piglets, has not been proven and continued work in these areas is planned. In addition, work will be conducted to determine whether or not caliciviruses isolated from shellfish are of human or animal origin. A CRIS search identified six calicivirus projects, of which only one was not by a NC-1007 member. That project is at Oregon State University, and the principle investigator indicates that they also have identified caliciviruses in the feces of calves, but they have not reported any animal or genetic studies.

Porcine respiratory coronaviruses (PRCV) are natural mutants of transmissible gastroenteritis virus (TGEV) that display trophism for the respiratory tract and immunologically cross-react with TGEV. In the past, NC-1007 researchers have conducted research on immunity to TGEV and PRCV in order to improve vaccines, and other control measures. Currently, control of TGEV focuses on elimination. Consequently, differentiation between TGEV and PRCV infection is important for the sale of breeding stock both nationally and internationally. Work by members of this committee resulted in molecular tests to differentiate the two viruses and to differentiate antibodies to the two viruses.6,7,8 However, the serologic tests have sometimes shown inconsistent results in differentiating TGEV/PRCV,8 and continued work on control and differentiation of the two viruses is needed and planned in this proposal. A CRIS search for porcine coronavirus identified projects at four NC-1007 stations, NADC (a close collaborator with IA) and Cornell. The Cornell project does not overlap with this proposal.

Bovine coronaviruses are associated with neonatal calf diarrhea, winter dysentery in adult cattle, and, more recently, pneumonia in feedlot cattle. Recent work indicates that there are antigenic differences between isolates from different disease syndromes and between isolates from the same syndrome,9,10,11 which may explain why the available vaccines for neonatal diarrhea are often ineffective. Only recently have coronaviruses been recognized as causal agents of bovine pneumonia,12,13 and little is known concerning their epidemiology, pathogenic mechanisms, and relationship to enteric coronaviruses. Past work as part of this project resulted in new and improved molecular and serological tests to identify, differentiate, and characterize enteric and respiratory coronaviruses, added to the knowledge base concerning their pathogenic mechanisms, immunology, and epidemiology, and helped establish their importance as causes of winter dysentery and pneumonia. Continued collaborative research in these areas is planned. A CRIS search yielded no overlapping projects with non-NC-1007 members concerning bovine enteric coronaviruses. Current USDA funded research on bovine respiratory coronavirus by non-NC-1007 member stations is being conducted at Louisiana State and by the NC-107 Technical Committee on bovine respiratory diseases, which includes six NC-1007 stations. By including the association between enteric and respiratory coronaviruses and expanding the scope of ongoing research, the work of NC-1007 will compliment that of NC-107.

Bovine torovirus is a cause of diarrhea in cattle, but because it cannot be grown in cell culture and is difficult to identify by electron microscopy, it has been recognized very infrequently. Recently, with the use of reverse transcription-PCR (RT-PCR), torovirus was detected in 36% of 118 fecal samples from Canadian cattle with diarrhea.14 Two members of this project have developed and are refining RT-PCR procedures for bovine torovirus, and collaborative research is planned to determine the prevalence of torovirus in the NC-1007 area as well as further characterize and study the pathogenic potential of torovirus strains.

In spite of extensive research and the availability of vaccines, rotaviruses continue to be one of the most common causes of diarrhea in calves and pigs. One of the most promising developments is the recent identification and characterization of an intestinal receptor for group A rotaviruses15,16 by NC-1007 collaborators. This has resulted in the development of an orally administered synthetic neoglycolipid that blocks virus attachment, and, in preliminary experiments, dramatically attenuates diarrhea and virus shedding by orally infected pigs. Continued collaborative research in these areas is planned. Other research includes collaboration by multiple stations to determine the incidence of non-group A rotaviruses, and to determine the genotypes of group A rotaviruses from calves with diarrhea. This information is not fully known and is needed for development of vaccines and other control methods.

Campylobacter spp (primarily C. jejuni) cause an estimated 2.4 million cases of gastroenteritis per year in the U. S.,1 yet their pathogenic mechanisms remain largely unknown. Several studies, including studies by participants in this project, have shown that Campylobacter spp. are commonly carried in the intestinal tract of asymptomatic swine and cattle. Recent findings by NC-1007 researchers that have contributed significantly to our understanding of pathogenic mechanisms used by C. jejuni include: identification of a gene, cibB, that encodes a type III secretion-like protein that is necessary for invasion of cultured mammalian cells and in vivo virulence; and demonstration that the catalase gene, katA, is necessary for survival within macrophages.17-19 Collaboration on the roles of these and other genes in virulence of C. jejuni will continue. A CRIS search for Campylobacter and cattle and pigs yielded a large number of projects concerning epidemiology, antibiotic resistance, and pre- and post-harvest control, but nothing that would overlap with this proposal.

Cooperative research on the pathogenesis of enterotoxigenic Escherichia coli (ETEC) resulted in the identification of multiple porcine intestinal receptors for K88 E. coli fimbriae; identification of a porcine intestinal receptor, intestinal mucin-type glycoprrotein (IMTGP), to which K88+ (F4) Escherichia coli binds; demonstration that expression of IMTGP strongly correlates with susceptibility of piglet to K88+ E. coli infection; and identification of the role of hemolysin.20-23 Preliminary results from a collaborative effort involving four NC-1007 diagnostic laboratories indicate that K99 (F5) and 987P(F6) have almost disappeared as swine pathogens. Recent studies in Korea24 and Canada indicate that the gene for EAST1, the heat-stable enterotoxin associated with enteroaggregative E. coli, is present in E. coli isolates from pigs and calves with diarrhea. However, the ability of EAST1 to induce diarrhea in livestock is unknown. In the new proposal, multistation projects to further define pathogenic mechanisms of ETEC adhesion, colonization, diarrhea induction, and sepsis are planned. In addition, ability of EAST1 to cause diarrhea will be investigated in swine. A CRIS search did not reveal any overlapping research.

