Isolation, Identification Of Pasteurella Multocida From Ducks And Pathologic Dynamics Observation On Ducklings

Pasteurella multocida (P.m) was a common pathogenic agent which can cause great economic losts to both livestock and poultry industry, especially to duck industry, as ducks was more susceptible to the bacteria. In this study, a strain of Pasteurella multocida was isolated and identified from a duck farm in Wuhan, HuBei province. 18 day-old duckings were experimentally inoculated the bacterita to study the pathogenesis. Different inoculation methods were applied to evaluate its virulence. The role of TNF-αand IL-1βin inflammation were discussed. All the work was for a better understanding the pathological damage mechanism of P.m. The research contents were as follows:1. Isolation and identification of the bacteria. Colony culture characterization, biochemical reactions, drug resistant test, and 16S rDNA sequence analysis were performed to this strain. The results revealed this strain was Pasterulla multocida type Fo, with highly sensitive to sulfonamides, macrolide antibiotics. The 16S rDNA partial sequence of PM strain shared 99% homology with other pasteurella multocida belonging to cattle, pig, sheep and chicken, whereas, shared less than 93% homology with Pasteurella haemolytica. The results indicated the close relationships of P. multocida isolated from different species, monophyletic were also found in these strains.2. Different inoculation methods were performed to evaluate the virulence of this strain. Subcutaneous inoculation and bronchial inoculation showed highly virulence to ducklings, and flipper injection showed medium virulence, whereas intragastric showed no virulence to ducklings. The results indicated that the infection of P.multocida in duckling was most likely not occurred through the alimentary tract. 3. Duplication model of P.m infection ducklings. 18^th day-old ducklings were experimentally inoculated with Pasteurella multocida through flipper injection, with dose of 2×10²CFU/0.5mL approximately. Acute hemorrhagic septicemia can be observed and lasting for 3 days, with clinical features of anorexia, chills, bradybasia and ptyalism in 10h post infection (p.i). Some died in 24h p.i, and the mortality reached peak in 48h p.i; Recovery phase appeared in 72h with clinical symptoms relieved. It was concluded that course of disease was more likely a transient response, with symptom of the respiratory tract injury.4. Gross necropsy and histological changes were observed within 4h, 10h, 24h, 40h, 48h, 7d post-inoculation, respectively. Results indicated that ducklings had lesions of hemorrhagic septicemia with widespread vascular damage. Necrosis focus diffused with large number of heterophilic granulocyte can be observed in liver, spleen, lung and other organs. Thymus lesions appeared at first phase of disease (4h p.i), while follicular lymphoid necrosis and depletion in bursa appeared at late phase of disease (40h p.i). Nervous system lesions, including edema, chromatolysis and perineural invasion were observed in brain. No visible lesions were noted in control group. This suggested that the pathogens first activated T-cell immune response and can induced immune dysfunction.5. White blood cell differential count result revealed the heterophilic granulocyte/lymphocyte ratio was higher than the control group in 8h, 10h, 17h and 24h post inoculation, and the difference was no significant in 40h post inoculation. Serum TNF-αlevel was tested by ELISA method, and the result showed no significant difference between inoculated group and control group in 2h, 4h, 6h, 10h, 11h, 17h, 24h, 40h, 48h, respectively. The distribution of IL-1βin spleen, bursa and lung in infected group was observed by SABC immunohistochemistry method. The IOD value of lung and bursa in infected group was significant higher than control group (P<0.01), and spleen in infected group was higher than control group (P<0.05). The high levels of IL-1βand no correlation with the release of TNF-αinto serum indicated that, when ducklings infected with P.multocida, the well documented iter in gram-negative bacteraemia:"LPS—TNF-α—IL-1β"wasn't as a phlogogenic factor in the infection. The same result presented in the study of bovine infectious pleuropneumonia induced by P.multocida.