Progress 10/01/02 to 09/30/07
Outputs
Molecular epidemiologic typing of L. intracellularis. New knowledge regarding infection and transmission of L. intracellularis was obtained using the variable number tandem repeat (VNTR) genetic typing technique. Though Lawsonia is antigenically conserved, there is genetic variation that exists between isolates. No variation was observed between isolates obtained from various clinical types of proliferative enteropathy within a flow, including acute, chronic, and subclinical samples. Slight variation between isolates from different geographic locations was detected, though those variations were no greater between isolates from different continents than between isolates from different Midwestern U.S. pig farms. Marked variation exists, however, between isolates from pig and certain non-pig sources. These variations may be used to track outbreaks occurring in pigs, horses, or other animals. In vitro activity of six antimicrobial agents against ten L. intracellularis
isolates from the U.S. and Europe. To achieve the best outcome for treatment and control of proliferative enteropathy, the selection of correct antimicrobials is a critical decision. However, little information on antimicrobial sensitivities against L. intracellularis is available because few strains have been successfully isolated worldwide. In this study, a tissue culture system was used to determine the minimum inhibitory concentrations of both intracellular and extracellular activity against 10 Lawsonia isolates from the United States (n=6) and Europe (n=4). Results of these in vitro studies can be used as a basis for evaluating treatment options in the field. Use of a recombinant protein FliC for development of an indirect ELISA for the diagnosis of proliferative enteropathy. Serological tests are the most economical tools for diagnosis of proliferative enteropathy in pigs. This study, utilizing the flagellar protein, FliC, is the first report to illustrate the potential use of a
recombinant protein of L. intracellularis in an indirect ELISA. Although the FliC-ELISA failed to demonstrate high DSn and low cross-reactivity, the advantage of this protein-based ELISA is that it required no culturing of live L. intracellularis for antigen preparation, therefore, enhancing the efficiency and ability to detect proliferative enteropathy worldwide. In vitro activity of chicken egg antibody (IgY) against L. intracellularis. Currently, methods to control proliferative enteropathy include long-term use of approved feed-grade antibiotics and vaccination. Furthermore, use of antimicrobial alternatives represents a more desirable management technique due to public health concerns over the potential antibiotic residues in meat and the risk of development of antibiotic resistance. In this study, Lawsonia-specific antibodies produced in chicken eggs were able to reduce the incidence of infection of cultured cells after challenge with L. intracellularis.
Impacts
Proliferative enteropathy is an important infectious disease caused by the obligate intracellular bacterium, Lawsonia intracellularis. Little is known of the pathogenic mechanisms for this organism. Furthermore, sensitive and specific methods for diagnosing proliferative enteropathy are not universally available. Characterization of the gene products from this organism will help to identify genes responsible for microbial virulence, or that will be useful in the development of diagnostic reagents and candidate recombinant vaccines against this organism. Development of a molecular typing database will enable further studies on the ecology and epidemiology of L. intracellularis.
Publications
- Wattanaphansak, S., T. Asawakarn, C.J. Gebhart and J. Deen. 2008. Development and validation of an enzyme-linked immunosorbent assay for the diagnosis of porcine proliferative enteropathy. J. Vet. Diagn. Invest. (In press).
- Pusterla, N., J.C. Higgins, P. Smith, S. Mapes and C Gebhart. 2008. Epidemiological survey on farms with documented occurrence of equine proliferative enteropathy due to Lawsonia intracellularis. Vet. Rec.
- Pusterla, N., S. Mapes, D. Rejmanek and C. Gebhart. 2008. Molecular detection of Lawsonia intracellularis in the feces of feral animals from equine farms with documented occurrence of equine proliferative enteropathy. Vet. Rec.
- Wattanaphansak, D. Dau, R. Singer and C. Gebhart. 2007. Activity of six antimicrobial agents against ten L. intracellularis isolates from the U.S. and Europe. 3rd Asian Pig Vet. Soc., Wuhan, China
- Asawakarn, T., S. Asawakarn, S. Wattanaphansak and C. Gebhart. 2007. Activity of chicken egg antibody (IgY) against L. intracellularis. 3rd Asian Pig Vet. Soc., Wuhan, China
- Feary, D.J., C. Gebhart and N. Pusterla. 2007. Lawsonia intracellularis proliferative enteropathy in a foal. Schweiz. Arch. Tierheilkund. 149:129-133.