Multiple NC-1007 stations contributed to the identification of Lawsonia intracellularis, reproduction of proliferative enteropathy (PE) in pigs using isolates from the U.S., and the development of molecular test to identify Lawsonia intracellularis in diagnostic samples. More recently two stations collaborated to improve molecular diagnostics,25 and that work continues. Traditionally recognized as a disease of pigs and hamsters, PE caused by L. intracellularis has been recognized in a wide variety of animals. Recently NC-1007 researchers have worked with others to identify L. intracellularis infection of horses, ratites and primates.26-28 In swine, PE has become especially important in high health and nucleus herds.26 Thus, it is important to determine if strains that affect different animal species are the same or different, in order to ascertain if other animals are sources of infection for pigs, and visa versa. Because it is an obligate intracellular bacterium that can be grown only in cell culture, almost nothing is known about its virulence mechanisms.25 Two NC-1007 stations are among only a handful of institutions worldwide that have isolates of the organism. Past work will provide a strong basis for the projected projects to explore cross-reactivity between isolates, virulence mechanisms, improved diagnostics, and development of a mouse laboratory model.

Currently, there are no effective treatments for Cryptosporidium parvum infection of humans or livestock. As part of this project, IL has discovered that plasma membrane vesicles and fractioned cell membranes significantly inhibit binding of C. parvum to host cells. They have isolated and partially purified the inhibitor, and will continue to develop this product as a preventative/therapeutic agent for C. parvum induced diarrhea.

Escherichia coli O157:H7 is an important food borne cause of human illness1 that is a common inhabitant of the digestive tract of healthy cattle. In order to prevent human illness, it is important to increase our understanding of the prevalence and ecology of E. coli O157:H7 in cattle and their environments. NC-1007 researchers have completed several studies to determine the prevalence of E. coli O157:H7 on cow-calf farms, feedlots, and in free ranging deer in contact with cattle, as well as studies to identify possible management factors that are associated with bovine infection.29-32 Collaboration is planned to share fingerprinting results for E. coli O157:H7 bovine isolates from the northwest and midwest in order to better understand the epidemiology of E. coli O157:H7 in cattle.

Salmonellosis also is an important food borne disease of humans1 that is often associated with contaminated beef and pork products. During the course of the last proposal, researchers at NC-1007 stations completed work to identify the prevalence and serotypes of Salmonella spp. in dairy cows at slaughter, their prevalence in cattle drinking water, and the effect of transportation and feed withdrawl on shedding of S. typhimurium by experimentally infected pigs.31,33-35 NC-1007 members also identified a change in antimicrobial resistance among S. enterica serovar Typhimurium (S. typhimurium) isolates from cattle due to the emergence of multidrug-resistant definitive phage type 104 (DT104) S. typhimurium and developed a PCR test to identify DT104 S. typhimurium.34,36,37 NC-1007 researchers in collaboration with the NC-1007 diagnostic laboratories will continue collaboration to monitor the Salmonella serotypes and their antimicrobial resistance patterns of isolates from cattle and swine so as to identify changes as they emerge.

Objectives

1. Define mechanisms of pathogen-host-environmental interactions in enteric and food borne diseases.
   
2. Develop and improve diagnostics, treatment, and preventative measures for enteric and food borne diseases.
   
3. Provide training and continuing education opportunities and dissemination of information to students, producers, consumers, veterinarians and diagnostic laboratories.
   
4. 
   

Methods

Objective 1: Define mechanisms of pathogen-host-environmental interactions in enteric and food borne diseases.

OH and AZ will survey shellfish and environmental water sources for potential food borne enteric caliciviruses and determine by sequence analysis their animal (swine, cattle) versus human origin. The pathogenicity of selected animal strains will be studied in calves or pigs by OH. OH and MI will identify and obtain bovine enteric calicivirus (BEC) and porcine enteric calicivirus (PEC) isolates from bovine and porcine samples or waste lagoon samples supplied by NC-1007 diagnostic laboratories (AZ, KS, SD, NE, IA, WA) or collaborators. OH will genetically characterize these isolates and, by experimental infection of pigs and/or calves, investigate their ability to cause diarrhea and viremia, and assess the impact of viremia on diarrhea induction. OH will attempt to adapt the BEC and PEC isolates to cell culture using techniques (intestinal contents in medium) previously successful for cultivation of PEC. In collaboration with IL, OH will identify the factors in intestinal contents that promote growth of PEC in cell culture. OH will attempt to create infectious clones of PEC (wild type and cell culture adapted) and determine the molecular basis for cell culture infectivity and virulence.

