Agroterrorism - University of Georgia College of Veterinary Medicine

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Poultryareas within the poultry house or plant. This studyexamined the use of PCR to identify those secondaryenrichments containing Salmonella. The uniqueSalmonella virulence gene invA was chosen as thetarget for development of a nested-PCR because ofits uniform distribution among Salmonella serotypes.Using PCR as a screen of primary enrichments forpresumptive Salmonella contamination, we improvedour efficiency at isolating Salmonella upon secondaryenrichment by 20 % and no false negatives wereobserved. This method will not only validate the useof secondary enrichment procedures, but also reducecosts and manpower for surveillance of Salmonella.Detection of Salmonella and Campylobacter inpoultry by PCR-ELISA. Contamination of retailpoultry by Campylobacter spp. and Salmonella entericais a significant source of human diarrheal disease.Isolation and identification of these microorganismsrequires a series of biochemical and serological tests.In this study, Campylobacter ceuE and Salmonella invAgenes were used to design probes in PCR-ELISA,as an alternative to conventional bacteriologicalmethodology, for the rapid detection ofCampylobacter jejuni, Campylobacter coli, andSalmonella enterica from poultry samples. ELISAincreased the sensitivity of the conventional PCRmethod by 100- to 1,000-fold.A restriction fragment length polymorphism basedpolymerase chain reaction as an alternative to serotypingfor identifying Salmonella serotypes. Twentyfourphase 1 flagellin and eight phase 2 flagellingenes could be differentiated among each other usingrestriction endonucleases in RFLP-PCR analysis.These genes comprise the major antigenic formulasfor fifty-two serotypes of Salmonella sp., whichinclude the common serotypes found in poultryand other important food animal species. With thisknowledge, ninety percent of the Salmonella serotypescould be identified using this double restrictionenzyme RFLP analysis.These multiplex PCR assays for detecting specificO and H antigen gene alleles can become a rapidand cost-effective alternative approach to serotypingfor the identification of common poultry Salmonellaserotypes. This multiplex PCR has now becomepart of diagnostic services offered to poultry industryfor identifying Salmonella serotypes.PI: Dr. John J. Maurer (jmaurer@vet.uga.edu)Co-PI: Dr. M. D. LeeAvian MycoplasmosisMycoplasma gallisepticum strain K5054 has beenfurther developed as a live vaccine strain. Apatent has been applied for, and we are in negotiationwith 3 companies for potential licensing. (Work incollaboration with Dr. Naola Ferguson).Techniques for molecular epidemiology of avianmycoplasmas continue to improve. Primers forthe mgc2 gene of M. gallisepticum and vlhA ofM. synoviae are about ready for field evaluation.PCR based on both of these genes are promisingas diagnostic tests; the PCR products have potentialvalue for preliminary identification of strains. Amplifiedfragment length polymorphism analysis showspromise as a definitive test for studying relationshipsamong strains. (In collaboration with MaricarmenGarcía, Yang Hong, Sharon Levisohn, David Yogev,and Dusan Bencina).Diagnostic services included 5765 cultures, 13,451M. gallisepticum HI tests, 13,606 M. synoviae HItests, 163 M. meleagridis HI tests, for a total of 27,219 HI tests conducted. There were 106 cases involvingMycoplasma fingerprinting.PI: Dr. S. H. Kleven (skleven@uga.edu)Co-PIs: Dr. W. D. Hall and Dr. V. LeitingInvestigation into factors affecting hatch-abilityand chick qualityThis project has been an ongoing study of disinfectantefficacy, and the effect of disinfectantuse in the hatchery on hatchability and chick quality.During this period, studies were conducted usingformaldehyde. This product has been studied in previousgrants and found to be detrimental to respiratoryepithelium at high levels. This study compareda constant rate infusion of a lower dose of formaldehydeto a higher dose given every 12 hours as waspreviously studied. This project also supported thecontinued work evaluating some problematic Pseudo-8Veterinary Medical Experiment Station
