Skim milk

Protein (100 μg) was mixed at a 1:1 v/v ratio with 2× sample buffer (62.5 mM Tris-HCl, pH 6.8; 2% SDS; 6 M urea; 10% β-mercaptoethanol; and 20% glycerol) and heated to 100 °C for 5 min. Samples were electrophoresed on 12% Tris-glycine SDS-PAGE gels and transferred to polyvinylidene fluoride membranes (GE Healthcare, Marlborough, MA, USA). Membranes were blocked with 5% skim milk (Thermo Fisher Scientific) or 3% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) in PBS (pH 7.4) containing 0.05% TWEEN^® 20 (PBST) for 3 h at RT. Primary antibodies (1:1000; against STAT1/3, JAK2, SOCS1, TYK2, TAK1, SHP-2, CSF-1R, NF-κB1, MyD88, ERK1/2, and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) were prepared in either 2% skim milk or 0.5% BSA in PBST overnight at 4 °C. Membranes were washed with PBST and treated with the HRP-linked anti-rabbit secondary antibodies (1:4000; Sigma-Aldrich) or goat anti-mouse IgG HRP conjugate (1:4000; Thermo Fisher Scientific) in 2% skim milk or 0.5% BSA in PBST for 2 h at RT. Subsequently, the membranes were developed using western Lightning^® Plus-ECL (Thermo Fisher Scientific) on Hyperfilm™ (GE Healthcare).

The plasma samples separated from mammal and bird blood were subjected to IFA assay to determine the seropositivity. JEV (Nakayama strain, courtesy of Dr Y. C. Chan) was cultured in Vero cells (ATCC CCL-81) in the biosafety level 3 (BSL3) laboratory at the Environmental Health Institute. Immunofluorescence slides were made, inactivated and transferred out of the BSL3 to BSL2 where the IFA was performed. Animal plasma was diluted 1:50 in 5% skim milk (Oxoid, Kansas, USA) and applied onto the JEV IFA slides. Incubation was performed in a humidifier at 37 °C for 30 min before washing with 1× PBS. Secondary antibody anti-bird Ig-FITC (Bethyl Laboratories, Texas, USA) or anti-pig Ig-FITC (Bethyl Laboratories) was applied onto the slides, depending on the animal sera tested. Incubation and washing were repeated before visualization of fluorescence was performed.

For Western blot analysis, the purified rCpEno was separated on 12% SDS-PAGE and electrotransferred to a PVDF membrane (Merck Millipore, Billerica, MA, USA) using a Trans-Blot® SD Semi-Dry Transfer Cell (Bio-Rad, Hercules, CA, USA) according to standard procedures. Induced pET28a (+) plasmid transformed BL21 (DE3) competent cells were selected as the control. The membranes were blocked with 5% skim milk (Oxoid, Basingstoke, Hants, UK) for 2 h at room temperature and incubated with His-Tag mouse mAb (1:1,000) (Cell Signaling Technology, Danvers, MA, USA) or serum isolated from cattle artificially infected with C. parvum (1:200) for 1 h, followed by incubation with anti-mouse IgG (whole molecule)-Peroxidase conjugate antibody (Sigma-Aldrich, Louis, MO, USA) or anti-bovine IgG (Sigma-Aldrich) at 1:2,000 dilution for 1 h at room temperature. Reaction bands were detected using a DAB Substrate Kit (Pierce).

The ability to produce and secrete hemolysins and hemolysin BL (HBL) was assessed by streaking bacterial cells on blood agar (Columbia agar containing 5% horse-blood, Oxoid, Basingstoke, UK) and sheep blood agar (Columbia agar containing 5% sheep-blood, Oxoid), respectively. Plates were incubated at 30 °C for 18 h. Hemolysin production was checked by visually evaluating the presence of a halo of incomplete hemolysis immediately around the colonies. HBL secretion was tested by observing the formation of an unusual discontinuous zone of hemolysis surrounding colonies [10 (link)]. Diarrheal toxin was detected in filtered culture supernatants by using the B. cereus enterotoxin-reversed passive latex agglutination (BCET-RPLA, Oxoid, Basingstoke, UK) kit accordingly to the manufacturers. The production of phosphatidylcholine-specific phospholipase C (PC-PLC) was evaluated by agar-diffusion assays by using 0.15% l-α-phosphatidylcholine (Sigma-Aldrich, Milan, Italy) [15 (link)]. Protease secretion was checked by seeding bacterial cells on 1.5% skim milk (Oxoid, Basingstoke, UK), followed by incubation at 37 °C for 18 h [16 (link)]. The presence of a clear degradation halo around colonies was indicative of the presence of proteolytic activities. Experiments were repeated three times in separate days.

ELISA plate binding assay was used to determine the binding activity of rCpEno to human plasminogen as described by Bao et al. [16 (link)]. Briefly, 10 μg/well of purified rCpEno was added into 96-well plate and incubated at 4 °C overnight. BSA (10 μg/well) was used as a negative control. After being washed three times with PBST, the plate wells were blocked with 5% skim milk (Oxoid) in PBST at 37 °C for 2 h. After washing, different concentrations of human plasminogen (0, 1, 5, 10, 15, 20, 25 and 30 μg/ml in PBST) (Merck Millipore) were added to each well and incubated at 37 °C for 2 h. Then the plates were incubated with rabbit anti-plasminogen polyclonal antibody (100 μl/well, 1: 3,000) (Abcam, MA, USA) at 37 °C for 1.5 h. After washing three times with PBST, the plate wells were incubated with goat anti-rabbit IgG-HRP (100 μl/well, 1: 5,000) (Sigma-Aldrich) at 37 °C for 1 h. Finally, the colour reaction was performed by adding soluble TMB substrate solution and terminated with 2 M H₂SO₄. Absorbance at OD450 was read using a spectrophotometer (BioTek).

The supernatant was obtained when the cells were mixed with lysis buffer at 4 °C for 1.5 h and then centrifuged (12,000×g, 10 min). The protein concentration was determined by Coomassie blue staining. An appropriate amount of loading buffer was added according to the volume, and the samples were boiled to denature proteins before and an appropriate amount was used for western blotting. Proteins were separated by SDS-PAGE, transferred to PVDF membranes (Bio-Rad, USA) and blocked with 5% skim milk (Oxoid, England) for 1.5 h at room temperature. The membrane was incubated with the primary antibody overnight at 4 °C, washed, incubated with secondary antibody for 1.5 h at room temperature, and washed again, and the immunoreactive protein bands were detected using a chemiluminescence detector (Beyotime, China).

Proteolytic bacteria were selected using nutrient agar media (Oxoid) plus skim milk as a protein source. nutrient agar media (Oxoid) were added with 1% skim milk (HiMedia, India), heated until homogeneous, and boiled. The media were sterilized at 121°C for 15 min. The media were stored at room temperature 28°C for 24 h and then at 4°C until used.