NC-1007 diagnostic laboratories (KS, SD, NE, IA, WA) will supply OH with fecal and nasal samples from feedlot cattle with bovine respiratory disease for identification and/or isolation of bovine torovirus (BoTV) and bovine coronavirus (BCV). OH will compare the ability of BCV isolates originating from different syndromes, such as calf diarrhea, winter dysentery, and respiratory disease, to cross-protect against one another and against isolates from the same syndrome. OH will examine the association between BoTV and the various above clinical disease entities including the bovine respiratory disease complex.

KS has isolated a coronavirus from a sheep, which have not been previously reported to be infected by coronavirus. NC-1007 diagnostic laboratories (SD, NE, IA, MI, WA) that identify coronaviruses in ovine samples by immunologic or molecular tests or by electron microscopy will send those samples to KS for isolation and characterization. KS will propagate ovine coronaviruses in HRT-18 cells in the presence of pancreatin and trypsin. The different open reading frames starting with the nucleoprotein gene will be amplified by RT-PCR and compared with corresponding sequences from other ruminant coronaviruses.

OH will continue to collaborate with NC-1007 members at IA, SD, KS, MI, and NE by characterizing and differentiating strains of porcine coronaviruses from outbreaks of "atypical transmissible gastroenteritis virus (TGEV)" and porcine respiratory coronavirus (PRCV) infection. OH will determine if the viruses are atypical strains of TGEV with reduced virulence or strains of porcine respiratory coronavirus that are shed in the feces. OH will study the pathogenicity of selected porcine coronavirus isolates in gnotobiotic or conventional TGEV/PRCV seronegative pigs, and they will analyze a portion of the S gene to identify the genetic changes that are associated with the biological differences seen clinically.

Other enteric viruses (or respiratory isolates of bovine and porcine coronaviruses) will be detected in fecal, nasal, lagoon or oyster samples by a combination of immune electron microscopy, ELISA and RT-PCR using antisera or primers designed by OH. Selected isolates will be partially sequenced to confirm their identity and relationships to one another and to reference virus strains to study their potential origin (human vs. animal) and to assess their molecular evolution in the field. In addition, using gnotobiotic pigs or calves OH will examine the pathogenicity and disease mechanisms for selected virus strains.

IL has identified a new asialoganglioside binding activity associated with viral protein 6 in double-layered particles supplied by OH that appears to be common to all group A rotaviruses. They will continue work to determine if this binding activity represents a new virus target that can be exploited for therapeutic purposes. Through its characterization of a porcine rotavirus receptor, IL has synthesized a relatively inexpensive neoglycolipid carbomimetic analogue of the active epitope of the intestinal receptor for group A rotavirus. This analogue almost completely blocks rotavirus binding to and infection of host cells in vitro. A preliminary trial indicated that the receptor analogue protects pigs from challenge by the OSU strain of rotavirus. IL and OH will collaborate to test the therapeutic efficacy of this neoglycolipid receptor analogue in protecting against various strains of porcine rotavirus in studies conducted at IL. Work on defining the relative activity of intestinal gangliosides, as compared to other enterocyte glycoconjugates, as natural receptors for porcine rotavirus will be continued by IL. In this approach, an inhibitor of in vivo ganglioside synthesis is used in a xenograft model to explore the natural preference of rotavirus to bind to intestinal gangliosides versus other glycocojugates. IL and OH will collaborate to determine the universality of this porcine enterocyte ganglioside receptor in recognizing group A rotaviruses. OH will provide a variety of porcine and bovine rotavirus strains and will create and provide rotavirus-like particles (VLP) with known P and G types for use by IL in trials to define the receptor specificity and for potential vaccine applications. IL and OH will continue collaboration to characterize the mechanism of action of the recently discovered rotavirus nonstructural protein (NSP4) produced in rotavirus-infected cells. NSP4 is thought to function as a viral enterotoxin that may be responsible for early development of diarrhea. OH will produce baculovirus-expressed rotavirus NSP4 and IL will probe for the existence of a cellular receptor for NSP4.

Using multiplex PCR developed by NADC, KS and SD will test Escherichia coli isolates for virulence factors of enterotoxigenic (ETEC) and Shiga-like toxin producing E. coli (STEC) that cause diarrhea and edema disease, respectively, in pigs. These virulence factors include F4, F5, F6, F41, and F18 pilus adhesions, and toxins that include heat labile (LT) enterotoxin, heat stable (ST) enterotoxins a (STa) and b (STb), and Shiga-like toxin 2e. IA, NE, and MN will send porcine E. coli isolates to SD or KS for pilus and toxin testing. After analyzing for ETEC associated pili and toxins, SD will send their E. coli isolates to KS where they will be tested for the EAST1 gene by PCR. EAST1 is a heat stable toxin that was first associated with enteroaggregative E. coli, which are a major cause of persistent diarrhea and decreased growth in children. Recently, the EAST1 gene has been reported to be common in E. coli isolates from pigs with diarrhea, but the ability of EAST1 to cause diarrhea in pigs is unknown. KS will identify isolates that have genes for pilus adhesions and EAST1, but no other toxins. After testing the isolates for pilus production by ELISA and fluorescent antibody staining, KS, NE, and SD will determine the relative importance of LT, STb, and EAST1 production by ETEC in causing diarrhea in piglets. This will be done by comparison of the pathogenicity of knockout mutants for each of these 3 toxins with that of complemented mutants and parent strains in gnotobiotic and conventional piglets. SD will also test strains, identified by themselves and KS, that possess incomplete repertoires of virulence determinants (pili but not toxins and vise versa) for virulence in gnotobiotic pigs. The purpose of these studies will be to identify novel pathogens and virulence factors (ie, strains that express enterotoxin genes but no known fimbriae but still cause disease likely produce novel fimbriae).

