Infectious coryza is a well-recognized and commonly encountered upper respiratory tract disease of chickens that is caused by the bacterium Haemophilus paragallinarum. The occurrence of recent outbreaks in North America has emphasized that the disease can be significant in meat chickens as well as layer chickens. In developing countries, coryza is commonly complicated by the presence of a range of other infections, resulting in severe disease and significant economic losses. Unusual forms of the disease, involving arthritis and septicemia, again associated with the presence of other pathogens, have been found in South America. Newly recognized bacteria such as Ornithobacterium rhinotracheale and phenotypic variant forms of both H.
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Haemophilus paragallinarum is the causal agent of infectious coryza, an economically important disease for the poultry industry. This bacterium secreted proteins of 25— kDa during its growth in brain heart infusion, tryptic soy broth, or Luria—Bertani glucose phosphate media, all lacking serum.
Some of these proteins were recognized by sera from chickens experimentally infected with H. A kDa protein was recognized by a serum pool from convalescent-phase pigs naturally infected with Actinobacillus pleuropneumoniae , and also by a rabbit polyclonal serum against Apx I as well as a rabbit serum against Mannheimia haemolytica leukotoxin, suggesting the presence of an RTX-like protein in H.
Infectious coryza IC is an acute respiratory disease of chickens caused by Haemophilus paragallinarum. The potential impact of IC on meat chickens has been emphasized by reports on economically important outbreaks in two regions of the USA [ 2 , 3 ]. Several factors have been associated with the pathogenicity of H. Page serogroup A and C mutants lacking HA are defective in tissue colonization [ 5 ].
The capsule has been associated with colonization, and considered a key factor in the lesions produced by this infection [ 6 ]; it also protects the microorganism against the bactericidal activity of chicken serum [ 7 ].
Crude polysaccharide extracted from H. Secreted antigenic proteins of H. Iritani et al. The heat-stable antigen was constituted by lipopolysaccharide LPS [ 10 ]. Inoculation of specific pathogen-free chickens with the heat-labile antigen protected them against infection with viable H. The characteristics and function of this antigen have not been elucidated yet. In this work, we report the presence of different immunogenic proteins of H.
Since H. Reference H. Soriano [ 11 ], were also used. To obtain the secreted proteins, H. All media were used without serum and supplemented with 0. Purity of the cultures was verified by inoculating samples on TS agar plates, streaked with a feeder culture of S.
After growth, H. Conditioned media were filtered through a 0. Protein concentration was measured by the Bradford method [ 12 ]. Non-inoculated media were processed in the same manner as culture supernatants and used as negative controls.
To obtain sera against H. These birds were obtained at 1 day of age, raised in isolation without vaccination and checked as negative to H. Birds were experimentally infected by infraorbital sinus injection with 0.
Chickens were maintained isolated and bled 10 days after clinical signs of IC were observed. To confirm that these signs were due to H. To obtain polyclonal sera against H. Animals received three boosts using Freund's incomplete adjuvant every 2 weeks. To obtain anti-Apx I polyclonal antibodies, Apx I protein was obtained from Actinobacillus pleuropneumoniae serotype 1 strain BC supernatants as described [ 14 ], checked as hemolysin by rabbit erythrocyte hemolysis, and inoculated into rabbits in the same manner as above.
To detect the immunoreactivity of H. Preimmune chicken and rabbit sera were used as negative controls. Blots were processed as described [ 15 ]. Immune recognition was observed using goat anti-chicken IgG, rabbit anti-pig IgG, or goat anti-rabbit IgG, all of them peroxidase-labeled diluted Reaction was revealed using diaminobenzidine hydrochloride and H 2 O 2.
The kDa secreted protein from H. Proteins were concentrated by precipitation with 2 volumes of cold methanol and resuspended in a minimum volume of 50 mM Tris—HCl buffer, pH 7. Immune recognition of the kDa protein was done as above, using polyclonal serum against the Apx 1 toxin, a serum pool from convalescent-phase pigs suffering from porcine pleuropneumonia, described previously [ 15 ], and polyclonal serum against Mannheimia haemolytica leukotoxin kindly donated by Dr. The kDa protein N-terminal sequence was determined after electroblotting the protein on a polyvinylidene difluoride membrane Bio-Rad.
