Source: MISSISSIPPI STATE UNIV submitted to
VIRULENCE PROPERTIES ASSOCIATED WITH CHANNENL CATFISH PATHOGENS
 
PROJECT DIRECTOR: Lawrence, M. :.
 
PERFORMING ORGANIZATION
COLLEGE OF VETERINARY MEDICINE
MISSISSIPPI STATE UNIV
MISSISSIPPI STATE,MS 39762
 
NON TECHNICAL SUMMARY: The external coat of Edwardsiella ictaluri is made up of long chains that contain repeating sugar units. This project will determine the role of the polysaccharide chains in disease and identify the genes that control their systhesis. This project will also develop genetic methods and experimental infection methods that can be used to idetify disease-causing genes in Flavobacterium columnare.
 
OBJECTIVES: (1) To genetically define and characterize genes involved in biosynthesis of the polysaccharide coat of Edwardsiella ictaluri; (23) To investigate the role of the E. ictaluri polysaccharide coat in disease pathogenesis by the development of isogenic mutant(s) and evaluation of their virulence; (3) To develop challenge models and genetic methods for the investigation of virulence genes associated with Flavobacterium columnare; (4) To conduct other appropriate studies to characterize virulence properties associated with veterinary bacterial pathogens.
 
APPROACH: A previously established conjugation/transposition system will be used to generate random kanamycin resistant Edwardsiella ictaluri mutants, which will be screeneed for O-side chain biosynthetic genes. Challenge models for Flavobacterium columnare in channel catfish will be developed, and gentic techniques allowing random and targeted mutagenesis in F. columare will be developed.
 
CRIS NUMBER: 0181933 SUBFILE: CRIS
PROJECT NUMBER: MISV-081310 SPONSOR AGENCY: CSREES
PROJECT TYPE: ANIMAL HEALTH PROJECT STATUS: TERMINATED MULTI-STATE PROJECT NUMBER: (N/A)
START DATE: Jul 1, 1999 TERMINATION DATE: Jun 30, 2005

GRANT PROGRAM: (N/A)
GRANT PROGRAM AREA: (N/A)

CLASSIFICATION
Knowledge Area (KA)Subject (S)Science (F)Objective (G)Percent
311371011004.2100%

CLASSIFICATION HEADINGS
KA311 - Animal Diseases
S3710 - Catfish
F1100 - Bacteriology
G4.2 - Reduce Number and Severity of Pest and Disease Outbreaks


RESEARCH EFFORT CATEGORIES
BASIC 100%
APPLIED (N/A)%
DEVELOPMENTAL (N/A)%

KEYWORDS: channel catfish; fish diseases; animal pathogens; virulence; bacterial diseases (animals); animal health; edwardsiella ictaluri; flavobacterium; lipopolysaccharides; bacterial genetics; coatings; gene analysis; biosynthesis; pathogenesis; mutants; aquaculture; kanamycin; mutagenesis; anti bacterial agents; resistant microorganisms

PROGRESS: Jul 1, 1999 TO Jun 30, 2005
We have reported the construction and phenotypic characterization of an isogenic Edwardsiella ictaluri O polysaccharide(OPS)negative mutant strain, 93-146 R6. This strain was shown to be OPS negative using SDS-PAGE with silver staining and immunoblotting, and it was found to be highly attenuated in channel catfish compared to wild type strain 93-146. We have cloned the mutation responsible for the OPS negative phenotype of 93-146 R6 along with flanking sequence from the E. ictaluri chromosome. The mutation was located in a gene encoding a UDP-glucose-4-epimerase, and the clone contained eight additional complete open reading frames and one partial open reading frame from the E. ictaluri OPS biosynthesis region. A total of approximately 12 kb from the E. ictaluri OPS biosynthesis region has been sequenced (GenBank #AY057452). E.ictaluri OPS was determined to be composed of N-acetylgalactosamine and galactose in a 2:1 ratio. Functional assays have shown that E. ictaluri OPS partially mediates resistance to catfish serum, but not to catfish neutrophils. Following immersion exposure of juvenile channel catfish, the OPS mutant 93-146 R6 is able to persist in catfish tissues for at least 14 days. Significant protection against challenge with virulent E. ictaluri is provided when channel catfish are vaccinated with a single dose of live 93-146 R6 by intraperitoneal injection, but not when channel catfish are vaccinated with live 93-146 R6 by immersion. Phenotypic comparisons of virulent and avirulent strains of Edwardsiella ictaluri indicated that hemolysin, an outer membrane protein, and core oligosaccharide may play a role in virulence. We have constructed an isogenic E.ictaluri mutant that fails to express the cell-associated hemolysin, and we have cloned the affected gene. Sequencing revealed that the gene is part of a two-component hemolysin similar to Edwardsiella tarda and Serratia marcescens hemolysins (GenBank #AY338755). The hemolysin mutant was not attenuated in catfish. We have also identified an E. ictaluri outer membrane protein that is preferentially expressed by virulent E. ictaluri isolates and cloned the gene encoding it. For development of a Flavobacterium columnare challenge model, we have constructed two plasmids for expression of green fluorescent protein in this species. The first plasmid, pMWFCgfp, is derived from pCP11, which is a F. johnsoniae-E. coli shuttle vector, and the second plasmid, pBBRFCgfp, is derived from broad host range plasmid pBBR1MCS4. Experiments for plasmid transfer into F. columnare are ongoing. We have constructed and reported a Pasteurella multocida strain with altered DNA adenine methylase (Dam) expression and showed that it is highly attenuated in mice. We have cloned and sequenced the P. multocida A:1 dam gene (GenBank #AF411317), and we have published a PCR method for detection of P. multocida.

IMPACT: 1999-07-01 TO 2005-06-30 Enteric septicemia of catfish (ESC), caused by Edwardsiella ictaluri, is the most serious of disease affecting the channel catfish industry. Little is currently known about how the pathogen causes disease. Identification of the bacterial factors (OPS, core oligosaccharide, hemolysin, and outer membrane proteins) responsible for causing disease will provide new potential targets for a vaccine or treatment to prevent ESC. We have shown that E.ictaluri LPS is essential for causing disease, but hemolysin activity is not. We have also determined that antigenic structures in E.ictaluri LPS are necessary for stimulating protective immunity against ESC. Flavobacterium columnare causes columnaris disease in fish. Currently, there is not a well-established protocol for conducting experimental challenges in catfish, which hinders research into the pathogenesis of columnaris disease. Our research has the potential to allow development of an effective challenge model. Pasteurella multocida is one of causative agents of bovine respiratory disease complex, and the Dam altered strain shows potential as a live attenuated vaccine. The PCR method we developed has the potential to be used as a diagnostic tool for detection of P. multocida.

PUBLICATION INFORMATION: 1999-07-01 TO 2005-06-30
Lawrence, M. L., M. Banes, and M. L. Williams. 2001. Phenotype and virulence of a transposon-derived lipopolysaccharide O side chain mutant strain of Edwardsiella ictaluri. J. Aquat. Anim. Health 13:291-299.

PROJECT CONTACT INFORMATION
NAME: Lawrence, M. L.
PHONE: 662-325-1195
FAX: 662-325-1031