SD and NE will determine the chain of events that enable ETEC to colonize the intestines of pigs. Issues to be examined include how the bacterium penetrates the fibrous glycocalyx and thereafter gains access to intestinal receptors. They will also examine whether enterotoxin facilitates receptor access by hydrating microvilli and, in effect, thus thinning the glycocalyx. Finally, they will examine whether bacterial attachment to enterocytes with or without enterotoxin production results in cytoskeletal rearrangements or stimulates other host cell activities. Isolated porcine enterocytes, intestinal tissue explants and ligated loop models will be used for these experiments. Effects of enterotoxin will be assessed after inoculation with exogenous LT or commercially available cholera toxin; bacterial binding studies will employ laboratory constructs and enterotoxin knockout mutant strains. Cellular morphology studies will employ electron and immunofluorescent microscopy. Comparison of protein expression in enterocytes with and without attachment of bacteria will be by two-dimensional PAGE followed by mass spectrum analysis of proteins, and perhaps sequencing to determine their identity.

IL will continue its work defining the mechanism of Cryptosporidium parvum sporozoite interaction with host cells using two approaches. One is to identify naturally occurring host enterocye surface molecule(s) that mediate sporozoite recognition and invasion and the other focuses on differential sporozoite gene expression that occurs as a direct consequence of host cell adhesion. IL is also in continuing epidemiologic and overland transport studies with C. parvum in collaboration with the Agricultural Engineering Department at the University of Illinois. These studies are investigating the stability of Cryptosporidium parvum oocysts in dairy fecal waste, its potential contribution as run-off to contamination of municipal drinking water and the use of vegetative buffer strips to control this overland transport. IL will also continue to investigate the mechanism of bisphosphonate inhibition of C. parvum infectivity in vivo, recently discovered by IL, in collaboration with Drs. Eric Oldfield (Department of Chemistry) and Roberto Docampo (Dept. of Pathobiology), U. of Illinois.

AZ and MN will exchange Lawsonia isolates and monoclonal antibodies to determine antigen cross-reactivity between strains and to identify neutralizing-sensitive epitopes of field isolates. N-terminal sequencing will be performed on neutralizing-sensitive epitopes for the eventual cloning of the gene(s) expressing the protein(s). In order to determine the relatedness of pig isolates to those from other species, NC-1007 diagnostic laboratories (IA, KS, MI, NE, SD, WS) will send intestinal samples from animals other than swine with PE to MN or AZ for isolation and/or characterization. NE will collaborate with MN on the development of a laboratory mouse model of proliferative enteropathy caused by Lawsonia intracellularis. The role of dietary factors in resistance and susceptibility to infection and disease will be evaluated. This laboratory model will provide basic information that will complement the data generated from the on-going whole-genome sequencing of L. intracellularis.

AZ will collaborate with WS to examine Campylobacter gfp constructs for expression in infected porcine enterocytes and macrophages. Genes downstream of the gfp-induced promoters will be sequenced and identified. Isogenic mutants will be constructed and examined for their role in virulence in in vitro assays and in the newborn pig model. WS will examine Campylobacter jejuni isolates obtained from various hosts (dogs, cats, cattle, swine, poultry) in AZ for their ability to secrete ciaB invasion proteins and for loss of flagella and motility.

Objective 2: Develop and improve diagnostics, treatment, and preventative measures for enteric and food borne diseases.

MI and WS will complete work on an assay in which an oligonucleotide-based DNA microarray is used as a detector for multiplex PCR products for group A rotavirus, coronavirus, bovine enteric calicivirus (BEC), and Cryptosporidium. Proof-of-concept experiments have been completed and MI will use this assay to determine the frequency of these four pathogens in samples from neonatal dairy and beef calves in Michigan, Indiana, and Wisconsin. All samples will be checked for all four agents by existing PCR-based assays. Potential variation between BEC strains will be determined by direct sequencing of PCR products. Factors potentially influencing frequency of occurrence of different combinations of pathogens (age, beef or dairy, herd size, location, clinical picture etc.) will be identified. This information will be used to develop recommendations to reduce economic losses resulting from infections of neonatal calves with these agents.

In collaboration with NC-1007 diagnostic laboratories (KS, MI, SD, NE, and IA), OH will evaluate the prevalence of group A and nongroup A rotaviruses in swine and cattle using RT-PCR and virus type-specific monoclonal antibodies. Serologic and RT-PCR techniques will be established and compared to type and monitor the presence of groups B and C rotaviruses. MI and OH will collaborate on determining the G (G6, G8, G10) and P (P1, P5, P11) genotypes of bovine rotavirus isolates using RT-PCR. KS has approximately 60 isolates of bovine, equine, porcine, and laprine rotaviruses, most of which are group A. KS will perform sequence comparisons of VP 4, VP6, and VP7 genes using primer pair information provided by OH. Genotyping of clinical samples over a period of several years will provide information on the stability or evolution of genotypes in the sample area, and it will be useful in assessing the efficacy of current immunization strategies. These studies will establish which rotavirus serogroups/serotypes are the most common in the field and provide valuable reagents. NE has developed a monoclonal antibody to group A rotavirus that recognizes group A rotaviruses from a variety of animal species. Using monoclonal antibody supplied by NE, KS will determine the sensitivity and specificity of the antibody for detection of group A rotaviruses in formalin-fixed, paraffin-embedded tissues from different animal species.