- Wattanaphansak, S. C. Gebhart and R. Singer. 2007. In vitro testing of antimicrobial agents for Lawsonia intracellularis. 2007. Proc. Am. Assoc. Swine Vet., Orlando, FL
- Wattanaphansak, S., R. Singer, R. Isaacson, J. Deen and C. Gebhart. 2007. In vitro assessment of the effectiveness of the disinfectant Stalosan-F against L. intracellularis. CRWAD, Chicago IL
- Gebhart, C. 2007. Proliferative enteropathy: past progress and potential prospects. (Invited Key-note Lecture). CRWAD, Chicago, IL
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Progress 01/01/06 to 12/31/06
Outputs
Molecular epidemiologic typing of L. intracellularis. New knowledge regarding infection and transmission of L. intracellularis was obtained using the variable number tandem repeat (VNTR) genetic typing technique. Though Lawsonia is antigenically conserved, there is genetic variation that exists between isolates. No variation was observed between isolates obtained from various clinical types of proliferative enteropathy within a flow, including acute, chronic, and subclinical samples. Slight variation between isolates from different geographic locations was detected, though those variations were no greater between isolates from different continents than between isolates from different Midwestern U.S. pig farms. Marked variation exists, however, between isolates from pig and certain non-pig sources. These variations may be used to track outbreaks occurring in pigs, horses, or other animals. In vitro activity of six antimicrobial agents against ten L. intracellularis
isolates from the U.S. and Europe. To achieve the best outcome for treatment and control of proliferative enteropathy, the selection of correct antimicrobials is a critical decision. However, little information on antimicrobial sensitivities against L. intracellularis is available because few strains have been successfully isolated worldwide. In this study, a tissue culture system was used to determine the minimum inhibitory concentrations (MICs) of both intracellular and extracellular activity against 10 Lawsonia isolates from the United States (n=6) and Europe (n=4). Results of these in vitro studies can be used as a basis for evaluating treatment options in the field. Use of a recombinant protein FliC for development of an indirect ELISA for the diagnosis of proliferative enteropathy. Serological tests are the most economical tools for diagnosis of proliferative enteropathy in pigs. This study, utilizing the flagellar protein,FliC, is the first report to illustrate the potential
use of a recombinant protein of L. intracellularis in an indirect ELIS. Although the FliC-ELISA failed to demonstrate high DSn and low cross-reactivity, the advantage of this protein-based ELISA is that it required no culturing of live L. intracellularis for antigen preparation, therefore, enhancing the efficiency and ability to detect proliferative enteropathy worldwide. In vitro activity of chicken egg antibody (IgY) against L. intracellularis. Currently, methods to control proliferative enteropathy include long-term use of approved feed-grade antibiotics and vaccination. Furthermore, use of antimicrobial alternatives represents a more desirable management technique due to public health concerns over the potential antibiotic residues in meat and the risk of development of antibiotic resistance. In this study, Lawsonia-specific antibodies produced in chicken eggs were able to reduce the incidence of infection of cultured cells after challenge with L. intracellularis.
Impacts
Proliferative enteropathy is an important infectious disease caused by the obligate intracellular bacterium, Lawsonia intracellularis. Little is known of the pathogenic mechanisms for this organism. Furthermore, sensitive and specific methods for diagnosing proliferative enteropathy are not universally available. Characterization of the gene products from this organism will help to identify genes responsible for microbial virulence, or that will be useful in the development of diagnostic reagents and candidate recombinant vaccines against this organism. Development of a molecular typing database will enable further studies on the ecology and epidemiology of L. intracellularis.
Publications
- McOrist, S. and C.J. Gebhart. 2005. Genus Lawsonia. In G.E. Garrity, et al, (ed.), Bergey's Manual of Systematic Bacteriology, 2nd ed. Williams and Wilkins, Baltimore, MD.