Although NAD-independent strains have been isolated [ 16 ], H. However, since the serum proteins could hinder the identification of the secreted bacterial proteins involved in IC pathogenesis, the bacterium was grown in media lacking serum. The ability of H. Kinetics of bacterial growth in different culture media.
A: Field isolate H. Media were supplemented with 0. Molecules released into the cellular environment or those integrated to the cell surface are a basic aspect of a microorganism's physiology.
These molecules, such as polysaccharides, proteins, LPS, perform different functions inside the host, i. For this reason extracellular proteins are regarded as part of a major virulence mechanism occurring in bacterial infections.
Although different bacterial components have been described to be involved in the pathogenesis of IC, the secretion of host-damaging proteins by this bacterium has been poorly studied. Secreted proteins of H. Several proteins were observed in all three cultures: of 20, 27, 30, 40, 50, 60, 65, 70, and kDa Fig. Proteins were not observed in non-inoculated media data not shown.
Culture media or environmental conditions inside a host can induce differences in the expression of proteins [ 18 ]. Probably the LBGP medium lacks a specific component that promotes the expression of the kDa protein. The electrophoretic pattern of H. Lane 1, molecular mass markers. B: Immunorecognition of secreted proteins from serogroup A by: chickens experimentally infected with H.
Arrows indicate the main protein bands and immune recognition bands. Considering that field chickens with IC signs could be infected with other bacterial pathogens besides H. Indeed, H. The main proteins displayed a similar immune recognition pattern when rabbit polyclonal sera against secreted proteins from H.
These proteins were not recognized by chicken or rabbit preimmune sera Fig. Similar results were obtained for both reference and field strains. The secreted proteins corresponding to a molecular mass of kDa from H. These precipitated proteins were recognized by sera of pigs convalescent from pleuropneumonia Fig. Apx I and leukotoxin are members of the RTX protein family, which has been considered an important virulence factor in different Gram-negative pathogens.
In summary, size, immune cross-reaction, and the way in which the H. Furthermore, in a previous report, Kuhnert et al. Although it has been suggested that a toxin released from H. B: Immune recognition of the H. The N-terminal amino acid sequence of H. This sequence did not present similarity in the N-terminal region with other RTX toxins, however, N-terminal amino acid sequence similarity is not a characteristic conserved by the members of this family [ 19 ].
The identity of the kDa protein as a possible RTX protein must be confirmed by the nucleotide sequence of the gene and the presence of glycine repeats. The different H. Blackall P. Matsumoto M. Yamamoto R. In: Diseases of Poultry , 10th edn. Calnek B. Barnes H. Beard C. McDougald L. Saif Y. Google Preview. Droual R. Bickford A. Charlton B. Cooper G. Channing S. Avian Dis. Google Scholar. Hoerr F. Putman M. Rowe-Rossmanit S.
The disease is characterized by a swelling of the face, inflammation of infra orbital sinuses and conjunctivae with clear or purulent discharge from the nostrils in the acute stage of the disease Blackall et al. In many parts of the world, IC has become a major problem that affects all ages of chickens of both indigenous native chickens and laying hens in poultry farms. As early as , Beach believed that IC was a distinct clinical entity. The aetiological agent eluded identification for a number of years, as the disease was often masked in mixed infection, and with fowl pox in particular Blackall et al. DeBlieck isolated the causative agent and named it Bacillus hemoglobinophilus coryzae gallinarum Blackall et al. Elliot and Lewis and Delaplane et al.
Infectious Coryza: Overview of the Disease and New Diagnostic Options
Serological classification of Haemophilus paragallinarum with a hemagglutinin system.
Antigens, prepared from 17 strains of Haemophilus paragallinarum by treatment with potassium thiocyanate followed by sonication, uniformly agglutinated glutaraldehyde-fixed chicken erythrocytes and formed specific hemagglutination inhibition antibodies in rabbits. Attempts were made to classify the strains into serotypes by a combination of cross-hemagglutination inhibition and cross-absorption tests, using the hemagglutinating antigens, designated as HA-L hemagglutinin, and their antisera. The cross-hemagglutination inhibition tests showed the existence of three distinct groups among the 17 strains. Further cross-absorption studies indicated that two of the three groups could be subdivided into three serotypes each, forming a total of seven serotypes, designated HA-1 through HA Classification based on the serotype-specific HA-L system was found to be superior in its wider and more clearly defined specificities to other previous classifications, which are based on the agglutination test. There appeared to be a correlation between serotypes and geographic origins of the strains.