Biochemical, antigenic and proteomic properties of isolates and strains of the causative agent of chicken infectious coryza Avibacterium paragallinarum (Biberstein and White 1969) Blackall et al. 2005

Infectious coryza (haemophilus infection) of chickens is a disease reported in many countries of the world. In the Russian Federation, there is no information both about the extent of the disease spread across the poultry farms and about the serotype diversity of the agent circulating in the country. The paper for the first time demonstrates biochemical properties and specifies antigenic relatedness of new Avibacterim paragallinarum isolates recovered from chickens with respiratory signs in Russia and in Belarus. The paper also shows results of creation of the Avibacterim paragallinarum subsection in the database of tested microorganisms’ mass-spectra which can be used as reference ones for the identification and protein profiling of the infectious coryza agent strains and isolates. The work aimed to determine biochemical and antigenic properties of A. paragallinarum isolates and to produce the species-specific mass-spectra as a tool for Avibacterim paragallinarum species identification and intraspecies differentiation. A. paragallinarum No. 29545 АТСС (serotype А1) form the collection of the Federal Centre for Animal Health (ARRIAH) served as a reference strain. Thirteen A. paragallinarum isolates were used in the study. The isolates were recovered from the pathological material collected from chickens with respiratory pathology (nasal exudates, contents of infraorbital sinus and conjunctival sac, lung tissues) in 2015. The isolates were inoculated onto Columbia agar supplemented with 5 % defibrinated sheep blood, together with a streak of Staphylococcus epidermidis . Pure cultures of the agent were grown on the serum agar containing NADP at 20 μkg/cm3 and 5 % of horse blood serum. The bacteria were cultured for 24-72 at high CO2 concentration and 37 °С. Bilateral antigenic relatedness of the reference strain АТСС No. 29545 and A. paragallinarum isolates was determined using slide agglutination test (SAT). Hemagglutination activity was assessed using hemagglutination inhibition test (HI). Homogeneity of the tested isolates’ serogroup was confirmed by PCR. Amplified 800 bp DNA fragments were indicative of the presence of serogroup A A. paragallinarum genome, 1000-1100 bp of serogroup В, and 1500-1600 bp of serogroup С. A. paragallinarum identification was performed using MALDI Autoflex III Biotyper mass-spectrometer (Bruker Daltonik GmbH, Germany). The resulted mass-spectra were recorded, processed and analyzed using FlexControl 3.4 software (Bruker Daltonik Gmb», Germany) according to MALDIBiotyper 2.0. UserManual, Version 2.0 SR1, Germany, 2008. Testing of biochemical properties indicated that all 13 isolates of A. paragallinarum form a diverse group. The A. paragallinarum isolates’ growth absolutely depended upon presence of blood serum in the culture medium. Saccharolytic activity of the A. paragallinarum isolates also varied. All isolates and the reference strain can utilize glucose and sucrose, but not lactose, trehalose and galactose. The property also varied for mannitol and mannose. As for antigenic properties, all tested isolates belonged to the same serogroup B that was also confirmed by real-time polymerase chain reaction method. Examination of proteomic properties of A. paragallinarum isolates revealed typical MS peaks, m/z 4768-4770 and 5347-5349, that were similar to those for the reference strain A. paragallinarum ATCC No. 29545. Protein profile mass-spectra were entered into MALDI Autoflex III Biotyper database to improve the reliability of A. paragallinarum species identification. Basing on the examined biochemical, antigenic and proteomic properties, three A. paragallinarum isolates were deposited inещ the strain collection of the Federal Centre for Animal Health (ARRIAH) as A. paragallinarum strain No. 1818, A. paragallinarum strain No. 5111, and A. paragallinarum strain No. 1116.