Progress 10/01/03 to 09/30/06
Outputs
It was our intent to identify and investigate virulence factors which are associated with the onset and prevalence of this disease. Objective 1: To assess the response of ETEC to BETA-lactam antibiotics and establish specific categories of antibiotic resistance. Infectious strains of ETEC were profiled by antibiotic susceptibility testing utilizing ten BETA-lactam antibiotics. Five of the F18 ETEC isolates were resistant to BETA-lactam antibiotics. Three of the K88 ETEC strains were resistant to BETA-lactam antibiotics. The Nitrocefin test results suggested BETA-lactamase production in the same eight ETEC isolates that were ampicillin resistant, as well as the positive control, E. coli pTem1. Comparison of plasmids extracted from clinical isolates revealed that each contained at least one plasmid with molecular sizes of >9kb. Objective 2: To identify factors associated with the resistance of ETEC to antibiotics and to correlate the factors with other resistance factors
such as toxin production. Electrophoretic analysis of whole cell lysates and outer membrane Lipopolysaccharides of ETEC strains from active infections in neonatal swine have led to molecular profiles of these Diarrheagenic strains. Whole cell lysates of ETEC and LPS from our K88 and F18 ETEC isolates were profiled by PAG electrophoresis. These profiles resolved the oligosaccharide moieties and lipid anchors of the LPS and allowed for the molecular distinction between K88 and F18 ETEC strains. Also, the specific verotoxin-SLT was identified in three of the strains tested. The composition of mammary secretions, with regard to the selective transport of immunoglobulins were found to change rapidly over the first 24 hours after partuition. Whole-cell lysates of these ETEC isolates were resolved by electrophoresis in SDS Polyacrylamide Gels. Colostrum samples from healthy sows at 6hr, 12hr, 24 hr post-partum, were used to probe immunoblots of the ETEC gels. Objective 3: To isolate those
resistance factors which are extra-chromosomal (plasmid-encoded) and to determine the DNA sequences of any identified resistance factors and to compare those sequences to sequences of known factors. The presence of plasmid DNA, confirmed by agarose gel electrophoresis, suggests that BETA -lactamase (the antibiotic resistance protein) is extra-chromosomally encoded. The AmpR (ampicillin resistance) gene of Escherichia coli and other gram-negative bacteria codes for a BETA-lactamase of approximately 27,000 daltons that catalyzes the hydrolysis of penicillins to penicilloic acids (Sutcliffe, 1978). Resistance in more than 50% of AmpR E. coli clinical isolates is due to a BETA-lactamase (Bush et al., 1997). Polymerase Chain Reaction amplified the bla1 and bla 8 primers for eight of the ten ETEC isolates and the positive control, E. coli pTem1. ETEC isolates can be differentiated by the presence or absence of the amplified products using bla 1 and bla 8 primers.
Impacts
During the past 10 years much change has occurred in the composition of NC's agriculture, including the vast growth in the state's swine industry. Between 1991 and 1996, the NC swine inventory increased from 2.7 million to approximately 8 million. In September 2002, NC ranked second nationally in swine production (NC Hog Inventory, 2002). The economic benefits of the growth of the swine industries have been significant. Colibacillosis or scours is caused by Enterotoxigenic (toxin producing) Escherichia coli (ETEC) strains. Scours is the leading cause of death in unweaned pigs, contributing to an estimated $35 million loss annually, nationwide. According to the National Animal Health Monitoring System (NAHMS), 15% of preweaning deaths were caused by scours. We are now, as a result of the characterization of these microorganisms, looking at ways to prevent this disease. The economic impact of this disease is profound and our research, under the auspices of this project,
has focused on understanding the mechanisms of this disease. Our studies so far strongly indicate that a complete characterization of ETEC and the onset of the diarrhea-causing disease, scours, must include an understanding of two very important events, both of which were the focus of our studies. The first was a focus on passive immunity of specific antibodies to ETEC stains passed from mother to offspring in the first 24 hours post-partum. The second focus was a study of bacterial adhesion to the mucous of the neonatal gut, and competitive binding between ETEC strains and normal microbial flora in the gut.
