Characterization of the hemolytic activity of Riemerella anatipestifer

Abstract

Riemerella anatipestifer infection causes serious economic losses in the duck industry worldwide. Acute septicemia and high blood bacterial loading in R. anatipestifer infected ducks indicate that R. anatipestifer may be able to obtain iron and other nutrients by lysing duck erythrocytes to support its rapid growth and proliferation in the blood. However, so far, little is known about the hemolytic activity of R. anatipestifer to duck erythrocytes. In this study, 29 of 52 R. anatipestifer strains showed hemolytic activity on duck blood agar, whereas all the tested dba⁺ (with hemolytic activity on duck blood agar) and dba^- strains created pores in the duck red blood cells, with 4.35-9.03% hemolytic activity in a liquid hemolysis assay after incubation for 24 h. The concentrated culture supernatants of all the tested R. anatipestifer strains and the extracted outer membrane proteins (OMPs) from dba⁺R. anatipestifer strains showed hemolytic activity on duck blood agar. These results, together with the median lethal dose (LD50) of some dba⁺ and dba^-R. anatipestifer strains in ducklings, suggested that there was no direct relationship between the hemolytic capacity of R. anatipestifer on duck blood agar and its virulence.

Keywords: Riemerella anatipestifer; culture supernatant; hemolytic activity; outer membrane proteins (OMPs); virulence.

Fig. 1.

All the R. anatipestifer strains tested generated pores in duck red blood cells, observed with phase contrast microscopy. Four dba⁺ strains 2, 3, 33 and 38, and five dba^- strains 6, 17, 18, 26 and 46 were respectively incubated with duck red blood cells at 37 °C for 12 h. The morphology of the duck red blood cells was observed under a Nikon Eclipse Ci phase contrast microscope. The observation through phase contrast microscopy showed that all the tested dba⁺ and dba^- R. anatipestifer strains generated pores in duck red blood cells, and the dba⁺ strains generated more pores in the duck erythrocyte membrane than the dba⁻ strains.

Fig. 2.

The culture supernatants of all the tested dba⁺ and dba⁻ R. anatipestifer strains generated many pores in the duck erythrocyte membrane, observed with phase contrast microscopy. The culture supernatants from five dba⁺ R. anatipestifer strains 1, 2, 3, 5 and 38, and four dba⁻ strains 6, 17, 18 and 26, were incubated with duck red blood cells for 12 h. The observation through phase contrast microscopy showed that the culture supernatants of all the tested dba⁺ and dba⁻ R. anatipestifer strains generated many pores in the duck erythrocyte membrane.