Methods for serotype classification of Haemophilus paragallinarum field isolates.

Abstract

Historically, the causative agent of infectious coryza has been identified as the NAD requiring bacterium Haemophilus paragallinarum and the implementation of an intensive vaccination program led to the effective control of this contagious upper respiratory infection. More recently, however, a decline in the protective capacity of a vaccine conditioned immune response was noted, with a number of contributing factors, including the emergence of a fast-growing NAD-independent bacterium, which has largely replaced the traditional NAD-dependent variety. As such, accurate, reproducible methods for determining and continually monitoring the type of infecting bacteria was necessitated. To address this need, strains of H. paragallinarum were evaluated according to both their phenotypic and their genotypic properties, in a combination serodiagnostic approach. A data bank of NAD-dependent H. paragallinarum reference strain and field isolate serovar-specific fingerprints was established on both a whole cell and outer membrane protein level. Visual comparative analysis of the qualitatively and quantitatively similar outer membrane protein patterns of all strains of NAD independency studied with the formulated data bank, indicate that the NAD-independent strains displayed profiles typical of serovar C-3. The outer membrane proteins have been identified as putative virulence determinants and, as such, were characterised according to their surface location, susceptibility to heat modification, functional role as endotoxins, sequence homology to structural membrane counterparts, and finally, their ability to induce an immune response. These studies represent novel efforts and form the foundation for identifying those antigens responsible for maintaining an infection in the host milieu. Ribotype analysis served as an adjunct to phenotypic observations, with the local NAD-independent field isolates being identified as serotype A. These contradictory outcomes call for the creation of a set of reference strains specific for NAD-independent isolates. The identification of restriction fragment length polymorphisms in the conserved 16S rRNA gene sequences indicate the potential application of this method for type assignment, requiring the recognition of a battery of versatile restriction enzymes to generate serovar-specific polymorphic profiles. The complexity of serotype allocation demands that a combination approach in which genotypic analyses complement phenotypic-based methods of haemagglutination inhibition and outer membrane protein profiling. The groundwork for implementation of such a system has been accomplished.