Antimicrobial susceptibility of Avibacterium paragallinarum isolates from outbreaks of infectious coryza in Dutch commercial poultry flocks, 2008-2017.

The objective of the present study was to determine the in vitro antimicrobial susceptibility of Avibacterium paragallinarum isolates from infectious coryza outbreaks in Dutch commercial poultry, from 2008 till mid-2017. By using a broth microdilution method, minimal inhibitory concentrations (MICs) of 15 antimicrobial agents were assessed, and MIC50 and MIC90 values were determined. Additionally, isolates were subjected to different PCRs for the presence of genes that may confer antimicrobial resistance. Besides field isolates, a set of reference strains, among which the nine Kume strains and one Page serovar strain, were included in the study. For broth microdilution testing a new growth medium, recently developed for susceptibility testing of Haemophilus parasuis, was used. The medium proved to be suitable for broth microdilution susceptibility testing of NAD dependent Av. paragallinarum as well; visible growth was obtained in growth control wells and accepting a deviation of one dilution step, MIC values were reproducible. Results of 44 field isolates originating from 25 outbreaks showed relatively good susceptibility to antimicrobial agents that are recommended for the treatment of infectious coryza in the Netherlands, except for tetracycline; circa 75% of the isolates were characterized by MIC values of tetracycline of ≥16 µg/ml. In almost a quarter of these isolates with high MICs of tetracycline, tet genes were detected. For the remaining isolates with elevated MIC values, the mechanism conferring resistance remains to be studied. Of most agents, low MIC values were determined for the nine Kume and one Page serovar reference strains, as well as negative PCR results for resistance genes, being concordant with agar diffusion results reported for these strains.

Unusual growth variants of Avibacterium paragallinarum.

Two isolates of haemophilic bacteria originally isolated in the 1980s from chickens were re-examined. The addition of a 10% sterile filtrate from an overnight culture of Staphylococcus epidermidis allowed growth of both isolates in solid and liquid media that were otherwise not capable of supporting the growth of these isolates. Using the modified media, genotypic and serotypic studies were performed, which confirmed both isolates to be Avibacterium paragallinarum, with one isolate being serovar A and the other serovar C. The unusual growth requirements of these two isolates reinforces the need for careful interpretation by diagnostic laboratories examining chickens showing signs of upper respiratory tract disease.

Haemophilus paragallinarum haemagglutinin: role in adhesion, serotyping and pathogenicity.

It is suggested that Haemophilus paragallinarum requires at least three haemagglutinins for adhesion during infection. This paper reports the partial purification and characterization of the HA-L haemagglutinin from H. paragallinarum strain 46-C3, a heat sensitive, trypsin sensitive haemagglutinin that has been shown to be the serovar specific haemagglutinin in this organism. Using the pl and molecular mass obtained, it was shown that this protein shares similarities with other types of adhesins found in Gram-negative bacteria. The haemagglutination assay conditions were optimized at pH 7.5 at 37 degrees C. It was also shown that activity is enhanced by the addition of Ca2+ and Mn2+ ions.

Haemophilus paragallinarum secretes metalloproteases.

Haemophilus paragallinarum secretes metalloproteases into different culture media lacking serum. Secreted proteins, concentrated by precipitation with 70% ammonium sulphate ((NH(4))(2)SO(4)) or methanol, displayed proteolytic activity at >100 kDa molecular mass in 10% polyacrylamide gels co-polymerized with porcine gelatin (0.1%). They were active in a broad pH range (4-9); pH 7.5 being the optimum. Protease activity was inhibited by 20 mmol EDTA/L and reactivated by calcium. The proteolytic activity was heat-stable at 40, 50, and 60 degrees C, but its activity diminished at 70 degrees C or higher. Secreted proteins partially degraded chicken immunoglobulin G (IgG) and cross-reacted with a polyclonal serum against a high molecular mass protease secreted by Actinobacillus pleuropneumoniae. Extracellular proteases could play a role in infectious coryza caused by H. paragallinarum.

Antigenic secreted proteins from Haemophilus paragallinarum. A 110-kDa putative RTX protein.

Haemophilus paragallinarum is the causal agent of infectious coryza, an economically important disease for the poultry industry. This bacterium secreted proteins of 25-110 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. paragallinarum. A 110-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. paragallinarum. H. paragallinarum secreted proteins could be important immunogens in the control of infectious coryza.