<OL> <LI> Define mechanisms of pathogen host environmental interactions in enteric and food borne diseases. <LI> Develop and improve diagnostics, treatment, and preventative measures for enteric and food borne diseases. <LI> Provide training and continuing education opportunities and dissemination of information to students, producers, consumers, veterinarians and diagnostic laboratories.
NON-TECHNICAL SUMMARY: KS and other NC-1007 members will work to more fully understand the epidemiology of enteric viruses and bacteria in causing diarrhea in livestock and the role of livestock in transmitting diarrheal disease to humans. Improved tests for detection of these agents will be developed. Research to understand how these agents cause diarrhea and how to control and prevent disease will be conducted.
APPROACH: AZ, IA, KS, MI, NE, SD, and OH will collaborate to identify and obtain isolates of bovine and porcine caliciviruses. These isolates will be used to determine their virulence in calves and pigs, and they will be genetically characterized and compared to human isolates to determine if livestock are a source of calicivirus infection in humans. KS, SD, NE, IA, WS, and OH will identify and attempt isolation of bovine toroviruses and coronaviruses and determine their importance in enteric and respiratory disease syndromes. Using RT-PCR, KS will cooperate with SD, NE, IA, MI, OH and WA to amplify segments of the genome of ovine coronaviruses and compare the sequences with those of coronaviruses from other species. IA, SD, KS, MI, and NE will collect samples from pigs with atypical TGE and OH will genetically characterize the isolates to determine if they are TGE viruses (TGEV) with reduced virulence or porcine respiratory coronavirus (PRCV) shed in the feces. SD, KS, IA, NE and MN will use multiplex PCR to analyze isolates of E. coli for virulence factors associated with production of diarrhea and edema disease in pigs. KS, NE, and SD will use PCR and animal studies to determine the importance of EAST1 toxin and other enterotoxins in causing diarrhea in pigs. IA, KS, MI, NE, SD, and WS will collaborate with MN and AZ to isolate and genetically characterize Lawsonia intercellularis from species other than pigs to determine their relatedness to isolates from pigs. OH and MI, in collaboration with IA, KS, SD, and NE, will use RT-PCR and virus type-specific monoclonal antibodies (Mabs) to determine the prevalence of group A and nongroup A rotaviruses in calves and pigs and determine the importance of different G and P genotypes in calf diarrhea. KS and OH will use RT-PCR to perform gene sequence comparisons of rotaviruses from different species of livestock. NE and KS will determine the sensitivity and specificity of a Mab to identify group A rotaviruses from livestock in formalin-fixed, paraffin-embedded tissues. IA, KS, NE, SD, and WS will provide samples to OH and MI so that they can validate RT-PCR and real-time PCR techniques for identification of bovine toroviruses and coronaviruses, and TGEV. OH will provide Mabs and RT-PCR information to the same stations for detection of toroviruses and coronaviruses and to survey their incidence in cattle. KS, MI, NE, and SD will assist OH in improving serologic, immunohistochemical, and RT-PCR tests for differentiating TGEV and PRCV. MI and KS will cooperate in evaluating new rapid immunomigration assays for identification of enteric pathogens. All NC-1007 members will participate in summarizing the information presented at their annual meetings into a format suitable for dissemination to livestock producers, veterinarians, scientists, and consumers. This information will be provided to the respective states' Cooperative Extension Service, producer groups, the editor of the KS Food Safety Newsletter, and the editors of the KS and NE veterinary newsletters. The group will hold 1 annual meeting in Washington, DC and invite members of Congress and consumer, industry, and commodity groups.
PROGRESS: 2002/10 TO 2007/09<BR>
The long term goals of studies on Escherichia coli O157 at KSU are to understand the ecology of the pathogen and its complex association with cattle, including the impact of the environment, and develop practical intervention strategies at the farm level. Major outputs of this work include: 1) fecal shedding of E. coli O157 is influenced by the immune status of the animal based on a time - dependent correlation between immunosuppression (induced by dexamethasone injection) and the concentration of E. coli O157 shed in the feces; however, persistent infection with Bovine Viral Diarrhea virus did not change either the level or duration of shedding in calves, 2) an in vitro fermentation system with a fecal microbial inoculum was developed to assess mitigation strategies and use of probiotics, 3) a method to quantify the levels of fecal shedding of E. coli O157 was developed ,4) feeding of distiller's grain, a byproduct of ethanol industry, was positively associated with prevalence of E. coli O157 in cattle, and 5) the common house fly (Musca domestica L.) was shown to be capable of transmitting the pathogen to cattle and may play a key role in dissemination in feed lots. The significance of the heat-stable enterotoxin produced by different pathotypes of E. coli known as EAST1 in post-weaning piglet diarrhea has been difficult to assess due to low amounts of the enterotoxin present in culture supernatants and rapid loss of biological activity during purification. Fusion proteins containing either ubiquitin or thioredoxin as the fusion partner with EAST1 were expressed in high yields. Unfortunately, the biological activity of EAST1 released by protease digestion of both fusion proteins was very low, which suggested that the two disulfide bonds necessary for biological activity of EAST1 had not formed in the cytoplasm. Promising results have bee obtained using a two cistron expression system containing a minicistron upstream of the astA gene, which facilitates formation of the translation complex and increased amounts of recombinant EAST1. A direct enzyme linked immunosorbent assay (ELISA) was developed to quantify the amount of his-tagged fusion proteins containing EAST1.