MI will continue collaboration with OH on developing and refining RT- PCR based detection and genetic characterization of bovine respiratory coronavirus and bovine torovirus infections. IA, KS, NE, SD, and WS will provide bovine fecal and respiratory tract samples to the two stations for validation of their tests.

KS, MI, NE, and SD will provide suspected PRCV specimens and paired sera to OH for further improvement of diagnostic assays (i.e., immunohistochemistry, RT-PCR, and blocking ELISA) to differentiate between TGEV and PRCV infected swine, and to survey the prevalence of these infections in swine. OH will provide monoclonal antibodies against TGEV to diagnostic laboratories at NC-1007 stations to detect TGEV or PRCV by immunohistochemistry in formalin-fixed intestine or lung tissues. IA, MI, and OH will apply RT-PCR techniques to detect TGEV or PRCV shedding from naturally or experimentally inoculated pigs.

OH will provide monoclonal antibodies to bovine coronavirus (BCV) or bovine torovirus (BoTV) or RT-PCR primer information to cooperating NC-1007 researchers to diagnose BCV and BoTV enteric and respiratory infections of cattle. Similarly, RT-PCR primer information will be shared with NC-1007 members to routinely diagnose bovine and porcine enteric calicivirus infections in cattle and swine respectively, and survey their prevalence in diarrhea cases and age-matched normal controls.

Real time PCR/RT-PCR assays will be developed by MI to detect group A rotavirus, bovine coronavirus and TGEV infections. NC-1007 diagnostic laboratories (IA, KS, SD, NE, WI) will provide samples to MI for validation of the tests. Real time PCR reduces the possibility of laboratory contamination and is usually more sensitive than a gel-based post PCR detection system. MI will develop a RT-PCR assay to detect TGEV in formalin fixed tissues. Currently, the test used most often for diagnosis of TGEV is the direct fluorescent antibody (DFA) test on small intestinal sections, which depends upon the availability of minimally autolyzed specimens. Immunohistochemistry is an alternative to DFA, if tissues are properly fixed and suitable antibodies are available. The results of RT-PCR will be compared to those obtained by immunohistochemical staining using monoclonal antibodies to TGEV provided by OH.

MI and KS will evaluate new rapid immunomigration assays for their usefulness in identifying enteric pathogens in cattle.

OH will collaborate with NE, IA, and MN to improve the efficacy of current rotavirus vaccines and develop new vaccines (virus-like particles, DNA vaccines, and new adjuvant and deliver systems). OH will use monoclonal antibodies to porcine immunoglobulins provided by IA, and monoclonal antibodies to bovine immunoglobulins provided by NE, to define the humoral and cellular immune responses to various group A rotavirus serotypes from swine and cattle. Comparisons of mucosal and passive immunity will be done using isotype-specific monoclonal antibodies in ELISA and ELISPOT, lymphocyte proliferation, assays to measure antibody secreting cell responses, lymphoproliferative responses, and cytokine profiles, respectively in normal, diseases, and vaccinated animals. OH will collaborate with IA and MN in comparing immunologic reagents and assays to quantitate humoral and cellular immune responses in swine. These studies will be invaluable in defining the role of the host immune responses in disease pathogenesis and for the evaluation of existing as well as novel rotavirus vaccine approaches.

SD and NE will collaborate to evaluate the efficacy of two anti-secretory drugs, racecadotril, an oral enkephalinase inhibitor, and zaldaride maleate, a calmodulin inhibitor, that are effective in treatment of diarrhea in humans, in treatment of infectious diarrhea in pigs. If either drug proves efficacious in preventing or greatly attenuating diarrhea, the consequences of the loss of ability to cause fluid secretions on microbial proliferation in the intestines will be examined. Gnotobiotic piglets will be inoculated with either ETEC strain 3030-2 (O157:K88ac; LT, STb), or the porcine rotavirus strain OSU. Anti-secretory drugs will be administered orally four times daily, and changes in animal weights will be used as an indicator of fluid loss. Consistency of feces will also be assessed. Following challenge and treatment, piglets will either be observed for 96 hours to determine the course of infection and disease, or euthanized after 24 hours and necropsied and tissues examined histologically for evaluation of the extent of lesions and/or concentrations of bacteria in the small intestine. Animal challenges will be done by SD, and histologic analyses by NE.

IL, in collaboration with Oh, will determine the in vivo deliver parameters necessary for optimal therapeutic efficacy of the new synthetic neoglycolipid carbomimetic receptor developed by IL. This will include: dosage, time, frequency of administration, delivery vehicle, intestinal survival, transport, and absorption kinetics, as well as activity against different field strains of porcine rotavirus. These studies will define the dosage parameters necessary for achieving maximum benefit of receptor therapy as a treatment for porcine rotavirus disease.

IL will investigate the use of bisphosponates as a therapeutic agent for treatment of cryptosporidiosis using an intestinal xenograph animal mode. Il has identified a colostrum- derived lipid that can block binding of C. parvum, and they will test the ability of this lipid to prevent colonization by C. parvum in intestinal xenographs and newborn calves.

