| In recent years, large scale of death continually attacked Charybdis japonica which were kept in the Zhoushan for temporarily rearing, the dying crab exhibited white haemolymph which looks like the milk, the symptoms resemble the "milk sickness" previously reported on swimming crab Portunus trituberculatus. To find out the cause of this disease, we conducted studies on pathological observation, pathogen isolation and identification, immune-related indexes detection, and the results are as follows:Pathological observation:(1) Charybdis japonica suffered from the "milk sickness" showed sluggish and loss of appetite, and, while removing the back carapace, white milky haemolymph could be seen in the cavity of the shell;(2)Histological section observation showed various degree of pathological changes in hepatopancreas, gill, muscle and myocardial tissue of diseased crab including: cell swelling, degenerating or even necrosing,some of the cell nucleus showed pycnosis, cataclastic disappear or dissolved, while some of the basal membrane ruptured, and the cell boundaries blurred;(3)Through electron microscopy, a large number of rod shaped pathogen with the similar size of WSSV were observed in haemolymph, hepatopancreas, gill and heart of the disease crab. Meanwhile,pathogen particles purified from the haemolymph of the disease crab by density gradient centrifugation turned out to be full of rod virus-like pathogen under negative staining electron microscope.Pathogens isolation:(1) Microbial culture: Hepatopancreas and haemolymph of disease crab were used to inoculate by on the TSB and TCBS plates, but failed to isolate any suspicious pathogenic bacterium;(2) PCR identification: we extracted the genome DNA from the tissue of diseased crab, and then used 3 pairs of WSSV specific primers to detect the samples, the results turned out to be WSSV positive;(3) Western Blot assay: we used WSSV specific antibodies to detect the purified pathogen particles via Western Blot assay, and specific immune binding strips were observed.(4) Recursive infection trial: The artificial mock infection trail found that, compared with the control, the group injected with fluid of diseased crabs exhibited apparent higher mortality, and, the mortality is positively correlated with the concentration of injected solution, in addition, we identified WSSV from the mock infected crabs, however, the artificial mock infection failed to replicate the symptoms of the "milk sickness".Detection of immune-related index: We detected the immune-related index of both the healthy and diseased crabs, we found that the activity of several enzymes including ACP,AKP, T-SOD and POD on the healthy crabs were higher than that on the diseased ones,meanwhile, of all the 12 diseased crabs detected in our trial, 3 of them exhibited lower T-AOC activity compared with the healthy,while the rest 9 showed higher T-AOC activity.From all the results mentioned above, we could see that compared with the healthy individuals, the diseased crabs showed varying degrees of rise on immune-related enzyme activities, thus suggest the invasion of pathogens has triggered a stress response immunity inside the host bodies.In summary, we identified high concentrations of WSSV from Charybdis japonica suffered from "milk sickness" by electron microscopy, PCR and western blot, recursive infection trial also showed that WSSV existed in the hemolymph of crab post-infected, thus suggest the relevance between WSSV and crab sickness; Given the result that recursive infection failed to replicate the symptoms of "milk sickness" on the dying crab, the correlation between WSSV and "milk sickness" need further prove. |
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