IMPACT: 2002/10 TO 2007/09<BR>
This work shows that the association of Escherichia coli O157:H7 with cattle, which in contrast to humans does result in disease is complex and determined by several factors including: 1) the immune status of the animal, 2) diet, 3) seasonal factors (e.g. the presence or absence of insect vectors such as flies), and 4) management practices. A method to quantify fecal shedding of E. coli O157:H7 and an in vitro fermentation system to assess mitigations strategies of probiotics were developed. The shedding level was higher in animals fed distillers grain, a byproduct of the ethanol industry. The work with the EAST1 enterotoxin represents an important step in development of an immunological assay for use in diagnostic laboratories and production of amounts sufficient for characterization of its biological activity. Participants: T.G. Nagaraja, L. Zurek, D. Renter, M. Sanderson and D.C. Robertson Target Audiences: Livestock producers (cattle and pork), veterinarians and consumers
PROGRESS: 2006/01/01 TO 2006/12/31<BR>
One goal of this project is to identify methods to control Escherichia coli O157:H7, which is a major food borne pathogen of humans, for which cattle are the primary reservoir. Our research indicates that most E. coli O157 in cattle are carried in the hind gut and that swabs from the rectoanal junction are superior to feces for diagnosis of carrier cattle. That monensin does not prolong fecal shedding and that feed (15%) and house flies (55%) are infected. Diets formulated to impact hindgut fermentation and probiotics were shown to reduce the prevalence of E. coli O157 in cattle. We also evaluated the prevalence of Salmonella fecal shedding in feedlot cattle pulled for respiratory disease and determined the impact of Salmonella on subsequent performance. Using standard culture methods and PCR, Salmonella was identified in 946 of 1,270 (74.5%) cattle pulled and treated. Although, Salmonella shedding was common there was no indication that shedding was associated with clinical or subclinical disease and shedding did not affect the subsequent health of the animals. Cattle treated with multiple antibiotics were more likely to shed Salmonella. Fifteen serotypes were isolated, of which Orion (48.6%), Anatum (20.8%), and Kentucky (9.1%) were the most common. Although the high prevalence of Salmonella in feedlot cattle feces may have public health consequences, the serotypes isolated from the cattle are different from those reported in national surveys of salmonellosis in humans. Another project is to study the expression and mechanism of action of EAST 1 enterotoxin produced by enteroaggregative E. coli, which is a cause of diarrhea in humans and domestic animals. E. coli expressing EAST 1 enterotoxin gene have been isolated from over 60% of humans, cattle, and pigs with diarrhea. However, the significance of the enterotoxins has been difficult to establish, partly because of the low yield by wild type E. coli. In an effort to increase the production of EAST 1, its gene, astA, was cloned into several high yield expression vectors, but none of the vectors increased production over that of wild type E. coli. Alternate approaches to increase EAST 1 production continue. Another project looked at the effect of EAST 1 on cultured intestinal epithelial cells using the cell line IPEC-J2 and intestinal tissues freshly harvested from piglets. Preliminary data indicates that EAST 1 enhances the effects of other enterotoxins, thus acting as a virulence factor. Results from the IPEC-J2 cells and the fresh pig intestines closely parallel each other indicating that the cell line is an excellent in vitro model to study the effects of EAST 1 and other enterotoxins.
IMPACT: 2006/01/01 TO 2006/12/31<BR>
This work identifies flies as a potential source of human infection by E. coli O157. It also demonstrates that probiotics and diet manipulations might be useful in decreasing E. coli prevalence in cattle, thus helping to decrease the likelihood of food contamination and transmission to cattle. The research also demonstrates that in most cattle Salmonella infection is subclinical and in most cases has little effect on the health of infected cattle. However, antibiotic treatment did increase fecal shedding, but not by serotypes usually associated with human salmonellosis. The work with EAST 1 enterotoxin will help us to understand the importance of the enterotoxins in causing diarrhea in humans, cattle, and pigs and to understand the mechanisms by which it works. This will help in prevention and control of E. coli diarrhea in people and livestock.
