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A NAD-dependent isolate 46 (C-3) of Haemophilus paragallinarum, which was previously demonstrated to be of high virulence, was transformed to NAD independence using a plasmid isolated from a naturally occurring NAD-independent isolate of H. paragallinarum. The transformation was performed by two different methods and the identity of all of the isolates, before and after transformation was confirmed using a H. paragallinarum-specific PCR test. The transformed NAD-independent serovar C-3 isolate and the wild-type serovar C-3 isolate were used to experimentally infect vaccinated layer chickens. It was shown that the transformation to NAD independence significantly altered the virulence of the serovar C-3 isolate that was used in the transformation experiment. The mechanisms responsible for a decrease in virulence are not clear, but may be related to the pathology of the transformed isolate in the sinus of the chickens.

The virulence of four South African field isolates of NAD-dependent Haemophilus paragallinarum, representing the four serovars known to occur in that country, was investigated. During this study an alternative challenge model for infectious coryza was used, in which the infectivity as well the virulence of different isolates could be evaluated. The challenge model consisted of the direct challenge, via intrasinus injection of one chicken in a row of interconnected layer cages, containing 10 chickens, which are subsequently infected by natural routes. A scoring system of the clinical signs was established in which a score is given to the ability of the isolate to produce clinical signs in the challenge birds. The mean daily disease score for the flock can be calculated and plotted on a graph to give a graphic representation of the disease profile. A mean disease score, calculated over a 20-day examination period can be calculated. Isolates can then be compared to each other, either graphically or by a comparison of the mean disease scores. It has been demonstrated using this scoring system that the South African serogroup C isolates appear to be more virulent than the South African serogroup A or B isolates. It was further established that the serovar C-3 isolate appeared to be the most virulent.

Naturally occurring NAD-independent variants of Haemophilus paragallinarum, which have been isolates from poultry showing clinical signs of infectious coryza, were used to determine their virulence using a newly developed challenge model for infectious coryza. It was established that the NAD-independent isolates belonging to a particular serogroup, were less virulent when compared to the virulence of the NAD-dependent isolates from the same serogroup. It was shown that the virulence of the NAD-independent isolates belonging to serogroup C and serogroup A were very similar to each other. This differs to the results obtained with NAD-dependent isolates reported on previously, in which the serogroup C isolates were found to be more virulent then the serogroup A isolates.

Two strains (Strains 39 & B) of Haemophilus paragallinarum were isolated and identified as serotype C by dot-blotting analysis in Taiwan in 1994. To identify their virulence and pathogenicity in Taiwanese local chickens, local chickens were inoculated via nostril with the isolates, and examined for 14 days. Clinical signs of swollen face, and nasal discharge were first seen at 24 hr after inoculation, and the infectivity of Strains 39 and B at 14 days after inoculation among the inoculated chickens was 50.0-71.4% and 55.6-77.8%, respectively. From the results, we concluded that the two bacterial isolates were pathogenic to local chickens with a maximum morbidity of 77.8%.

A survey of infectious coryza of chickens was performed in West Jawa of Indonesia between 1987 to 1988 by the detection of hemagglutination-inhibition (HI) antibody against Haemophilus paragallinarum (Hpg) in non-vaccinated healthy laying (12 farms, 196 chickens) and native (8 farms, 197 chickens) chickens. HI antibodies against Hpg were detected not only in the native chickens but also in the laying chickens, regardless of the district, and were observed in 70% (14/20) of farms and 19% (73/393) of chickens. HI antibodies against serotype A were detected from 11 farms (55%) and 11% (45/393) in chickens. Those against serotype C were detected from 5 farms (25%) and 8% (30/393) in chickens. Three Hpg strains were isolated from different chickens affected with infectious coryza. Two of them were identified as type A and the other as type C by the rapid plate agglutination test. These results demonstrated that the outbreaks of infectious coryza caused by serotype A and C strains had occurred in Indonesia.

