Figures & data
Figure 1. Proteolytic activity in 10% polyacrylamide gels copolymerized with 1% bovine casein. Cell-free culture supernatants of G. anatis strains were precipitated with 1 volume of methanol and 20 µg dissolved protein was loaded into each well. Lane 1, 250900 isolate; lane 2, 249101 isolate; lane 3, F149T G. anatis reference strain. 1b: Effect of pH on proteolytic activity. Gels were incubated at pH 6 to 10 (lanes 1, 3, 5, 7, 9, 249101 isolate; lanes 2, 4, 6, 8, 10, 250900 isolate).
Figure 2. Temperature effect. The same conditions, isolates and sample amount as in b. The first sample (lane 1) was not incubated while the other samples were incubated at (lane 2) room temperature, (lane 3) 37°C, (lane 4) 40°C, (lane 5) 50°C, (lane 6) 60°C, (lane 7) 70°C and (lane 8) 80°C during 10 min prior to electrophoresis. 2a: 250900 isolate; 2b: 249101 isolate.
Figure 3. Inhibitory effect on the proteolytic activity. The same conditions, isolates and sample amount as in b. Gels were incubated in 50 mM Tris–HCl buffer (pH 9.0) with protease inhibitors: 10 mM EDTA (lanes 3, 4), 5 mM p-hydroxymercuribenzoate (lanes 5, 6) and 5 mM phenylmethylsulfonylfluoride (lanes 7, 8). A sample without inhibitor was used as control (lanes 1, 2). Lanes 1, 3, 5, 7, 250900 isolate; lanes 2, 4, 6, 8, 249101 isolate. 3b: Immunoblot analysis of CP from G. anatis reference strain (lanes 1 and 3) and A. pleuropneumoniae (lanes 2 and 4) reacted with the polyclonal serum raised against the purified protease of A. pleuropneumoniae serotype 1 (lanes 1 and 2) or with preimmune rabbit serum (3 and 4).
Figure 4. Chicken IgG degradation by CP from G. anatis. Twenty micrograms of CP from field isolates or reference strain were mixed with 10 µg chicken IgG and incubated at 37°C during 24 h (lanes 2, 5 and 8), 48 h (lanes 3, 6 and 9) and 72 h (lanes 4, 7 and 10). Lane 1, control chicken IgG incubated during 72 h; lanes 2 to 4, G. anatis reference strain; lanes 5 to 7, 249101 isolate; lanes 8 to 10, 250900 isolate.