PROGRESS: 2005/01/01 TO 2005/12/31<BR>
Enteroaggregative Escherichia coli heat-stable toxin 1(EAST1) is a low-molecular weight E. coli heat-stable enterotoxins that is associated with enteroaggregative E. coli (EAggEC), which is recognized as a cause of diarrhea for travelers, persistent diarrhea and failure to thrive in young children, and chronic diarrhea in debilitated adults. EAST1 is also found in E. coli isolated from cattle and pigs with diarrhea and systemic disease; however, the role of EAST1 in diseases of cattle and pigs is unknown. We attempted to characterize the biological activity of EAST1 in a transformed cell line derived from the small intestine of pigs. Exposure of the intestinal epithelial cells to EAST1 resulted in a direct and rapid effect that is consistent with the induction of profuse watery diarrhea. The response to EAST1 was greater than that evoked by heat-stable toxin a, a recognized virulence factor in many strains of E. coli that cause diarrhea in calves and pigs.
IMPACT: 2005/01/01 TO 2005/12/31<BR>
These results show that EAST1 has the potential to induce diarrhea in livestock. It is not uncommon to isolate from pigs and calves with diarrhea E. coli that carry the gene for EAST1, but not other virulence factors. Further research to more fully explore the role of EAST1 in causing diarrhea in calves and piglets is warranted.
<BR><BR>PROGRESS: 2004/01/01 TO 2004/12/31<BR>
We examined 25 soil samples from 2 feedlots and 19 soil samples from a small farm with diarrhea in adult cattle for bovine coronavirus (BCV). Six of the 19 farm samples were positive by antigen-capture ELISA, but they were negative by virus isolation and reverse transcriptase-polymerase chain reaction (RT-PCR) suggesting that BCV is inactivated in the soil. We investigated the efficacy of 4 disinfectants to inactivate BCV in the presence and absence of organic material. The disinfectants were bleach, ethanol, Virkon S, and DF-2ood by Sandia National Laboratory. The effect on virus viability was determined by growth on human rectal tumor 18 cells. The effects on virus proteins and RNA were determined by Western blot and RT-PCR. One minute exposure to 12.5% DF-2ood, 10% bleach, 70% ethanol, and 1% Virkon S resulted in loss of viability of BCV. RNA was more stable and needed longer and higher concentrations for complete inactivation. We compared the BCV antigen-capture ELISA to the Syracuse Bioanalytical BCV antigen-capture ELISA by testing 25 bovine fecal samples with both ELISAs. There was agreement in 19 samples that were negative with both tests. The other 6 samples were weakly positive with the KSU ELISA and negative with the Syracuse ELISA. We conclude that the two ELISAs differ in their analytical sensitivity. Additional studies based on experimental infections are in progress to resolve these differences.
IMPACT: 2004/01/01 TO 2004/12/31<BR>
Our results indicate that soil is not a reservoir for BCV and that infected cattle are the major source of infection for other cattle. Also, all of the tested disinfectants can be used to effectively disinfect buildings that have housed affected cattle. Because the human pathogen SARS virus is a related coronavirus, these disinfectants hold promise for disinfection of the environment of people infected with SARS virus. Our studies on the sensitivity and specificity of various tests to detect BCV will help to improve diagnosis.
PROGRESS: 2003/01/01 TO 2003/12/31<BR>
We have been collaborating with Sandia National Laboratories in testing unique decontamination formulations to inactivate coronaviruses. Bovine coronavirus is being used as a safe substitute for SARS-like coronaviruses. Bovine coronavirus was completely inactivated after 3 minutes contact with the compound. Studies to assess shorter contact times and the ability of the decontamination agent to inactivate coronavirus in the presence of organic compounds are in progress. Because of concerns that antibiotic usage in livestock can contribute to development of antibiotic resistance by bacteria that cause human infections, development of non-antibiotic feed additives to replace currently used feed-grade antibiotics is important. We evaluated the effects of mannanoligosaccharides (MANN) and sodium chlorate (SC) on growth of pigs before and after challenge with Salmonella enterica serovar Typhimurium and the effect of the 2 compounds on fecal shedding of S. typhimurium. Pigs were fed the test diets 14 days before and after S. typhimurium challenge and the results compared to pigs fed carbadox or no feed additives. At the levels fed, MANN and SC depressed feed intake and average daily gain. After challenge, the weight gain and feed efficiency of pigs fed carbadox, but not MANN or SC, were significantly better than those fed the control diet. Seven days after challenge, the S. typhimurium fecal shedding scores of the MANN group were significantly decreased in comparison to the other groups. However, 14 days after challenge there was no difference between groups. Rectal temperatures after challenge returned to normal sooner and serum insulin-like growth factor values decreased the least in the carbadox group.
IMPACT: 2003/01/01 TO 2003/12/31<BR>
The coronavirus experiments will result in safe compounds that will effectively and rapidly destroy environments contaminated with human and animal coronaviruses, thus decreasing transmission of these viruses to new hosts. Development of non-antibiotic replacements for feed grade antibiotics will help livestock producers to maintain high production parameters and to produce healthy livestock while not worrying about possibly contributing to antibiotic resistance in human pathogens.