- McOrist, S. and C.J. Gebhart. 2005. Porcine proliferative enteropathies. In B.E. Straw, et al. (ed), Diseases of Swine, 9th ed. Iowa State University Press, Ames, IA.
- McOrist, S., C. J. Gebhart and B. Bosworth. 2006. Evaluation of porcine ileum models of enterocytes infection by Lawsonia intracellularis. Can. J. Vet. Res. 70:155-159.
- Wattanaphansak, S.W., T. Asawakarn, C.J.Gebhart and J. Deen. 2006. Development and validation of an ELISA for the diagnosis of PPE. Proc. 19th IPVS, Denmark.
- Wattanaphansak, S.W., T. Asawakarn, C.J.Gebhart and J. Deen. 2006. Use of the recombinant protein FliC for development of an elisa for the diagnosis of PPE. Proc. 19th IPVS, Denmark.
- Guedes, R.M.C. and C. Gebhart. 2006. Enterocyte proliferation and apoptotic changes in ileum samples from pigs experimentally infected with Lawsonia intracellularis. Proc. 19th IPVS, Denmark.
- Gebhart, C. 2006. Lawsonia intracellularis infections (Invited Key Note Lecture). Proc. 19th IPVS, Denmark.
- Kinsley, K, N. Winkelman, and C. Gebhart. 2006. Elimination of Lawsonia intracellularis from pigs fed carbadox. Proc. Am. Assoc. Swine Vet., Kansas City, MO.
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Progress 01/01/05 to 12/31/05
Outputs
Lawsonia intracellularis is an obligate intracellular bacterium and the causative agent of proliferative enteropathy in pigs as well as a variety of other species. Characterizations of gene products from this microorganism may assist studies to identify its biology and identify proteins important in its pathology. With a bioinformatics approach for the identification of putative virulence genes, 10 coding sequences of Lawsonia were selected. Nucleotide sequences of each coding region were amplified, cloned and expressed in E. coli. Western immunobloting analysis showed that some of the expressed proteins immuno-reacted with both polyclonal rabbit antibody against whole cell Lawsonia and swine-exposed Lawsonia sera. However, some recombinant proteins reacted slightly with normal swine sera. Sensitive and specific methods for diagnosis, prevention and treatment of the proliferative enteropathy are poorly defined or unavailable. The recent completion of the complete
nucleotide sequence of Lawsonia intracellularis by our group provided an opportunity to investigate methods for enhanced in vitro and/or axenic growth of the organism. We evaluated various methods of biochemical augmentation and modification of culture conditions for enhanced growth of Lawsonia intracellularis in cell culture. Enhancement of growth in various types of cell cultures (cell types; monolayer and suspension) was evaluated. We also attempted to establish appropriate culture conditions for axenic growth of Lawsonia intracellularis. Despite these efforts, Lawsonia intracellularis continues to be an obligate intracellular organism, culturable only in cells in vitro. Recent analysis of variable number tandem repeat regions (VNTR) in the Lawsonia intracellularis genome has provided a means for the differentiation of isolates. This method was demonstrated to be stable, reproducible, and applicable to samples from field outbreaks of proliferative enteropathy. Using this
information we were able to further evaluate the utility of VNTR typing of Lawsonia isolates from a variety of sources and gain insight into the epidemiology of this disease. We successfully obtained a VNTR profile from the commercially available LI vaccine. This profile is unique to that of the other isolates or field outbreaks that have been evaluated thus far. We further evaluated the VNTR profiles of various samples from field outbreaks, both acute and chronic. All the isolates we have typed this far continue to produce unique VNTR profiles.
Impacts
Proliferative enteropathy is an important infectious disease caused by the obligate intracellular bacterium, Lawsonia intracellularis. Little is known of the pathogenic mechanisms for this organism. Furthermore, sensitive and specific methods for diagnosing proliferative enteropathy are not universally available. Characterization of the gene products from this organism will help to identify genes responsible for microbial virulence, or that will be useful in the development of diagnostic reagents and candidate recombinant vaccines against this organism. Development of a whole cell ELISA as an alternative serological test for the diagnosis of proliferative enteropathy will enable more laboratories to perform Lawsonia serology testing with higher throughput capabilities. Establishment of mechanisms to enhance growth of Lawsonia in cell culture will allow others to more easily cultivate this fastidious organism, leading to development of even more sensitive and specific
methods for diagnosis, prevention and treatment of proliferative enteropathy.
