Du 530

B. pertussis strain UT25 (UT25Sm1) (53 (link)) was cultured on Bordet-Gengou (BG) agar (Remel) (54 ) supplemented with 15% defibrinated sheep blood (Hemostat Laboratories) for 48 h at 36°C. B. pertussis was then transferred from BG plates to three flasks containing 12 ml of modified Stainer-Scholte liquid medium (SSM) (37 (link)). SSM cultures were not supplemented with cyclodextrin [heptakis(2,6-di-O-methyl)-β-cyclodextrin)] and were grown for ∼22 h at 36°C with shaking at 180 rpm until the optical density at 600 nm (OD₆₀₀) reached 0.5 on a 1-cm-path-width spectrophotometer (DU 530; Beckman Coulter). For infection of NSG mice, cultures were then diluted to provide a challenge dose of 2 × 10⁷ CFU in 20 μl. For growth of Pseudomonas aeruginosa strain PAO1, Pseudomonas Isolation Agar (Difco) was used. Bordetella bronchiseptica RB50 and Escherichia coli TOP10 were cultured on lysogeny agar (10 g of NaCl, 5 g of yeast extract, 10 g of tryptone) at 36°C for 18 h. In vitro cultures of bacteria were grown and then filtered through 5-µm-pore-size syringe filters (Minisart; Sartorius). The total number of input and output bacteria were determined through serial dilutions and plate counts on the appropriate medium, and the percent recovery of each species was determined.

PBCV-1 vLysin isolated from ultra-purified virions was treated with anti A561L^D4 antibody (AB) produced in mice (800-fold dilution). After 1 h incubation, to remove vLysin neutralized with anti-A561L^D4 antibodies from the reaction, magnetic beads (MB) coupled with anti-mouse Ig (RayBiotech, GA, USA, cat # 801–103) were added to the reaction and the supernatant fraction was separated from the MB following the manufacturer’s instructions. The resultant supernatant fraction that presumably had vLysin activity neutralized and removed was mixed with NC64A cells at concentration 6 × 10⁷ cells/mL and incubated for 1 h. SDS was added to the treated cells to 1% concentration (final) and incubated for 1 min at room temperature, then NC64A cells were centrifuged for 15 min at 16,000× g, the supernatant fraction was removed and chlorophyll release was measured using a spectrophotometer (Beckman DU 530) at λ = 420. Untreated PBCV-1 vLysin was used as a positive control. NC64A cells treated with pre-bleed serum and 1% SDS served as a negative control.

Blood specimens were taken from the caudal vein in heparinized tubes and centrifuged at 3,500 g for 10 min; serum samples were separated and stored at −20°C until analysis. The analyzed serum parameters such as alanine aminotransferase (ALT) were determined using an automatic analyzer (Beckman Coulter DU @ 530, automatic biochemical analyzer, USA).
Hemoglobin (Hb) concentration was evaluated by Sahli's hemometer [24 ] and hematocrit (Hct) concentration was determined using the microhematocrit method [25 ]. Red blood cells (RBC) and white blood cells (WBC) were counted using the improved Neubauer hemocytometer [25 ], and for differential leucocyte counts, freshly prepared blood smears were stained with modified Giemsa's stain [26 (link)] and observed under a microscope. The mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were calculated as stated by Mahfouz and Sherif [27 (link)].

Peripheral blood samples were collected in tubes coated with EDTA and were stored at − 80 °C. Genomic DNA was extracted from whole blood using the Universal Genomic DNA Extraction Kit (TaKaRa Bio Inc., Japan). DNA concentration was measured with the UV/VIS spectrophotometer (DU530, Beckman Instruments, USA). Four tag-SNPs (rs2881766, rs9383951, rs9340799 and rs3020449) were selected from The Single Nucleotide Polymorphism database (dbSNP). A multiplexed SNP MassEXTEND assay was designed by Sequenom MassARRAY Assay Design V3.0 Software (Agena Bioscience Inc., USA). And SNP genotypes was detected using Sequenom MassARRAY RS1000 [10 , 11 (link)]. The primers of the selected SNPs were listed in Additional file 1: Table S1. Data was analyzed with Sequenom Typer Software (version 4.0, USA).