Publications
- Ibrahim S. A., J. W. Allen, and A. R. Byers . Effect of calcium on autoaggregation behavior of Lactobacillus reuteri. (abstract accepted) The 232nd ACS National Meeting, San Francisco, CA, September 10-14, 2006
- Ibrahim, S. A., J. W. Allen, and A. R. Byers. AGFD 107 Effect of calcium on autoaggregation behavior of Lactobacillus reuteri. (abstract accepted) The 232nd ACS National Meeting, San Francisco, CA, September 10-14, 2006
- Allen*, J W and A Byers. Enterotoxigenic Escherichia coli Infections from Neonatal Swine. Annual meeting of Southern Association of Agricultural Scientists, Feb., 2006, Orlando, FL
- Allen*, J W and A Byers. Antibody Response In Porcine Colostrum With Specificity To Enterotoxigenic E. coli. Annual meeting of Southern Association of Agricultural Scientists, Feb., 2006, Orlando, FL
- John W. Allen*, Andrea R. Byers. Antibody Response in Porcine Colostrum with Specificity to Enterotoxigenic E. coli, 14th Biennial ARD Research Symposium, April, 2006, Atlanta, GA
- John W. Allen*, Andrea R. Byers. Enterotoxigenic Escherichia Coli Infections from Neonatal Swine, 14th Biennial ARD Research Symposium, April, 2006, Atlanta, GA
- Allen, John W. and Andrea Robertson Byers. Characterization of Enterotoxigenic Escherichia Coli From Neonatal Swine in NC.. Journal of Animal Science, 2006. submitted
- Ibrahim, S.A., J. W Allen and A. R. Byers. Effect of calcium on autoaggregation behavior of Lactobacillus reuteri. Milchwissenschaft - Milk Science International, 2006. submitted
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Progress 01/01/05 to 12/31/05
Outputs
This project has involved characterization of isolates of Enterotoxigenic Escherichia coli (ETEC) from active infections in neonatal swine in North Carolina. It is our intent to identify and investigate virulence factors which are associated with the onset and prevalence of this disease. We are studying strains types, antigenic make-up, toxin production, adhesion factors, antibiotic resistance factors and any other aspect which may lead to an understanding of this disease and to an incite into its prevention. Electrophoretic analysis of whole cell lysates and outer membrane Lipopolysaccharides of ETEC strains from active infections in neonatal swine have led to molecular profiles of these Diarrheagenic strains. These profiles have led to the identification of a number of molecular components (virulence factors) which are possible candidates for vaccines. These profiles have also identified non-ETEC strains which were isolated from active infections. Characterization
of these non-ETEC strains have identified an atypical, infectious strain of E. coli and an EHEC strain, both of which are being studied. Sows secrete large amounts of immunoglobulins, particularly in the colostrum, and the newborn nursing pig absorbs them intact into its blood through a nonselective absorption mechanism which takes place during the first few days of the pigs life. The composition of mammary secretions, with regard to the selective transport of immunoglobulins, changes rapidly over the first 24 hours after partuition. Isolates of ETEC from active infections in neonatal swine in North Carolina were collected, strain-typed and supplied by the Rollins State Diagnostic Laboratory, Raleigh, North Carolina. Whole-cell lysates of these ETEC isolates were resolved by electrophoresis in SDS Polyacrylamide Gels. Colostrum samples from healthy sows at 6hr, 12hr, 24 hr post-partum, were used to probe immunoblots of the ETEC gels. Anti-swine IgG and IgA conjugated to peroxidase,
served as second antibody. The study revealed a complex pattern of specificity and duration of response to various antigenic epitopes over the initial post-partum period.