Objective 3: Provide training and continuing education opportunities and dissemination of information to students, producers, veterinarians, and diagnostic laboratories.

In addition to its annual technical report, the NC-1007 committee will assemble a lay document describing major advances in knowledge and technology through research by participating states, with livestock producers, veterinary professionals, and consumers as the target audience. The document will be formatted for dissemination in printed form or over the World Wide Web. Representatives from each participating station will be responsible for submitting pertinent information to the NC-1007 chair, who will appoint an editing committee to assemble information of the highest perceived public interest from individual stations for inclusion in the document. After completion, the document will be emailed to individual station representatives who will disseminate it and pertinent in-state research findings through their respective states Cooperative Extension Service Agriculture Extension and other producer/consumer education entities for their information and use.

This committee will volunteer to provide an annual presentation to the Enteric Diseases Committee at the annual meeting of the American Association of Veterinary Laboratory Diagnosticians.

This committee will solicit conference grant funding from the USDA NRI to support expansion of attendance at its annual meeting to include graduate students, who will be invited to present research findings and to participate in research discussions. This activity will serve as a training experience for young scientists, ensuring their preparation to assume leadership roles in infectious disease research of importance to animal agriculture. Arizona and South Dakota will author the conference grants in the first year of activity. Other stations will participate on a rotating basis.

During one year of this projects 5-year cycle, this committee will hold its annual meeting in Washington, DC. This meeting will be coordinated with that of NC-229 and other animal disease focused committees of like plans. The meeting in Washington, DC will include a symposium with a specific outreach focus. Commodity groups, industry, and congressional delegation representatives will be invited to attend and participate in discussions relative to establishment of goals in enteric disease research and food safety, and the enhancement of research activity to reach those goals. The Washington, DC meeting will enable committee members and invited guests to visit congressional delegations and for committee members to confer with USDA grant program directors.

Enteric disease and food safety researchers from participating stations will disseminate research findings within their respective states through cooperation with Agricultural Extension and other producer/consumer education entities.

Measurement of Progress and Results

Outputs

• <B>Objective 1: </B> <OL> <li>Determination of the incidence and roles of caliciviruses and toroviruses in causation of enteric and respiratory diseases of swine and cattle. <li>Determination of the relatedness of porcine and bovine caliciviruses and toroviruses to those of humans and those found in shellfish, water, and waste lagoons. <li>Publication of results of the survey to determine the virulence genes carried by enterotoxigenic E. coli (ETEC) isolated from swine and the changes that have occurred in the last 15 to 20 years. <li>Determination of the importance of the EAST1 enterotoxin of E. coli in causing diarrhea in swine. <li>Determination of the roles of virulence genes of C. jejuni in causing diarrhea. <li>Identification of natural and synthetic compounds for prevention and control of C. parvum and assessment of the benefits of vegetative buffer strips to control overland transport of C. parvum. <li>A mouse model for the study of L. intracellularis infection will be developed and the effects of various feed ingredients in controlling clinical disease will be studied. </ol> <li><B>Objective 2: </b> <ol> <li>Improved molecular and immunologic tests and typing methodologies for caliciviruses, toroviruses, rotaviruses, and coronaviruses will be developed. <li>The prevalence and genotypes of rotoviruses and coronaviruses affecting cattle and swine in the NC-1007 region will be determined and summarized yearly. <li>Development of receptor-blocking compounds for treatment of C. parvum and rotaviruses will be completed. <li>Data concerning E. coli O157:H7 molecular subtypes and Salmonella serotypes and antimicrobial resistance patterns of isolates from participating stations and diagnostic laboratories will be gathered and summarized. </ol> <li><B>Objective 3: </b><ol> <li>The findings presented at the annual NC-1007 meetings will be summarized and distributed through lay channels to veterinarians, researchers, veterinary diagnosticians, livestock producers, and consumers. <li>Funds to support increased participation by graduate students in the NC-1007 project and annual meetings will be solicited from the USDA NRI. <li>One of the NC-1007 annual meetings will be held in Washington, DC to better inform lawmakers, producers, and consumers of the importance of enteric diseases and NC-62 research.

Outcomes or Projected Impacts

• <b>Objective 1:</b><ol> <li>Identification of naturally occurring and synthetic compounds to prevent and treat C. parvum infections in cattle and humans. <li>Research on use of vegetative buffer strips to control overland transport of C. parvum will result in reduced water contamination by cattle and reduce exposure of humans and livestock. <li>Identification of the virulence factors carried by enterotoxigenic E. coli affecting swine will allow swine producers and veterinarians to better control colibacillosis in swine. <li>Understanding the effects of virulence genes of C. jejuni will help in control and prevention of food borne infections of humans. <li>Development of a mouse model for L. intracellularis infection will allow more efficient research into methods for control of the disease in swine. </ol> <Li><b>Objective 2: </b><ol> <li>Improved molecular and immunologic tests and typing methodologies for caliciviruses, toroviruses, rotaviruses, and coronaviruses will allow faster and more accurate diagnosis of the causes of enteric disease in livestock. <li>Knowledge of the prevalence and genotypes of rotaviruses and coronaviruses in the NC-1007 region will help in production of more efficacious vaccines to protect livestock. <li>Development of receptor-blocking compounds for C. parvum and rotaviruses will for the first time allow successful treatment of diarrhea caused by these agents. <li>The continued development and use of non-infectious rotavirus- and calicivirus-like particles will improve understanding of virus-receptor interactions and mucosal immune responses and help in development of improved vaccines. <li>Knowledge concerning E. coli O157:H7 molecular subtypes and Salmonella serotypes and antimicrobial resistance patterns will aid in treatment of Salmonella infections of livestock and improve our understanding of the epidemiology of these human food borne pathogens, which will help prevent human infection. </ol> <li><b>Objective 3:</B><ol> <li>Distribution of NC-1007 findings through lay channels will better inform all concerned parties of the importance of enteric diseases of swine and cattle and of food borne diseases that can be contacted through consumption of pork and beef. <li>Dissemination of research findings of the NC-1007 committee will help improve the health of cattle and swine and of humans consuming beef and pork.

