Pathogen Biology Of Mass Mortiality Of Blood Clam Tesillarca Granosa And Immunological Characteristics Of Host Blood Cells

Blood clam, Tegillarca granosa, is an important economic shellfish. But diseases of blood clam have become increasingly severe issue. The mass mortalities occurred frequently in recent years and resulted in large losses. Therefore, it is very important to investigate the cause of mass mortalities and study immunological prevention for promoting the healthy development of the blood clam aquaculture. In this study, the causes of mass mortality of blood clam in the Yueqing Bay were investigated using epizoological survey methods and experimental study, and the immunological features of hemocyte cell from the blood clam were studied using light microscopy, electron microscopy, flow cytometry and enzyme technology. In addition, an aspartate racemase molecule was identified in the hemocyte cell of blood clam.1. A series of mass mortalities of the cultured blood clam, Tegillarca granosa, occurred in the Yueqing Bay of China from2005to2009. An obligate intracellular prokaryote, designated as rickettsia-like organisms (RLO), was frequently found in the moribund or dead blood clam sample during ultrastructural examination. These organisms were usually round, ellipsoid or occasionally dumbbell-shaped, ranged from approximately0.28to0.71μm in size and had a trilaminar cell wall. Two reproductive modes of organisms, transverse binary fission and budding, were observed. The organisms are able to form eosinophilic inclusions. Most inclusions were found within epithelial and connective tissues of the mantle, gills and digestive tube. The biological and morphological characteristics indicate that these organisms may belong to the family Rickettsiaceae. RLOs exhibited significant pathogenicity. Cytopathological examinations revealed extensive necrosis and destruction in the infected cell. The degree of tissue destruction was positively related to the number of RLO inclusions in the tissues, and the cytopathological effects were positively related to the number of intracellular RLOs. RLOs and their inclusions were discovered throughout different epizoological areas and in different periods of time. The infection intensity of the RLOs was positively correlated with the mortality rate of clams. Therefore, RLO infection might be associated with mass mortalities of cultured blood clams in the Yueqing Bay.2. Light, electron microscopics and flow cytometer studies were conducted to characterize morphology and structure and phagocytosis of haemocytes in blood clam Tegillarca granosa. Based on cell size, and microstructure and granular types of cell, blood clam haemocytes were divided into red granulocyte (89.67%), basophil granulocyte (7.05%) and hyalinocyte (3.28%), respectively under light microscopy. The haemocyte densities in healthy blood clam haemolymph were assessed3.29±0.82×106cell/mL. Three kinds of haemocytes were respectively recognized by transmission electron microscope:granulocyte type Ⅰ, granulocyte type Ⅱand agranulocyte. It was observed under scanning electron microscopy that haemocytes appear smooth-surface and some of them have knot-like projections in cell surface. APIZYM assays on enzymatic activities of19enzymes in haemolymph were performed and, of these,13and10enzymes were detected, respectively in serum and haemocyte cell. There were marked increases in the levels of ALP, ACP, ELIP (C8) and AGLA in serum and haemocytes after stimulated by zymosan. Observation of phagocytosis in vitro revealed that phagocytic response in red granulocyte to zymosan and there was no significant phagocytic response in basophil granulocyte and hyalinocyte. It was observed that basophilic granulocyte accumulation and hyalinocyte adsorption after stimulated by Aeromonas hydrophila. The study of environmental stress factors on phagocytosis activity revealed that the temperature and salinity have significant influence on phagocytosis activity. The phagocytosis percentage of blood clam reached the highest values with optimum growth temperature (13-30℃) and salinity (20.0-26.2ppt).3. An aspartate racemase (AR) gene was cloned and identified from blood clam. ORF sequence length of blood clam AR gene is1017bp, encoding338amino acids, predicted molecular weight of37.1kDa and theoretical isoelectric point of8.62for the basic protein. Sequence analysis revealed homology of amino acid sequence between the blood clam AR and Burnt-end Ark AR. Compared with sea urchin and sea squirt AR, homology of amino acid sequence has reached51%and41%. Cluster analysis showed that blood clams AR is able to cluster all reported AR. Blood clam AR contains a5’sphate pyridoxal (PLP) binding motif. It is suggested that the role of this molecule requires the presence of PLP.