Impacts
During the past 10 years, much change has occurred in the composition of North Carolina's agriculture, including the vast growth in the state's swine industry. Between 1991 and 1996, the NC swine inventory increased from 2.7 million to approximately 8 million. In September 2002, North Carolina ranked second nationally in swine production (N.C. Hog Inventory, 2002). The economic benefits of the growth of the swine industries have been significant. Colibacillosis or Scours is caused by enterotoxigenic (toxin producing) Escherichia coli (ETEC) strains. In addition to producing toxins, these ETEC strains have the ability to adhere to the wall of the small intestine by means of hair-like structures known as pili on the surface of the bacteria. Several types of pili have been identified in ETEC strains producing colibacillosis in swine; the four major ones have been designated K88, K99, F18, and 987P. In North Carolina, the F18 and K88 strains are most prevalent. The
combined presence of pili or fimbriae and enterotoxins enable these pathogenic E. coli to adhere and multiply in very large numbers on the surface of the small intestine, and to secrete the enterotoxins that cause severe digestive alterations leading to clinical diarrhea, dehydration, and high mortality rates in neonatal swine. An understanding of the molecular mechanisms of this disease and of the mechanisms of resistance to treatment is the focus of this study and has direct impact on swine production and the profit margin of this vital North Carolina industry.
Publications
- J. W. Allen, A. Robertson-Byers, Characterization of Enterotoxigenic Escherichia coli, Annual Meeting of the American Society for Microbiology, May, 2005. Washington, DC #05-GM-A-3630-ASM.
- John W. Allen, Andrea R. Byers, Antibody Response in Porcine Colostrum with Specificity to Enterotoxigenic E. coli, ASAS Southern Sectin, Sarasota, FL Feb. 2006.
- John W. Allen, Andrea R. Byers, Diarrheagenic Escherichia Coli Infections from Neonatal Swine, ASAS Southern Section, Sarasota, FL, Feb. 2006.
- John W. Allen, Andrea R. Byers, Antibody Response in Porcine Colostrum with Specificity to Enterotoxigenic E. coli, 1890 Land-Grant Universities, Association of Research Directors, Inc., 14th Biennial Research Symposium, Atlanta, GA, April, 2006
- John W. Allen, Andrea R. Byers; Enterotoxigenic Escherichia Coli Infections from Neonatal Swine, 1890 Land-Grant Universities, Association of Research Directors, Inc. 14th Biennial Research Symposium, Atlanta, GA, April, 2006.
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Progress 01/01/04 to 12/30/04
Outputs
The intent of this project has been to characterize isolates of Enterotoxigenic Escherichia coli (ETEC) from active infections in neonatal swine in North Carolina. It is our intent to identify and investigate virulence factors which are associated with the onset and prevalence of this disease. We are studying strains types, antigenic make-up, toxin production, adhesion factors, antibiotic resistance factors and any other aspect which may lead to an understanding of this disease and to insight into its prevention. The objectives are: (1) To assess the response of ETEC to beta-lactam antibiotics and establish specific categories of antibiotic resistance. Infectious strains of ETEC were profiled by antibiotic susceptibility testing utilizing ten beta-lactam antibiotics. All five of the F18 ETEC isolates were resistant to beta-lactam antibiotics. Three of the K88 ETEC strains were resistant to beta-lactam antibiotics. (2) To identify factors associated with the resistance
of ETEC to antibiotics and to correlate the factors with other resistance factors such as toxin production. Lipopolysaccharide (LPS) or endotoxin is a virulence factor associated with the outer membrane of gram negative bacteria. Whole cell lysates of ETEC and LPS from our K88 and F18 ETEC isolates were profiled by PAG electrophoresis. These profiles resolved the oligosaccharide moieties and lipid anchors of the LPS and allowed for the molecular distinction between K88 and F18 ETEC strains. Also, the specific verotoxin-SLT was identified in three of the strains tested. (3) To isolate those resistance factors which are extra-chromosomal (plasmid-encoded) and to determine the DNA sequences of any identified resistance factors, and to compare those sequences to sequences of known factors. The presence of plasmid DNA was confirmed by agarose gel electrophoresis, which suggests that beta-lactamase (the antibiotic resistance protein) is extra-chromosomally encoded. The AmpR (ampicillin
resistance) gene of E.coli and other gram negative bacteria codes for a beta-lactamase of approximately 27,000 daltons that catalyzes the hydrolysis of penicillins to penicilloic acids. Polymerase Chain Reaction amplified the bla 1 and bla 8 primers for eight of the ten ETEC isolates and the positive control, E.coli pTEM 1. While this establishes that beta -lactam antibiotic resistance is prevalent among K88 and F18 strain types of ETEC in NC, it is unclear if there is a direct correlation between antibiotic resistance and toxin production or other virulence factors. Our studies so far strongly indicate that a complete characterization of ETEC and the onset of scours must include an understanding of two very important events which will be part of our studies (1) a focus on passive immunity of specific antibodies to ETEC stains passed from mother to offspring in the first 24 hours post-partum. (2) a study of bacterial adhesion to the mucous of the neonatal gut and competitive binding
between ETEC strains and normal microbial flora in the gut. Passive immunity to ETEC strains in the colostrum of sows has been identified in the form of specific antibodies to ETEC as shown in Western Immunoblotting.
