Isolation of Actinobacillus pleuropneumoniae from layer hens showing clinical signs of infectious coryza.

Actinobacillus pleuropneumoniae is the causal agent of porcine pleuropneumonia, which is a highly contagious respiratory disease that affects swine nearly exclusively. An isolate with characteristics of some Pasteurellaceae family members (Gram-negative bacterium, pleomorphic, and NAD-dependent) was isolated from layer hens showing clinical signs of infectious coryza. This bacterium presented hemolysis on rabbit red blood cell agar plates, and PCR amplification and sequencing of its 16S rDNA gene indicated 99% identity with A. pleuropneumoniae serotypes 3 and 7. The presence of a putative apxIIA gene was also determined by PCR. A single, smooth colony of this bacterium inoculated in five, 7-day-old chicken embryos via the yolk sac route induced 100% mortality. However, inoculation into 10-wk-old, specific-pathogen-free chickens induced only light facial swelling, and reisolation of the inoculated bacterium was negative.

Identification of iron-acquisition proteins of Avibacterium paragallinarum.

When Avibacterium paragallinarum reference strain 0083 (serovar A) was grown in an iron-restricted culture medium, the expression of the 60, 68 and 93 kDa outer membrane proteins increased as compared with normal media. Sera of chickens experimentally infected with Av. paragallinarum recognized these iron-restriction induced proteins, suggesting their expression in vivo. The three outer membrane proteins were identified as transferrin receptor and iron transport proteins by mass spectroscopy and a search in sequence databases. As these proteins have been reported to be regulated by the Fur protein in many bacteria, we investigated, through molecular methods, the presence of the fur gene in Av. paragallinarum. A candidate fur gene of Av. paragallinarum was amplified by polymerase chain reaction using complementary primers to conserved regions of fur gene sequences from members of the Pasteurellaceae family. The nucleotide sequence of the cloned gene, from ATG to TAA stop codon, was 453 base pairs in length and the deduced amino acid sequence showed 94% identity with Fur sequences of Actinobacillus pleuropneumoniae and Haemophilus ducreyi. The Av. paragallinarum deduced Fur protein (17.8 kDa) amino acid sequence contains the N-terminal helix-turn-helix DNA-binding domain and the two iron-binding sites in the C-terminal end, typical of other described Fur proteins. The study of iron-restriction-induced proteins and the mechanism regulating their expression could lead to an understanding of the responses of Av. paragallinarum to survive in an iron-restricted environment on host mucosal surfaces.

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.