Enteric Diseases of Swine and Cattle: Prevention, Control and Food Safety

NON-TECHNICAL SUMMARY: Diarrheal diseases are economically important causes of production losses to livestock producers. Without continued research, food safety and diarrheal diseases of livestock will likely continue at the present rate or increase. This project will evaluate pathogenic mechanisms, diagnostic assays, and educational approaches to reduce the risks associated with enteric diseases of pigs and cattle.

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APPROACH: Studies will be performed to assess the ability of a probiotic treatment to ameliorate E. coli induced edema disease in pigs. Pigs will be fed the probiotic bacterial strain prior to challenge with virulent strains of E. coli. Uisng molecular techniques, we will complete the analysis of pulse field gel electrophoresis (PFGE) patterns of the E. coli O147 strains and assess if other techniques technique, such as multilocus enzyme electrophoresis, place strains of E. coli O147 into similar families based on dendogram analysis. We will construct isogenic E. coli F18+ strains with the long-term goal of evaluating these constructs in piglets to determine the role of enterotoxins in the clinical manifestations of edema disease. We will continue to evaluate the mucosal immune response of pigs following onset of bacterial induce colitis. Groups of vaccinated and control pigs will be challenged and the local host response will be assessed using cellular immunology and molecular biology approaches. Using a fluorescent bead assay, we will work to develop a diagnostic approach that will allow for a more rapid detection of pigs infected with Salmonella species or Brachyspira hyodysenteriae.

