Evaluation of Pasteurella multocida serotype B:2 resistance to immune serum and complement system

Abstract

Members of gram-negative bacteria family Pasteurellaceae, include a large number of important economically human and veterinary pathogens. Organisms belonging to the family can colonize in mucosal surfaces of the respiratory, alimentary, genital tracts and cause diseases in various mammals, birds, and reptiles. Hemorrhagic septicemia is an acute disease of cattle and buffaloes in tropical countries caused by Pasteurella multocida serotype B:2. In the present study, the possible bactericidal activity of immune calf sera in the presence and absence of complement system was investigated. The results showed that P. multocida B:2 is highly resistant to positive serum, containing high levels of IgG and IgM obtained from calves after vaccination, and complement activity in normal fresh calf serum. This organism also grew rapidly in the normal fresh calf serum and the mixture of positive serum as well as normal fresh calf serum. As a control test an E. coli strain was subjected to the same experiment and found completely sensitive to the bactericidal activity of complement in calf and guinea pig fresh sera. Results were indicative of the presence of inhibitory mechanism(s) in P. multocida B:2 against bactericidal activity of immune calf serum and complement system.

Keywords: Cattle; Complement; Hemorrhagic septicemia; Pasteurella.

Fig. 1

Bactericidal activity of bovine serum on P. multocida. Data were the average of two assays ± standard deviation. An index of < 1 would indicate a bactericidal effect. The bactericidal index was defined as CFU mL^-1 after incubation for 1 hr divided by CFU mL^-1 at time zero. All three groups C1, D1, and E1 showed roughly the same growth rate and there was no significant difference between these average growth indexes (p > 0.05). A1: Immune serum + normal calf serum, B1: Heated immune serum + heated normal calf serum, C1: Immune serum + PBS, D1: Heated immune serum + PBS, E1: Normal calf serum + PBS, F1: Heated normal calf serum + PBS, and G1: PBS. Each well of a 96-well microtitre plate contained 25 μL of immune serum from a calf with high level of IgG and IgM antibody (Immune.Ser), with or without 25 μL of fresh normal calf serum (NC.Ser) or 25 μL heated Immune.Ser with or without 25 μL heated NC.Ser or 25 μL fresh NC.Ser or 50 μL heated NC.Ser or 50 μL PBS. A 50 μL suspension of P. multocida B:2 was added to each well and then incubated for 1 hr at 37 ˚C. Sample of 10 µL from undiluted, and dilutions of 10^-1 and 10^-2 were taken before and after incubation and were plated out on serum bovine albumin (SBA) and then incubated overnight

Fig. 2

Bactericidal activity of calf serum and guinea pig complement on E. coli. Data were the mean of two assays ± standard deviation. An index of < 1 would indicate a bactericidal effect. There was significant difference between D2 and C2 growth indexes mean (p < 0.05). There was no significant difference between C2 and E2 growth indexes (p > 0.05). A2: Fetal calf serum, B2: Heated fetal calf serum, C2: Normal calf serum, D2: Guinea pig complement, and F2: PBS. Each well of a 96-well microplate contained 50 μL of fetal calf serum (Fetal Calf Ser), heated fetal calf serum (Heat.Fetal Calf Ser), fresh normal calf serum (NC.Ser) or heated calf serum or guinea pig complement or PBS. A 50 μL of a suspension of E. coli was added to each well and then incubated for 1 hr at 37 ˚C. Sample of 10 μL from undiluted, and dilutions of 10^-1 and 10^-2 were taken before (time zero) and after incubation and were plated out on SBA and then incubated overnight. The bactericidal index was defined as CFU mL^-1 after incubation for 1 hr divided by CFU mL^-1 at time zero