Publications
- S. McOrist and Gebhart, C.J. 2005. Proliferative Enteropathies. In B. Straw, et al, (ed.), Diseases of Swine, Ninth Edition. Blackwell Publishing, Ames, IA.
- Wattanaphansak, S., C.J. Gebhart, M. Olin, and J. Deen. 2005. Measurement of the viability of Lawsonia intracellularis. Can. J. Vet. Res. 69:265-271.
- Deprez, P., K. Chiers, C.J. Gebhart. 2005. Lawsonia intracellularis infection in a 12-month-old colt in Belgium. Vet. Rec. 157:774-776.
- Beckler, D.C., N.L. Weber and C.J. Gebhart. 2005. Typing of Lawsonia intracellularis isolates by analysis of variable number tandem repeat profiles. Proc. Am. Assoc. Swine Pract., Toronto, Canada
- Wattanaphansak, S., T. Asawakarn, J. Deen, and C. Gebhart. 2005. Development of an enzyme-linked immunosorbent assay for the diagnosis of porcine proliferative enteropathy. Proc. Allen D. Leman Swine Conf. vol. 32, St. Paul, MN
- Asawakarn, T., A. Nuntaprasert, K. Kaur, V. Kapur, S. Wattanaphansak, and C. Gebhart. 2005. Expression of recombinant Lawsonia intracellularis proteins. Proc. Allen D. Leman Swine Conf. vol. 32. , St. Paul, MN
- Wattanaphansak, S., T. Asawakarn, J. Deen, and C. Gebhart. 2005. Development of an enzyme-linked immunosorbent assay for the diagnosis of porcine proliferative enteropathy. Rushmore Conference, Sept. 29-Oct.2, Rapid City, SD
- Asawakarn, T., A. Nuntaprasert, K. Kaur, V. Kapur, S. Wattanaphansak, and C. Gebhart. 2005. Expression and immunogenicity of proteins encoded by the genome of Lawsonia intracellularis. Rushmore Conference, Sept. 29-Oct.2, Rapid City, SD
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Progress 01/01/04 to 12/31/04
Outputs
Proliferative enteropathy is an important infectious disease caused by the obligate intracellular bacterium, Lawsonia intracellularis. We have begun to characterize the mechanism of pathogenesis of this bacterium by reconstructing its metabolism based on its genomic sequence. We have identified numerous genes involved in its pathogenicity and are beginning to clone, express, and purify these proteins for further study. We evaluated the effectiveness of hyper-immunized chicken eggs for controlling an experimental L. intracellularis infection in growing pigs. A molecular epidemiological typing system based on variable number tandem repeats was developed for differentiating isolates of L. intracellularis. Results of typing studies demonstrated that the test is stable, reproducible, and sensitive. We also optimized two methods, flow cytometry and direct counts with specific fluorescence stains, for determination of the viability of Lawsonia under various conditions. We
have evaluated the comparative agreement of two Lawsonia serology tests for serological diagnosis of field cases of proliferative enteropathy. Finally, we have provided various training and continuing education seminars on proliferative enteropathy to various producer groups, veterinary groups, and academic institutions.
Impacts
Information derived from the metabolic reconstruction of L. intracellularis may be used to define necessary nutritional components for enhanced cell culture or cell-free growth of the organism. Characterization of the gene products from this organism may assist studies to identify the mechanism that influences the progression of infection and identify proteins for use in sero-diagnosis of PE Specific egg yolk antibodies, at a dose of 2 kg/ton of feed, are capable of controlling the reduction in ADG during a Lawsonia infection. Egg yolk antibodies are cheaper and less controversial feed additives given the current concerns surrounding feed-grade antibiotics. Analysis of VNTR profiles appears to be a useful tool for distinguishing between strains of Lawsonia. The assay proved to be robust and gave identical results on repeat analysis. This method of rapidly detecting Lawsonia, and tracing specific isolates, may allow rapid identification of the source and transmission
pattern of this organism through epidemiological investigations.
