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Infectious coryza, an acute upper respiratory tract infection of chickens, is caused by the bacterium Haemophilus paragallinarum. The heamagglutinin antigen plays a key role in serotyping, immunity and pathogenicity. The two serotyping schemes, the Page and Kume schemes, are both performed using haemagglutination inhibition (HI) tests. The most widely used serotyping scheme, that of Page, groups H. paragallinarum isolates into three serovars. A, B and C. Considerable attention has also been paid to the role of the heamagglutinin antigens in pathogenicity and as protective antigens. For Page serovar A organisms, there is a close relationship between HI titre and both protection and clearance of the organism from the nostrils of chickens with purified haemagglutinin from a serovar A organism. However, until this report, neither the protein sequence nor the gene encoding the heamagglutinin has been identified. The gene encoding the heamagglutinin of H. paragallinarum, hagA, has been identified and the full-length nucleotide sequence has been determined. A ~39 kDa protein, recognised by an anti-haemagglutinin monoclonal antibody, mab4D, was purified from H. paragallinarum strain 0083 and the N-terminal sequence obtained. The full-length nucleotide sequence was obtained by inverse PCR and the deduced amino acid sequence of the protein encoded was shown to be similar to outer membrane proteins of closely related organisms in the HAP (Haemophilus, Actinobacillus and Pasteurella), especially the P5 protein of H. influenzae. The hagA gene was cloned into a His-tag expression vector and over-expressed in Escherichia coli strain M15(pREP4). The identity of the purified recombinant protein was confirmed by haemagglutination of chicken red blood cells and reactivity, in a Western blot, with the monoclonal antibody specific for the serivar A haemagglutinin. A strain survey of eleven serotyping reference strains of H. paragallinarum revealed that the degree of sequence variation of the HagA protein was limited. Close examination of amino acid sequence differences at each point of variation in the H. paragallinarum haemagglutinin protein revealed that none of these changes correlated with recognised serovar groupings, except at position 88, where an arginine (charged residue) in serovar B is replaced with a leucine, phenylalanine or serine (non-polar residues) in serovar A or C. However, this change alone cannot account for the serological groupings of this organism. During the initial attempts to isolate the gene encoding the haemagglutinin antigen, a novel protein (ORF3) distinct from the haemagglutinin was unexpectedly isolated. This protein is distinct from the haemagglutinin in terms of nucleotide and deduced amino acid sequence, predicted secondary structure and haemagglutination activity. The ORF3 gene was similarly cloned into a His-tag expression vector and over-expressed in Escherichia coli strain M15(pREP4). The identity of the purified recombinant protein was confirmed by reactivity, in a Western blot, with the HP1 polyclonal antiserum. The ORF3 deduced amino acid sequence is closely related to a number of lipoproteins from Gram-negative organisms, especially a lipoprotein of H. influenzae and the lipoprotein-34/NlpB from E. coli. The ORF3 protein, in addition to the HagA protein, induces strong immune responses in rabbits, as shown by Western blotting of whole cell lysates of H. paragallinarum with strain HP1 (0083) polyclonal antiserum, thus indicating their role as potential vaccine candidates against infectious coryza. Therefore, subunit vaccine trials using His-tagged recombinant HagA and ORF3 were established to investigate the role these proteins may have as vaccine candidates. Protection was not afforded in terms of prevention of clinical signs or colonisation of the organism in the nasal cavity of the chickens vaccinated. The type of adjuvant and number of boost immunisations did not seem to greatly affect protection. However, the rate of recovery from clinical signs was improved slightly for birds vaccinated with rORF3 with aluminium hydroxide. This suggests that either a conformational epitope (i.e. a specific secondary structure) or a post-translational modification of this protein provides protection against infectious coryza.
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