Progress 10/01/01 to 09/30/06
Outputs
In a previous report we described the use of porcine intestinal epithelial cell lines to demonstrate adhesive activity of porcine enterotoxigenic E. coli (ETEC) strains under in vitro conditions to cells relevant to natural disease. Bacterial binding to porcine cell line IPEC-J2 and/or IPEC-1 was mediated by fimbrial adhesins including K88ab, K88ac and K88ad; F41 and F18. Similar binding was not observed with K99 adhesins. These binding observations suggest that the two porcine cell lines may be valuable and convenient tools for the preliminary screening of plant-derived compounds for capability to blocking bacterial adhesion in the intestines of susceptible pigs. Recently, we have extended our cell culture work to establish that expression of E. coli heat labile enterotoxin (LT) from porcine ETEC can augment bacterial adhesion to the porcine cell lines. Further, E. coli strains expressing LT a non-toxic derivative thereof can block the binding of wild-type ETEC to
IPEC-J2 cells in a competitively exclusive manner. The work of this study, together with animal inoculation studies also previously reported by us strongly suggest that LT toxin may have dual roles in pathogenesis: production of diarrhea and increasing the cell adhesion ability of bacteria expressing the toxin.
Impacts
The long term objective of this project is to identify compounds, including those extracted from native plants that are useful in controlling diarrhea in pigs or other animals infected with diarrhea-causing pathogens. A short-term goal is to develop inexpensive assays for screening candidate compounds, including the bacteria to epithelial cell adhesion assays reported this year. The experiments reported indicate that LT expression is important in bacterial colonization, but also provides a relatively simple tool for screening plant extracts for at least some anti-enterotoxic capabilities. Screening methods can and will be devised to test extracts for anti-adhesive and anti-colonization enhancement (LT-mediated) activity.
Publications
- Koh, SY, S George, V Brozel, D Francis, R Moxley and RS Kaushik. 2006. Porcine Intestinal Epithelial Cell Lines as an in vitro Model for Studying Pathogenesis of Enterotoxogenic Escherichia coli. Abstract, ASM North Central Branch 66th Annual Meeting.
- Mateo, KS, PR Hardwidge, W Zhang, R Kaushik, and DH Francis. 2006. Effect of heat-labile enterotoxin expression on the ability of enterotoxigenic Escherichia coli to colonize the IPEC-J2 porcine epithelial cell line. Abstract, ASM North Central Branch 66th Annual Meeting.
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Progress 01/01/05 to 12/31/05
Outputs
A major challenge in the identification of compounds useful for prevention or treatment of diarrheal disease is the development of simple and inexpensive methods for screening candidate compounds. Such a method has not been available for screening compounds that block the binding of enteric bacteria to enterocytes, and was the objective of the current investigation. Attachment of bacteria to enterocytes is an early step in the development of enteric disease. Bacterial colonization by porcine enterotoxigenic Escherichia coli (ETEC) is mediated by fimbrial adhesins, the most common of which are F4(K88), F5(K99), F6(987P), F18, and F41. In this study, we investigated the adherence of ETEC strains expressing the various adhesins to two porcine intestinal epithelial cell lines, IPEC-J2 and IPEC-1. These cell lines were derived from jejunum and small intestine respectively, from one-day-old pigs. E. coli expressing adhesins K88ab, K88ac, K88ad, K99, F41, 987P, and F18 were
used for adherence studies. Different strains of E. coli expressing these fimbriae were incubated with IPEC-J2 or IPEC-1 cells in the presence of mannose to inhibit non-specific binding and bound E. coli cells were observed under a phase contrast microscope. E. coli expressing K88ac or K88ad equally bound to IPEC-1 and IPEC-J2, but E. coli expressing K88ab more heavily adhered to IPEC-1. E. coli expressing F41 heavily bound to both epithelial cell lines. E. coli expressing F18 strongly bound to IPEC-1 but did not adhere to IPEC-J2 cells. E. coli expressing K99 bound poorly to either cell line, but bound heavily to human epithelial tumor cell line Int-407. E. coli strains G58-1 and K12 which express no fimbrial adhesins failed to bind to either cell line. E. coli that expressed 987P also failed to bind either cell line. These findings show that the porcine intestinal epithelial cell lines may be useful for screening compounds for ability to block bacterial adherence to porcine
intestinal epithelium.