Milestones

(0):ctive 1:</b><ol> <li>Caliciviruses and toroviruses need to be obtained from cattle and swine during years 1 to 3 of the project so that pathogenicity studies can be carried out during years 4 and 5. <li>Strains of ETEC with and without enterotoxin and fimbriae genes need to be constructed by year 3 so that pathogenicity studies can be carried out in years 4 and 5. <li>Strains of E. coli that carry the gene for EAST1, but no other enterotoxins, need to identified to that knock out mutants can be prepared during year 3 and pathogenicity studies for swine can be carried out during years 4 and 5. <li>Development of knockout mutants of C. jejuni needs to be accomplished during the first 2 years of the project so that the effects of the mutations can be studied during the remainder or the project.</ol>

(0):ctive 2:</b><ol> <li>Microchip array protocols for detection of PCR products, real time PCR assays for rotavirus and coronaviruses, and RT-PCR assays will be need to be finished by the end of the first or second year so that the tests can be validated and data collected. <li>Development of receptor-blocking compounds for C. parvum and rotaviruses needs to be completed by the end of the third year so that work to bring these compounds into general use can begin. </ol>

(0):ctive 3:</b><ol> <li>The lay documents detailing NC-1007 research findings need to be prepared and distributed yearly within a few months of the annual meeting. <li>If a meeting is to be held in Washington, DC, the plans for the meeting need to be finalized by the end of the third year. </ol>

(0):0

Projected Participation

View Appendix E: Participation

Outreach Plan

The results of this project will be published in peer reviewed journals and communicated to the public through lay channels. A major portion of the outreach will be through activities outlined in objective 3.

Organization/Governance

The Research Technical committee shall consist of one technical committee representative from each cooperating agency as appointed or otherwise designated by the respective organization, an administrative advisor appointed by the Association of North Central Experiment Station Directors, and a representative of the Cooperative State Research, Education, and Extension Service (CSREES). The executive committee, consisting of the chair and secretary, is responsible for overall coordination of the project and will serve two-year terms. The chair, in consultation with the administrative advisor, will notify the technical committee members of the time and place of meetings, prepare the agenda, preside at meetings of the technical committee and executive committee, prepare the annual report, and send it to the administrative advisor within 60 days of the annual meeting for approval and distribution. The chair will also appoint an editing committee which will prepare a lay document of the research findings presented at the annual meeting and distribute the document to the committee representative from each station. The secretary will record the minutes of the annual meeting, and send copies to the administrative advisor within 60 days of the annual meeting for approval and distribution. The secretary will also keep and update a list of members, their addresses, phone numbers, and email addresses.

Literature Cited

1. Mead PS, Slutsker L, Dietz V, et al. Food-related illness and death in the United States. Emerg Infect Dis 1999;5:607-625.

2. Guo M, Chang KO, Hardy ME, Zhang Q, Parwani AV, Saif LJ. Molecular characterization of a porcine enteric calicivirus genetically related to Sapporo-like human calicivirueses. J Virol 1999;73:9625-9631.

3. Van der Poel Wim HM, Vinje J, van der Heide R, Herrera M-I, Vivo A, Koopmans MPG. Norwalk-like calicivirus genes in farm animals. Emerg Infect Dis 2000;6:36-41.

4. Saif LJ, Bohl EH, Theil KW, Cross RF, house JA. Rotavirus-like, calicivirus-like, and 23 nm virus-like particles associated with diarrhea in young pigs. J Clin Microbiol 1980;12:105-111.

5. Guo M, hayes J, Cho Ko, Parwani AV, Lucas Lm, Saif LJ. Comparative pathogenesis of tissue culture-adapted and wild-type Cowden porcine enteric calicivirus (PEC) in gnotobiotic pigs and induction of diarrhea by intravenous inoculation of wild type PEC. J Virol 2001;75:9239-9251.

6. Vaughn EM, halbur PG, Paul PS. Use of nonradioactive cDNA probes to differentiate porcine prespiratory coronavirus and transmissible gastroenteritis virus isolates. J Vet Diagn Invest 1996;8:241-244.

7. Sestak K, Zhau Z, Shoup DI, Saif LJ. Evaluationof the baculovirus-expressed S glycoprotein of transmissible gastroenteritis virus (TGEV) as antigen in a competition ELISA to differentiate porcine respiratory coronavirus from TGEV antibodies in pigs. J Vet Diagn Invest 1999;11:205-214.

8. Kim L, Chang K-O, Sestak K, Parwani A, Saif LJ. Development of a reverse transcription-nested polymerase chain reaction assay for differential diagnosis of transmissible gastroenteritis virus and porcine respiratory coronavirus from feces and nasal swabs of infected pigs. J Vet Diagn Invest 2000;12:385-388.