monas aeruginosa isolates that had caused severe chickquality problems as a result of hatchery contamination.PI: Dr. Jean E. Sander (jsander@uga.edu)Co-PIs: Dr. J.L. Wilson and Dr. J.J. Maurerbeing utilized to identify whether amino acid changesin locations outside of the hypervariable region ofsegment A might play a role in pathogenesis. Thesequence data obtained will be compared to previouslypublished full length sequences.PI: Dr. Holly Sellers (hsellers@uga.edu)Detection, Isolation, and Characterization ofAvian VirusesThe objectives of this proposal are to providediagnostic virology services for the U.S. poultryindustry, conduct applied research on current aviandisease isolates from the field, and improve detectionand isolation methods for monitoring avian viruses.During FY03 we processed 327 accessions; 677samples submitted for virus isolation; 296 virus isolationsmade from samples submitted; 381 negativesamples (no virus isolated).PI: Dr. Holly Sellers (hsellers@uga.edu)Epidemiological Studies on Infectious BursalDisease Virus Field Isolates in the SoutheasternUnited StatesDespite widespread vaccination, infectious bursaldisease virus (IBDV) continues to cause economiclosses to the poultry industry. Within serotype1 there are classic, variant, and very virulent viruses.The U.S. poultry industry is most affected by thepresence of antigenic variants that can be responsiblefor vaccination failures. The VP2 gene of IBDV hasbeen the target for molecular classification of the virussince it is the major host protective antigen responsiblefor inducing serotype-neutralizing antibodies.Advancements in nucleic acid technology have ledto the identification and classification of antigenicvariants by RT-PCR/RFLP analysis. The objectivesof this proposal are to conduct an epidemiologicalstudy of IBDV field isolates from the southeasternU.S. Field isolates will be chosen for the study basedon unique RFLP patterns obtained using the currentIBDV typing system at PDRC.During FY03, full-length genome sequencing ofIBDV field isolate 9109 and cell culture adaptedEdgar was completed and phylogenetic analysis iscurrently being performed. Sequence analysis isDevelopment and Characterization of InfectiousLaryngotracheitis (ILT)RecombinantVirusDifferentiation of Infectious LaryngotracheitisVirus strains is one of the goals in our laboratory.We have developed a PCR-RFLP assay wherethe glycoprotein E was amplified by PCR and theamplification product was digested with restrictionenzymes. This assay allowed discrimination of vaccinesand vaccine subpopulations. A second generationof PCR-RFLP assays has been developed basedon initial sequencing of glycoprotein I, and earlygenes ICP4 and ICP27. Amino acid substitutionsspecific to backyard flock isolates were detected in theglycoprotein I. These mutations were not present onany of the vaccines analyzed, or in outbreak relatedfield isolates. Sequencing of the ICP4 and ICP27genes allowed differentiation of two field isolatesfrom vaccine strains. Therefore we have developeda system for ILTV strain differentiation by first usingglycoprotein I to identify wild type strains: ICP27 toidentify vaccine related isolates, and glycoprotein Eto identify vaccine subpopulations.Because vaccine strains are one source of outbreaks inthe field, a second goal of this project was to comparethe virulence and transmission of the chicken embryoorigin (CEO) ILTV vaccine strain and CEO viral subpopulations.The objective behind this work was todetermine if viral subpopulations within the vaccineare more attenuated. Clinical signs and transmissionfrom inoculated to contact birds were recorded. Differencein the transmission and replication betweenCEO vaccine subpopulations was observed. Measuredby the appearance of clinical signs during earlystages of infection, one of the subpopulations transmitsat a faster rate than the second subpopulation.From this data we have concluded that one of thevaccine subpopulations delays latency while the otherenters latent infection faster. Ongoing studies fromPoultryVeterinary Medical Experiment Station9
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Agroterrorism - University of Georgia College of Veterinary Medicine

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