Albertyn, J.Bragg, R. R.Van Heerden, E.De Smidt, Olga2015-08-112015-08-112001-11http://hdl.handle.net/11660/797English: Haemophilus paragallinarum causes an acute respiratory disease of chickens known as infectious coryza (IC), a disease first recognized as a distinct entity in the late 1920's. Since the disease proved to be infectious and primarily affected nasal passages, the name "infectious coryza" was adopted (Blackall, 1989). Infectious coryza may occur in both growing chickens and layers. The major economic effect of the disease is an increased culling rate in meat chickens and a reduction in egg production (10-40%) in laying and breeding hens. The disease is limited primarily to chickens and has no public health significance (Yamamoto, 1991). The most common clinical signs are a nasal discharge, conjunctivitis, and swelling of the sinuses and face. Various sulfonamides and antibiotics are useful in alleviating the severity and course of infectious coryza; however, none of the therapeutic agents has been found to be bactericidal. Relapse often occurs after treatment is discontinued, and the carrier state is not eliminated (Yamamoto, 1991). All the commercially available bacterins against IC, consist of inactivated broth cultures of a combination of two or three different serotypes. Although vaccines against IC have been used in South Afr ica since 1975, it became apparent in the 1980s that the vaccines were becoming less effective in controlling the disease (Bragg et al., 1996). This could have been due to the emergence of a previously unknown serovar, or even serogroup and the possibility of changes in the population dynamics. Vaccine efficiency is therefore a problem and an alternative to available vaccines is needed. Capsules have long been associated with virulence properties of bacteria. The role that the capsule play in the virulence of bacterial species related to H. paragallinarum has been investigated by several workers (Kroll et al., 1988; Inzana et al., 1993; Boyce and Adler, 2000). Mutation, deletion or allelic exchange of gene/s involved in the transport of capsule polysaccharides in related species like Haemophilus influenza, Actinobacillus pleuropneumoniae and Pasteurella multocida, resulted in organisms with reduced virulence. The noncapsulated mutants of Actinobacillus pleuropneumoniae reported by Inzana et al. (1993) showed extreme stability and induceda protective immune response without any symptoms of disease. This not only proves the capsule�s involvement in virulence of bacteria but also offers the opportunity to investigate the possibility of producing live vaccines. The aim of this study was an attempt to understand the genetic organization of the capsular genes of H. paragallinarum in comparison to related HAP organisms and the possibility of producing a mutant lacking the capsule. The goals were: 1. Isolation and cloning of the capsule transport gene locus. 2. Sequencing and characterization of the locus 3. Transplacement of a gene/s to produce a noncapsulated mutant of H. paragallinarum.Afrikaans: Die bakterium H. paragallinarum veroorsaak 'n akute lugweginfeksie in hoenders genaamd "infectious coryza". Kommersiële entstowwe verskaf nie meer effektiewe beskerming teen die siekte nie en alternatiewe moontlikhede word bestudeer. Die moontlikheid van 'n lewende entstof word oorweeg deur die kapsel van H. paragallinarum as 'n virulensiefaktor te bestudeer. Die kapseltransport geenstreek bestaan uit vier oopleesrame, hctABCD, 3792 basispare in geheel, met begin- en termineringkodons wat oorvleuel. Die twee gene hctA (648bp) en hctB (798bp) is op proteïenvlak met die voorgestelde proteïne van verwante organismes vergelyk en het 'n ooreenkoms van meer as 80% getoon. Hierdie homologie, asook gekonserveerde streke binne elke geen, ondersteun die hipotese dat die hctA-geen vir 'n ATP-bindingproteïen kodeer en hctB vir 'n integrale membraanproteïen. 'n Laer homologie (>60%) bestaan tussen hctC (1164bp) en ander voorgestelde C-gene asook hctD (1188bp) in vergelyking met die D-gene van verwante spesies. Die vermoede dat HctC optree as 'n membraan-geassosieerde proteïen met ankers wat die membraan oorbrug, en dat hctD vir 'n buite-membraanproteïen met porien-eienskappe kodeer, word deur die homologie-studies ondersteun. Al vier proteïne saam vorm 'n ABC-vervoerkompleksstelsel in H. paragallinarum, verantwoordelik vir die vervoer van kapsulêre polisakkariede na die seloppervlak. Die genetiese inligting aangaande die transportgene van H. paragallinarum is gebruik om delesiekonstrukte saam te stel. In die konstrukte is 'n transportgeen of -gene met 'n tetrasiklienweerstand-kasset vervang en na H. paragallinarum selle getransformeer. In die eerste delesiekonstruk (hctD::Tet+) is al die gene vervang en in die tweede konstruk (hctAD::Tet+) is slegs die hctA-geen met die tetrasiklien-kasset vervang. Die liniêre produkte is na H. paragallinarum selle getransformeer en deur die proses van geenvervanging is isogeniese mutante sonder kapsulêre materiaal verwag. Bo alle verwagting was daar geen lewensvatbare bakterieë nie. Hierdie was slegs die eerste stappe in die rigting van 'n lewende entstof en daar is steeds 'n aantal invloede wat ongedefinieerd is.enMolecular cloningHaemophilus infectionsPoultry -- DiseasesCharacterizationTransportCapsuleHaemophilus paragallinarumDissertation (M.Sc. (Microbiology and Biochemistry))--University of the Free State, 2002DissertationUniversity of the Free State