9. Kapil S, Richardson KL, Maag TR, Goyal SM. Characterization of bovine coronavirus isolates/from eight different states in the USA. Vet Microbiol 1999;67:221-230.

10. Chouljenko VN, Lin XQ, Storz J, Kousoulas KG, Gorbalenya AE. Comparison of genomic and predicted amino acid sequences of respiratory and enteric bovine coronaviruses isolated from the same animal with fatal shipping pneumonia. J Gen Virol 2001;82:2927-2933.

11. Kourteis AB, Gelinas AM, Dea S. Genomic and antigenic variations of the HE glycoprotein of bovine coronaviruses associated with neonatal calf diarrhea and winter dysentery.

12. Storz J, Lin XQ, Purdy CW, Chouljenko VN, Kousoulas KG, Enright FM, Gilmore WC, Briggs RE, Loan RW. Coronavirus and Pasteurella infections in bovine shipping fever pneumonia and Evans criteria for causation. J Clin Microbiol 2000;38:3291-3298.

13. Lanthrop SL, Wittum TE, Brock KV, Loerch SC, Perino LJ, Bingham HR, McCollum FT, Saif LJ. Association between infection of the respiratory tract attributable to bovine coronavirus and health and growth performance of cattle in feedlots. Am J Vet Res 2000;61:1062-1066.

14. Duckmanton L, Carman S, Nagy E, petric M. Detection of bovine torovirus in fecal specimens of calves with diarrhea from Ontario farms. J Clin Microbiol 1998;36:1266-1270.

15. Rolsma MD, Kuhlenschmidt TB, Gelber HB, Kuhlenschmidt MS. Structure and function of a porcine enterocyte ganglioside receptor for group A rotavirus. J Virol 1998;72:9079-9091.

16. Kuhlenschmidt TB, Hanafin WP, Bergner DW, Chang KO, Saif L, Kuhlenschmidt MS. Identification of a VP6 asialoganglioside binding activity in group A rotaviruses. (submitted)

17. Konkel ME, Kim BJ, Rivera-Amill V, Garvis SG. Identification of proteins required for the internalization of Campylobacter jejuni into cultured mammalian cells. Adv Exper Med Biol 1999;473:215-224.

18. Ziprin RL, Young CR, Byrd JA, Stanker LH, Hume ME, Gray SA, Kim BJ, Konkel ME. Role of Campylobacter jejuni potential virulence genes in cecal colonization. Avian Dis 2001;45:549-557.

19. Day WA, Sajecki JL, Pitts TM, Joens LA. Role of catalase in Campylobacter jejuni intracellular survival. Infect Immun 2000;68:6337-6345.

20. Billey LO, Erickson AK, Francis DH. Multiple receptors on porcine intestinal epithelial cells for the three variants of Escherichia coli K88 fimbrial adhesin. Vet Microbiol 1998;59:203-212.

21. Francis DH, Grange PA, Zeman DH, Baker DR, Sun R, Erickson AD. Expression of mucin-type glycoprotein K88 receptors strongly correlates with piglet susceptibility to K88(+) enterotoxigenic Escherichia coli, but adhesion of this bacterium to brush borders does not. Infect Immun 1998;66:4050-4055.

22. Sun R, Anderson TJ, Erickson AK, Nelson EA, Francis DH. Inhibition of adhesion of Escherichia coli K88ac fimbria to its receptor, intestinal mucin-type glycoproteins, by a monoclonal antibody directed against a variable domain of the fimbria. Infect Immun 2000;68:3509-3515.

23. Moxley RA, Berberov EM, Francis DH, Xing J, Moayeri M, Welch RA, Baker DR, Barletta RG. Pathogenicity of an enterotoxigenic Escherichia coli hemolysin (hlyA) mutant in gnotobiotic pigs. Infect Immun 1998;66:5031-5035.

24. Choi C, Kwon D, Chae C. Prevalence of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 gene and its relationship with fimbrial and enterotoxin genes in E. coli isolates from diarrheic piglets. J Vet Diagn Invest 2001;13:26-29.

25. Zhang P, Gebhart CJ, Burden D, Duhamel GE. Improved diagnosis of porcine proliferative enteropathy caused by Lawsonia intracellularis using polymerase chain reaction-enzyme- linked oligosorbent assay (PCR-ELOSA) Mol Cell Probes 2000;14:101-108.

26. Lawson GHK, Gebhart CJ. Proliferative enteropathy. J Comp Pathol 2000;122:77-100.

27. Klein EC, Gebhart CJ, Duhamel DE. Fatal outbreaks of proliferative enteritis caused by Lawsonia intracellularis in young colony-raised rhesus macaques. J Med Primatol 1999;28:11-18.

28. Lavoie JP, Drolet R, Parsons D, Leguillette R, Sauvageau R, Shapiro J, Houle L, Halle G, Gebhart CJ. Equine proliferative enteropathy: a cause of weight loss, colic, diarrhoea and hypoproteinaemia in foals on three breeding farms in Canada. Equine Vet J 2000;32:418-25.

Attachments

Land Grant Participating States/Institutions

AZ, IA, IL, KS, MI, MN, ND, NE, OH, SD, WA

Non Land Grant Participating States/Institutions