Impacts
The long term objective of this project is to identify compounds that are useful in controlling diarrhea in pigs or other animals infected with diarrhea-causing pathogens. A short-term goal is to develop inexpensive assays for screening candidate compounds, including the bacteria to epithelial cell adhesion assays reported this year.
Publications
- Kaushik R, S George, A Young, and D Francis. 2005. Lectin binding profile of porcine intestinal epithelial cell lines. Abstract. Conference of Research Workers in Animal Diseases, Annual Mtg. St Louis MO.
- Koh S, S George, V Brozel, D Francis and R Kaushik. 2005. Porcine Intestinal Epithelial Cell Lines as an in vitro Model for Studying Pathogenesis of Enterotoxigenic Escherichia coli. Abstract. Conference of Research Workers in Animal Diseases, Annual Mtg. St Louis MO.
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Progress 01/01/04 to 12/31/04
Outputs
In order to enhance our ability to assess benefits of anti-diarrhea therapy, efforts are underway to improve an enterotoxigenic E. coli (ETEC) infection model in gnotobiotic pigs utilizing F18-fimbriaed E. coli. We are also perfecting a method to quantify bacterial colonization of intestinal epithelium. Reports by other investigators suggest that clinically significant disease generation following experimental challenge of pigs with F18+ ETEC is inconsistent. Factors that may be responsible for inconsistency include environmental induction of receptor expression and heritable resistance due to defective genes responsible for receptor expression. Germ-free piglets were inoculated with five bacterial stains reflective of the major constituents of the normal intestinal flora, weaned to a solid diet (or both), then after acclamation, challenge-inoculated with an F18+ ETEC strain. Upon euthanasia 18 hrs post-inoculation, pigs were assessed for ETEC colonization and
diarrhea, and tested for the defective fucosyltransferase (FUT-1) gene believed to render pigs resistant to F18+ ETEC. Either bacterial inoculation or weaning to solid feed marginally enhanced F18 receptor expression as determined by a brush border bacterial adherence test. One FUT-1-intact, and no FUT-1-defective pigs developed disease and became heavily colonized with ETEC. The ill pig received neither the bacteria representative of the intestinal flora, nor the weanling diet. These results suggest that the genetics of receptor expression and/or environmental induction of receptor expression may be more complex than originally thought. Methods for the quantitative assessment of bacterial colonization of the small intestine are under development. We have perfected procedures to stain and visualize colonized bacteria by immunofluorescence on paraffin-embedded histological sections using antibodies to somatic antigens. We are currently exploring techniques to assess proportionate
coverage by the bacteria of the epithelial surface.
Impacts
The long term objective of this project is to identify compounds that are useful in controlling diarrhea in pig or other animals infected with diarrhea-causing pathogens. Several such studys were performed in previous years. This year, our efforts were to improve upon available models of diarrhea and methods of assessing the extent of disease (pathogen colonization in the gut)in the infected animal. We made progress in developing a model to study F18+ E. coli infection in pigs. We also began a project to more accurately determine the extent of bacterial colonization in the intestines of infected animals.
Publications
- Boots, R.E. and Francis D.H. 2004. Factors Influencing F18+ Escherichia coli Receptor Expression in Weanling Pigs., Department of Veterinary Science, South Dakota State University, Brookings, SD. Proceedings, 84th Annual Meeting, Conference of Research Workers in Animal Diseases.
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Progress 01/01/03 to 12/31/03
Outputs
Loperamide has exhibited efficacy in the treatment of diarrhea of various etiologies in humans. This antidiarrheal agent acts primarily through activation of opioid receptors in the gastrointestinal tract. Previously, we tested the drug for its ability to attenuate diarrhea and dehydration in piglets resulting from enterotoxigenic E.coli (ETEC) strains expressing heat labile enterotoxin (LT) and heat stable enterotoxin type b (STb; E. coli strain 3030-2; serotype O157:K88). Loperamide did not alter either diarrhea or dehydration in this model. Neither did it affect intestinal colonization by the bacterium. During the present reporting period, we tested the ability of loperamide to attenuate the effects of an ETEC strain expressing the other common enterotoxin, STa. Neonatal piglets were challenge-inoculated with ETEC strain 1194 (O141: 987P; STa). The drug was administered to pigs per os at 0.5 mg/kg every 8 hours beginning 12 hrs before bacterial challenge. Pigs were
euthanized when the effects of dehydration became apparent in positive control pigs not receiving loperamide. Experimental observations were similar to those made regarding challenge with strain 3030-2. Loperamide had no appreciable effect on the diarrhea or dehydration in piglets challenged with strain 1194. To establish a model for better testing the effects of individual enterotoxins (LT and STb) on diarrhea and disease in young pigs, enterotoxin positive and negative isogenic E. coli strains were constructed from a K88+, enterotoxin negative E. coli strain. Plasmid pBR322 was used to clone and express LT and STb enterotoxins. Pathogenesis of each construct was assessed in one-week-old gnotobiotic pigs (6 pigs/treatment), along with a control strain that received only the plasmid vector. All of the isogenic strains were able to colonize the epithelium of the small intestines of K88 receptor-positive pigs. However, only enterotoxin-producing strains caused appreciable diarrhea.