Impacts
In order to reduce the incidence of foodborne illnesses related to enterotoxigenic Escherichia coli (ETEC), the aim of this project is to study the mechanisms of resistance of scours against selected antibiotics commonly used for treatment of the pathogen. These results potentially can be used to improve antibiotic efficiency by identifying the chemical and biological points where the antibiotics ability to kill or retard the growth of the pathogen may be strengthened.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
Work has just begun on this project. Strain types of enterotoxigenic E. coli (ETEC) are associated with gastrointestinal diseases in humans and gastrointestinal diseases in swine. However, in gastrointestinal infections from newborn swine in North Carolina, there were only two strain types, K88 and F18, both of which produce heat-stable and heat-labile toxins. The F18 strains are also associated with edema disease, which produces the verotoxin or shiga-like toxin. Objective 1: To assess the response of enterotoxigenic Escherichia coli (ETEC) to beta-lactam antibiotics. The disk diffusion method was used for antibiotic susceptibility testing in this study. Antibiotic disk diffusion tests were used to determine zones of inhibition of antibiotics in bacteria. When a filter paper disk impregnated with a chemical is placed on agar, the chemical will diffuse from the disk into the agar. Objective 2: To identify factors associated with the resistance of ETEC to antibiotics.
beta-lactamases are the main cause of bacterial resistance to penicillins and cephalosporins. Definitive identification of these enzymes is only possible by gene or protein sequencing. However, simple beta-lactamase detection and typing tests can be valuable in the clinical laboratory. These include direct tests for beta-lactamase activity in fastidious gram-negative bacteria such as E. coli, tests for extended-spectrum beta-lactamases and tests for chromosomal beta-lactamases. ESBL production was tested with double disc tests using ceftazidime, amoxicillin clavulanic acid, and cefotaxime. Chromosomal beta-lactamases were tested also with double disc method using cefotaxime and imipenem. Objective 3: To detect the genetic code of any identified resistance factors. Plasmid DNA isolation was used to determine if the enterotoxigenic Escherichia coli had plasmids and if the factors responsible for the antibiotic resistance were possibly encoded on those plasmids. Closed circular forms,
nicked circular, and linear duplex forms of plasmid DNA of the same molecular weight, migrate through agarose gels at different rates. Polymerase Chain Reaction assays can be used to determine if the ETEC isolates carried genes for E. coli enterotoxins of the heat labile or heat stabile types, Shiga type 2 (associated with edema disease), and pili of the K88 and F18 types (Moon et al., 1995). However, the enterotoxin genes and fimbrial types for these particular ETEC isolates have already been determined by the Rollins Diagnostic Laboratory. PCR in this experiment was used to determine if the plasmid DNA isolated from the ETEC isolates migrated at the same rate and yielded the same beta-lactamase amplification products as the positive control plasmid, pTem1.
Impacts
In order to reduce the incidence of foodborne illnesses related to enterotoxigenic Escherichia coli (ETEC), the aim of this project is to study the mechanisms of resistance of scours against selected antibiotics commonly used for treatment of the pathogen. These results potentially can be used to improve antibiotic efficiency by identifying the chemical and biological points where the antibiotics ability to kill or retard the growth of the pathogen may be strengthened.
Publications
- No publications reported this period
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