Isolation And Identification Of Bacterial Pathogen And The Immune Responses Of Giant Salamander To Inactivated Aeromonas Hydrophila

Chinese giant salamander (Andrias davidianus), namely Wawa-yu, is one of the rare animals of China and is currently listed in annex I of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) and in class II of the national list of protected animals in China, which is widely distributed in Sichuan?Hubei?Hunan?Guizhou and Shanxi provinces, etc. In the last decade, the artificial breeding and aquaculture of this species has gained considerable development and promoted the rapid development of the industry of farming giant salamander. Alongside the increase of intensive aquaculture of this species, diseases are becoming more and more serious and have caused huge economic loss.In this study, a strain of bacterium was isolated from the liver of diseased Chinese giant salamander and identified by the Biolog Microbial Identification System and 16S rDNA sequence analysis. Healthy Chinese giant salamanders were experimentally infected with the bacterial suspension. Thus, a bacterial strain named YZO1 was isolated from diseased giant salamander. Identification by the Biolog Microbial Identification System,16S rDNA sequence and phylogenetic analysis demonstrated that the bacterial pathogen YZOl is Plesiomonas shigelloides, which has been recovered and confirmed from the experimentally infected animals. The susceptibility test to antibiotics demonstrated that the bacterial strain YZOl was susceptible to ceftriaxone, cefotamine, chloramghenicol and zinacef, etc.Formalin-killed Aeromonas hydrophila cells (F-AH) were used as immunogen to immunize healthy giant salamander(Andrias davidianus) via intraperitoneal injection, the periphery blood samples were collected at d 1, d 4, d 7, d 14, d 21 and d 28 post-immunization for the measurement of the responses of immune-related parameters, including the blood cell counting, classification, phagocytic percentage, phagocytic index and serum agglutinating antibody assay, etc. Compared with the control group, the results showed, that the number of hematocytes in peripheral blood of immunized animals increased significantly at d 4, d 7, d 14 and d 21 post-immunization, in which, the number of erythrocytes and leucocytes reached the maximum value of 7.83×107/ml and 6.74×106/ml at d 4 post-immunization, respectively, the neutronphil percentage reached the maximum value of 28.60% at d 4 post-immunization; The percentage of monocytes reached the maximum value of 10.53% at d 7 post-immunization. Meanwhile, the phagocytic percentage (PP) and phagocitic index (PI) increased significantly and reached the maximum value of 34.09% and 3.73 at d 4 post-immunization, respectively. The percentage of lymphocyte and the serum agglutinating antibody titers increased significantly, and both were peaked with 75.30% and 1:426.67 at d 21 post-immunization, respectively. The result of challenge test with live Aeromonas hydrophila revealed that the relative percent survival (RPS) of immunized group was 69.23%. All the results here demonstrated that the F-AH enhanced the immunity of giant salamander by increasing the number of erythrocytes and leucocytes in peripheral blood, improving the activity of phagocytes and producing the specific antibody.The formalin inactivated Aeromonas hydrophila (1×10 cfu/ml) was prepared and mixed with propolis-adjuvant, freund's-adjuvant and aluminum-adjuvant to immune healthy giant salamander via intraperitoneal injection, respectively, and no-adjuvant group and control group were set up. The periphery blood samples were collected from the immunized giant salamanders at d 1, d 4, d 7, d 14, d 21, d 28 and d 35 post-immunization for the study of the serum agglutinating antibody assay. The challenge test with Aeromonas hydrophila was performed at d 35 post-immunization. Compared with the control group, the results showed that, the serum agglutinating antibody titers of the propolis-adjuvant group peaked with 1:469.33 at d 21 post-immunization and the Freund's-adjuvant group reached the maximum value of 1:448, the aluminum-adjuvant group reached the maximum value of 1:362.67 at d 28 post-immunization, and the no-adjuvant group was peaked with 1:384. The result of challenge test with Aeromonas hydrophila showed that the relative percent survival of the propolis-adjuvant group, freund's-adjuvant group, aluminum-adjuvant group and the no-adjuvant group were 88.9%,77.8%,72.2%and 61.1%, respectively. Thus, the results demonstrated that the immune protection of bacterial inactivated vaccine could be improved by the immunoadjuvants, especially the propolis-adjuvant.