Piglets inoculated with the LT+ strain became significantly dehydrated within 18 hrs (as assessed by change in blood packed cell volume, and serum total protein: 20%, p=0.012; and 25%, p=0.002, respectively), while those inoculated with the STb strain did not. This investigation suggests that the isogenic strains created are virulent in gnotobiotic pigs and may represent a good model for studying the effects of individual enterotoxins on the pathogenesis of enterotoxigenic E. coli infection.
Impacts
Development of isogenic constructs relative to LT and STb enterotoxin genes will facilitate future studies in the pathogenesis of enterotoxigenic E. coli infections and the evaluation of various therapies directed at the effects individual enterotoxins.
Publications
- Zhang, W., Freeling, J., Berberov, E., Moxley, R. and D. Francis, D. 2003. Development of a model for the assessment of the contributions of E. coli enterotoxins to the pathogenesis porcine colibacillosis. Proceedings, 84th Annual Meeting, Conference of Research Workers in Animal Diseases.
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Progress 01/01/02 to 12/31/02
Outputs
Loperamide has exhibited efficacy in the treatment of diarrhea of various etiologies in humans. This antidiarrheal agent acts primarily through activation of opioid receptors in the gastrointestinal tract (Daly, J.W., and J. Harper. 2000. Loperamide: novel effects on capacitative calcium influx. Cell Mol Life Sci 57:149-157). We tested the drug for its ability to attenuate the effect of enterotoxigenic E.coli (ETEC) infections in two-week-old gnotobiotic pigs. Twelve pigs from the same litter were divided into four groups and subjected to treatments as follows: Group A: 4 pigs; inoculated with 3 x 10(9) ETEC strain 3030-2 (O157: K88, LT, STb) and treated with loperamide. Group B: 2 pigs; retained germ free, but inoculated with loperamide. Group C: 4 pigs; inoculated with 3 x 10(9) ETEC strain 3030-2, but not treated with loperamide. Group D: 2 pigs; inoculated with non-pathogenic E. coli strain G58-1, but not treated with loperamide. The drug was administered to pigs
per os at 0.5 mg/kg every 8 hours beginning 12 hrs before bacterial challenge. Pigs were euthanized when the effects of dehydration became apparent in positive control pigs (Group C), 12 hours after inoculation. Pigs were weighed before inoculation and at the conclusion of the experiment. Blood was drawn at the time of weighing for the assessment of serum protein concentration and hematocrit. Change in weigh, hematocrit, and serum protein concentration were used as indicators of dehydration. Bacterial colonization of the intestines was assessed by quantitative culture of ileum, and analysis of duodenum, jejunum and ileam histological sections was done following processed with Wright-Giensa strain. Positive control pigs (Group C) were significantly dehydrated when compared with negative control animals (Groups B and D), but were not significantly more dehydrated than pigs treated with loperamide then infected with the same ETEC strain (Percentage of prechallenge weight at euthanasia:
mean positive controls = 0.79; negative controls = 1.22; P=0.001). At least one histologic section from each positive control pigs exhibited segmental or confluent epithelial colonization of the intestinal epithelium by bacteria. No such colonization was observed in negative control pigs. The intestinal epithelium of loperamide treated, ETEC challenged pigs was colonized as extensively as that of the positive control pigs, and the mean number of colony forming units of bacteria (CFU) per gram of ileum was greater 1,790,000,000 V.S. 960,000,000; P=0.03. These observations suggest that loperamide had no protective effect against the dehydration caused by ETEC in piglets and may have allowed for increased proliferation of the bacteria in the intestines.
Impacts
Loperamide, an anti-diarrheal drug, was not shown efficacious in two week old pigs infected with diarrhea-causing E. coli suggesting that other treatment methods should be sought for pigs with colibacillosis.
Publications
- No publications reported this period
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