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PROGRESS: 2002/10 TO 2007/09<BR>
OUTPUTS: Experiments were performed to evaluate the mechanisms of protection afforded to pigs following vaccination against disease caused by Brachyspira hyodysenteriae. Experiments were also performed to evaluate means to control post-weaning diarrhea in pigs following infection with E. coli. Experiments were also performed to evaluate the transmission of enterotoxigenic E. coli between pigs. Several graduate students were trained during this period of time. It was demonstrated that pigs could be protected from developing swine dysentery using a parenteral vaccine delivered by the intramuscular route. The mechanism of protection was associated with the induction of a protective T cell response. While induction of an antibody response played a role in the immune-mediated protection, it was not sufficient. <BR> PARTICIPANTS: Andrea Dorn was a research associate that work on this project. She assisted with immunological assays, cultivating bacterial strains, monotoring the pigs, and managing the laboratory. Raquel Hontecillas was a graduate student that work on this project. She prepared vaccines, performed immunological and molecular biology assays to assess the immune response of pigs, and monitored pigs after infection. Any Helgerson was a graduate student, performed bacteriological assays, isolated E. coli from pig feces, performed PCR analysis, and monitored pigs following challenge. <BR> TARGET AUDIENCES: Target audiences would include pork producers, swine extension scientists, veterinary immunologists and microbiologists. Knowledge would have been inparted to students, technicians, and other faculty during informal classroom presentations and laboratory exercises. In addition, scientific presentations were given at national meetings. <BR> <BR>
IMPACT: 2002/10 TO 2007/09<BR>
The results of these studies indicated that a parenterally administered vaccine could be used to provide protection against a mucosal disease. In addition, the disease seemed to be controlled by CD4 T cells as opposed to to an antibody response. The dietary inclusion of colicin E1 decreased the incidence and severity of post weaning diarrhea caused by F18-positive enterotoxigenic E. coli and improved the growth performance of the piglets. Additionally, the reduced incidence of post weaning diarrhea due to dietary colicin E1, lowered the levels of expression of the genes for interleukin 1beta and tumor necrosis factor beta in ileal tissues from these animals. The dietary inclusion of colicin E1 may be an effective alternative to conventional antibiotics in the diets of weaning pigs for the prevention of post weaning diarrhea caused by F18-positive enterotoxigenic E. coli. Serogrouping of edema disease isolates from the Iowa State University Veterinary Diagnostic laboratory recovered between 1996 and 2000 indicated that 42% belonged to serogroup O147. Our data suggest that these strains may be a common serotype of edema disease-causing E. coli in the United States.
<BR> <BR> PROGRESS: 2004/01/01 TO 2004/12/31<BR>
Escherichia coli O157:H7 is only occasionally isolated from healthy swine, but some experimentally infected animals shed the organism in their feces for 2 months. Potential explanations for the paucity of naturally occurring infections in swine, as compared to cattle, include an reduced ability to be transmitted, need for a high infectious dose, or management practices that affect maintenance of the organism in the intestinal tract. These studies examined the ability to transmit E. coli O157:H7 from infected to naive pigs. The study indicated that pigs shedding less than 10e4 E. coli O157:H7 per gram feces transmitted the organism to 50 percent (6 of 12) naive pigs. The infectious dose of E. coli O157:H7 for young pigs was 6 x 10e3 CFU. These results suggest that swine do not have an innate resistance to E. coli O157:H7. Recently, however, E. coli O147 strains have been recovered from outbreaks of edema disease across the U.S. We hypothesized that these isolates are clonal and have evolved from O147 ETEC by the acquisition of the stx2e gene. To this end, 53 strains of E. coli O147 (1996 to present) were genotypically compared to 21 strains recovered between 1974-1995. Among the recent isolates, 38 of 53 showed greater than 80% similarity and 37 of these 38 contain the virulence genes f18, sta, stb and stx2e. Isolates recovered prior to 1996 had less than 55% similarity to the new strains. ETEC virulence genes were present in 13/21 of these strains. Preliminary data for the clpX gene indicated that banding patterns of recent O147 isolates were similar to some of the older ETEC strains. Overall, the data suggests that the recent E. coli O147 isolates are closely related to one another and likely evolved from older strains of ETEC O147. Because of the need to reduce the risk of food-borne pathogens in the food supply, it is necessary to monitor livestock herds and flock for the presence of multiple pathogens. Many herds are serologically monitored for the presence of antibodies to specific agents (e.g., Salmonella enterica, PRRSV). In an attempt to design an assay that could be used to more rapidly evaluate serum samples for antibodies to given agents, we utilized a liquid-phase microbead assay system that would permit the detection of antibodies to multiple antigens in a single reaction tube. Using the Luminex 100 System, endotoxin prepared from five different serotypes of S. enterica were prepared and linked to separate sets of fluorescent microbeads. Using reference rabbit antisera against specific salmonella serovar antigens, the microbead assay was found to be highly specific and had a 20- to 200-fold increase in sensitivity relative to an ELISA assay performed with the same reagents. The microbead-based assay was also able to detect serum antibodies specific for the serotype antigens of Salmonella enterica. Serum samples were obtained from vaccinated, experimentally infected, and naturally infected pigs. Results indicated that the microbead assay could be used to reliably detect serum antibodies to the target antigen, that the required sample volume was low (0.1 mL), and that multiple antigens could be combined in a single reaction tube.
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IMPACT: 2004/01/01 TO 2004/12/31<BR>
Because pigs do not have an innate resistance to colonization by E. coli O157:H7, pigs could serve as a reservoir host for this zoonotic pathogen. Salmonellosis is a significant food-borne illness and there are multiple serotypes that can cause human disease. The results of these studies indicate that development of rapid, multiplexed immunoassays to identify pigs/herds exposed to S. enterica is possible.
<BR> <BR> PROGRESS: 2003/01/01 TO 2003/12/31<BR>
Work was initiated on a project that would allow for more rapid detection of porcine exposure to various serovars or serogroups of Salmonella enterica. For this project, bacterial endotoxin was successfully extracted from six separate isolates of Salmonella enteric representing six separate serogroups. The techniques for successfully conjugating the bacterial endotoxin moleculae to fluorescent beads were refined. We performed comparative assays to ascertain if the fluorescent bead assay was more sensitive than traditional ELISA for the screening of positive serum samples. The results of these tests indicated that the fluorescent beads assay employing the Luminex 100 system was nearly 100 times more sensitive than the traditional ELISA in detecting serum antibodies. Studies were conducted to assess the potential for farm staff or other individuals to mechanically transmit enterotoxigenic Escherichia coli (ETEC) from infected to susceptible weaned pigs during direct pig contact and to determine biosecurity measures that will prevent such transmission. One hundred and twenty-five 19- to 21-day-old weaned pigs, culture-negative for ETEC M1823B, were randomly allocated to six treatment groups housed in five separate isolation rooms. Inoculated Pigs were offered 1.36 x 10e10 to 8.92 x 10e10 colony forming units of E coli mixed in strawberry gelatin on two occasions. Pen Sentinels were housed with Inoculated Pigs. A caretaker fed pigs, checked waterers, and directly contacted each group of pigs for 10 minutes daily for 10 consecutive days. The caretaker contacted Inoculated Pigs and moved directly to Direct Sentinels, recontacted Inoculated Pigs, washed hands twice, changed outerwear, then contacted Hand-wash Sentinels. The caretaker then recontacted Inoculated Pigs, showered, changed outerwear, and contacted Shower Sentinels. Non-exposed pigs had a separate caretaker. Escherichia coli M1823B was isolated from all 20 Inoculated Pigs, all five Pen Sentinels, 20 of 25 Direct Sentinels, and 23 of 25 Hand-wash Sentinels. The 25 Shower Sentinels and 25 Non-exposed Pigs remained culture-negative for M1823B. Summary: A fluorescent bead assay will allow for rapid identification of swine serum samples with antibodies to a wide range of Salmonella enterica serogroups relative to the current methods employed. Mechanical transmission of E. coli by animal handlers occurred readily and was not prevented by hand washing or changing outerwear clothing. Showering and wearing clean clothing did prevent the mechanical transmission of E. coli.
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IMPACT: 2003/01/01 TO 2003/12/31<BR>
Methods were evaluated to prevent transmission of and to detect food-borne pathogens. Effective transmission of infectious agents between facilities was prevented when personnel showered and changed clothing. Diagnostically, detection of food-borne pathogens is slow allowing suspect food products to reach market. Using serum samples, this project demonstrated that a more rapid (i.e., 12 to 18 hr) method of surveillance is feasible.