The Immunological Responses And Protection In Chinese Giant Salamander, Andrias Davidianus Immunized With Inactivated Iridovirus

Chinese giant salamande（rAndrias davidianus）, namely Wawa-yu, is one of rareanimals in China, and has high consumpion, research and medicinal values, which iscurrently listed in annex I of the Convention on International Trade in EndangeredSpecies of Wild Fauna and Flora (CITES) and in class II of the national list ofprotected animals in China. Chinese giant salamander iridovirosis is a newly emergedinfectious disease and has caused huge economic losses. The causative pathogen hasbeen identified as the giant salamander iridovirus (GSIV). Vaccination has beenproven to be an effective method in fish for preventing diseases. This study wasdesigned to investigate the immunization dose of invactivated vaccine, differentadjuvents, the immunological responses and protection in Chinese giant salamander.The aim of this study is to establish a technology for the prevention and control ofgiant salamander iridovirosis. The contents and results of the study are listed asfollows:1. Eighty giant salarmanders were seperated randomly into four groups andimmunized with different dose of inactivated GSIV. Then, the periphery bloods fromthe Chinese giant salamanders were collected at7,14,21,28days after immunization.At30days post-immunization,100μl of live GSIV was injected intraperitoneally intothe animals (1.0ⅹ106TCID50ml-1per animal) in each group. Mortality wasmonitored over a period of14days, and protective efficency was evaluated. Theresults demenstrated that the Chinese giant salarmanders immunized with1.0ⅹ106TCID50ml-1inactivated vaccine obtained a relative survival of72%. The results ofthis experiment provide a basis for determining the immunizing dose of GSIVinactivated vaccine.2. In this study, the immunological responses and protection in Chinese giantsalamander intraperitoneally immunized with-propiolactone inactivated GSIV areconducted. Periphery blood and tissues from the Chinese giant salamanders were collected at day1,2,4,7,14,21and28post immunization. Then, red and whiteblood cell counting and classification, phagocytic activity, neutralizing antibodytitration, immune-related gene expression and determination of the relative percentsurvival were evaluated. The red and white blood cell counts showed that the numbersof erythrocytes and leukocytes in the peripheral blood of immunized Chinese giantsalamanders increased significantly at days4and7post-immunization (P＜0.01).Additionally, the differential leucocyte count of monocytes and neutrophils weresignificantly different compared to the control group; the percentage of lymphocyteswas70.45±7.52%at day21. The phagocytic percentage and phagocytic index was38.78±4.33%and3.75±0.52at day4post-immunization, respectively, which wereboth significantly different compared to the control group (P＜0.01). The serumneutralizing antibody titre increased at day14post-immunization and reached thehighest titre (341±9.52) at day21. The quantitative PCR analysis revealed that theimmunization significantly up-regulated the expression of immune related genesTLR-9and MyD88in the first two weeks after immunization. The challenge testconducted at day30post-immunization demonstrated that the immunized groupproduced a relative survival of72%.3. Aluminum gel and Freund’s complete adjuvant were added in the GSIVinactivated vaccine respectively. PBS diluted vaccine and unimmunized groupconsisted of the two control groups. The antibody titres were analyzed byneutralization assay during1～6weeks after immunization. The challenge test wasconducted at day60post-immunization, mortality was monitored over a period of14days, and protective efficency was evaluated. The result showed that the group ofFreund’s complete adjuvant produced a higher antobody titer （640±15.52）,significantly higher than the aluminum gel and control groups, and could sustained fora longer time, especially. The relative survival reached90%, which was higher thanthe aluminum gel (84%). The test for the GSIV vaccine provides useful informationfor the selection of different adjuvants in the application of GSIV inactivated vaccinein practice.