The pathogenicity and presence of serovar-specific hemagglutinating (HA) antigens of Haemophilus paragallinarum serovar B reference strains 0222 and Spross and field isolates 24268 and 24317 were investigated. Chickens challenged with all strains except strain 0222 showed clinical signs of infectious coryza. For all four strains, challenged chickens had intrasinus lesions and were colonized by the challenge organism. Before and after hyaluronidase treatment, strains 0222, 24268, and 24317 showed HA activity against formaldehyde-fixed chicken erythrocytes but not against fresh chicken erythrocytes. Strain Spross expressed HA activity only after treatment. In cross-hemagglutination-inhibition tests, the four serovar B strains cross-reacted with each other but not with serovar A and C strains. Cross-adsorption tests indicated that strain 24317 has a wider range of HA antigens than the other two strains. Our results indicated that H. paragallinarum serovar B strains are pathogenic for chickens and that they possess six different HA antigens, one of which is specific for serovar B strains.

Twenty-seven Australian avian Haemophilus isolates were tested for their ability to cause infectious coryza in specific pathogen-free chickens. All 15 isolates, identified as H. paragallinarum, produced infectious coryza, whereas all 12 H. avium isolates were nonpathogenic, but spread to in-contact chickens.

Studied were a total of sixteen cases with the clinical picture of coryza. Leading role in eleven of them had Haemophilus, in one the signs of chronical cholera and pox were seen, and in three there was a Pseudomonas aeruginosa infection. All cases were serologically positive for CRD. By their needs of nutrient growth factors the isolated Haemophilus strains belonged to H. paragallinarum, and they proved pathogenic for 8-week-old birds and poults that were about to lay their first eggs, killing 4-7-day-old chick embryos up to the 48 h hour. Tests for resistance to therapeutic means with the use of the disk-diffusion method revealed sensitivity to streptomycin, tetracycline, chloramphenicol, gentamycin, erythromycin, spectinomycin, furazolidon, imekil, cosumix, trimetoprim, and sulfadoxin. Good and very good therapeutic and prophylactic effects were produced with the use of sulfotin, cosumix, linkospectin, and trimedin coupled with aerosol disinfection with rivanol, the birds being present on the premise. It is stated that with regard to differential diagnosis coryza caused by Haemophilus paragallinarum should not be confused with fowl cholera, pox, CRD, and Pseudomonas aeruginosa infection which may run their course in a mixed infection.

Lesions induced in chickens by an encapsulated or nonencapsulated strain of Haemophilus paragallinarum were investigated. In terms of lesion severity, major differences in pathogenicity were observed between the encapsulated and nonencapsulated variants. The principal lesion manifested by the encapsulated variant was an acute catarrhal inflammation of the upper respiratory tract, mainly of the nasal cavity and paranasal sinus. Infiltration of a large number of mast cells into the lamina propria of the mucous membrane of the nasal cavity was also characteristic. Numerous organisms were found on the cilia or on the surface of the epithelial cells of the nasal mucosa. Chickens with these histologic lesions had severe clinical signs of coryza, and organisms were recovered in high numbers from the nasal cavity and paranasal sinus. In contrast, chickens that were given the nonencapsulated variant did not have clinical signs of coryza, and the organisms were recovered in low numbers from the inoculated sites; slight histopathologic lesions were observed in the nasal mucosa at postinoculation day 1. Mast cell infiltration in the chickens inoculated with the encapsulated variant indicated that mast cells may be responsible for producing clinical signs of coryza via the activation of pharmacologic mediators. Adherence to and colonization of the encapsulated variant on the nasal mucosa seems to be a first step of the infection.

Adhesion of Haemophilus paragallinarum to chicken embryo fibroblasts (CEF) in vitro was investigated in correlation with its virulence. Under the scanning electron microscope, the organisms were seen adhering to CEF exposed to the bacteria. By transmission electron microscopy, the organisms appeared to attach to the plasma membrane of CEF by their fuzzy material. They were enclosed in membrane-limited vesicles and appeared morphologically intact. Specific fluorescence was seen in the cytoplasm of CEF in later stages of infection. There was a good correlation between pathogenicity of the organisms for chickens and their ability to adhere to CEF. This correlation was reinforced by the fact that the ability of a pathogenic strain to adhere to the mucosal surface of the chicken trachea was demonstrated by scanning electron microscopy.