Publications
- Gebhart, C.J. and R.M.C Guedes. 2004. Lawsonia intracellularis. In C.L. Gyles, et al, (ed.), Pathogenesis of Bacterial Infections in Animals, 3rd ed. Blackwell Publishing, Ames, IA.
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Progress 01/01/03 to 12/31/03
Outputs
Proliferative enteropathy (PE) is an important infectious disease caused by the obligate intracellular bacterium, Lawsonia intracellularis. We have begun to characterize the mechanism of pathogenesis of this disease by evaluating the progression of L. intracellularis infection and resultant mucosal immune response in pigs. We have completed sequencing and analysis of the genome of this organism and have identified numerous genes involved in pathogenicity. We developed a real-time high throughput PCR technique to detect and quantify L. intracellularis in fecal samples and then compared results obtained to those of conventional PCR and a slide immunoperoxidase test. These tests will provide diagnosticians an efficient means of monitoring shedding of L. intracellularis from infected pigs. A hamster model was developed and used to evaluate the efficacy of hyper-immunized chicken eggs for passive protection against a L. intracellularis infection. Production of local
passive immunity may be utilized along with antimicrobials and vaccination to control or eliminate PE in swine populations. We also evaluated the efficacies of each FDA-approved feed antimicrobial labeled for control of PE and found each to be efficacious in controlling clinical signs and performance loss, but none eliminated LI from infected pigs. Finally, we evaluated the development of immunity to L. intracellularis in pigs fed carbadox as a preventive medication or as a treatment medication. Producing L. intracellularis-free pigs with a carbadox medication program may enable eradication of L. intracellularis from swineherds.
Impacts
A better understanding of the progression and timing of L. intracellularis infection in pigs is essential to improve methods of control and prevention of PE. The availability of the genomic sequence will greatly enhance our understanding of L. intracellularis metabolism, its interaction with host intestinal epithelial cells, and the ability of this bacterium to induce cell proliferation and lead to the development of superior diagnostic tests for PE in pigs and other species. Production of passive local immunity using hyper-immunized chicken eggs may be utilized along with strategic antimicrobial usage and vaccination to control or eliminate PE in swine populations. Also, egg antibodies represent a less controversial feed additive given the current concerns surrounding feed-grade antibiotics. The real-time PCR assay offers a high throughput test for detecting LI in feces of infected pigs. It is as specific as conventional PCR, but more sensitive. Evaluation of feed
additives labeled by the FDA for use in treatment and/or control of PE in pigs will be useful to swine practitioners when applying in-feed PE control measures in the field. Strategic carbadox medication offers producers a management tool to abbreviate natural infections of L. intracellularis while allowing natural immunity to develop
Publications
- Guedes, R.M.C. and Gebhart, C.J. 2003. Preparation and characterization of polyclonal and monoclonal antibodies against Lawsonia intracellularis. J. Vet. Diag. Investi. 15:438-446.
- Guedes, R.M.C. and Gebhart, C.J. 2003. Comparison of pure culture of Lawsonia intracellularis and intestinal mucosa homogenate as challenge models for porcine proliferative enteropathy. Vet. Microbiol. 93:159-166.
- Guedes, R.M.C. and Gebhart, C.J. 2003. Onset and duration of fecal shedding, cell-mediated and humoral immune responses after challenge with a pathogenic isolate or a commercial vaccine of Lawsonia intracellularis. Vet. Microbiol. 91:135-145.
- Guedes, R.M.C., Winkelman, N.L., and Gebhart, C.J. 2003. Relationship between the severity of porcine proliferative enteropathy and the infectious dose of Lawsonia intracellualaris. Vet. Rec. 153:432-433.
- Marsteller, T.A., Armbruster, G., Bane, D.P., Gebhart, C.J., et al. 2003. Monitoring the prevalence of Lawsonia intracellularis IgG antibodies using serial sampling in growing and breeding swine herds. J. Swine Health and Prod. 11:127-130.
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