Development of selective culture media for efficient isolation of Avibacterium paragallinarum from chickens

Infectious coryza (IC) caused by Avibacterium paragallinarum (AvP) is an upper respiratory disease in chickens and incurs a significant economic impact on laying hens. Control of IC requires reliable bacterial isolation, but AvP is a fastidious bacterium, and the currently used methods yield poor isolation. To address this need, we developed selective media for the efficient growth and isolation of AvP. Several basal media supplemented with various growth factors were explored, and Mueller Hinton agar (MHA) supplemented with fetal bovine serum (FBS) plus NAD yielded optimal AvP growth, eliminating the need for nurse bacteria. This medium (named MSN) was further supplemented with vancomycin and crystal violet to produce two selective media, named MSNV and MSCV, respectively, to inhibit commensal Gram-positive bacteria that reside in the upper respiratory tract of chickens. MSNV and MSCV were compared with the standard isolation methods used in veterinary diagnostic laboratories for AvP isolation using samples from IC-positive and IC-negative flocks verified by clinical observation and AvP-specific RT-PCR. The two selective media significantly increased the isolation of AvP from clinical cases (59.4% for MSNV and 46.9% for MSCV) compared to the conventional method (37.7%). In addition, MSNV and MSCV successfully isolated the recently discovered non-pathogenic AvP variants, which were difficult to obtain using the standard methods. Notably, both media drastically reduced background bacteria and improved the purity of AvP isolates. These results demonstrate the efficacy and usefulness of the selective media for AvP isolation, which will enhance IC diagnosis, antimicrobial susceptibility testing, and vaccine development.IMPORTANCEInfectious Coryza is an economically important disease in poultry, resulting in poor growth and a significant reduction in egg production. The causative agent (AvP) of the disease is difficult to isolate from clinical samples and often requires a nurse bacteria to provide essential nutrients for optimal growth. In addition, the currently used methods for isolation are inefficient and often result in contamination by residential bacterial species. The selective media (MSNV and MSCV) developed in this study solve these problems by eliminating the need for nurse bacteria and by inhibiting Gram-positive bacteria normally present in the respiratory tract of chickens. The media not only increase isolation efficiency but also improve the purity of the isolates. These advancements will facilitate IC diagnosis and the development of vaccines for effective control of this major poultry pathogen.