Identification of immune cells interacting with Vibrio spp. and its in vitro post-phagocytic killing mechanism of haemocytes in the penaeid shrimp, Penaeus indicus H. Milne Edwards.

Immune cells were identified and their interaction towards Vibrio alginolyticus, V. parahaemolyticus and V. anguillarum was studied in vitro in the penaeid shrimp, Penaeus indicus. Haemocytes were divided into agranulocytes, semi-dense granulocytes and dense granulocytes according to their morphology. Agranulocytes (100%) and 0.3-0.7% of granulocytes were actively involved in coagulation. Granulocytes were involved in in vitro phagocytosis and encapsulation of foreign materials. Phagocytosis was enhanced by prior opsonization of bacteria with cell-free shrimp haemolymph. Semi-dense granulocytes were phagocytic towards V. alginolyticus with and without opsonization at the rate of 91.1% and 83.1%, respectively (P < 0.05). Granulocyte death observed after 2 h with opsonized haemolymph was 26.1%. About 64.5% of dense granulocytes and 23.2% of semi-dense granulocytes were actively involved in encapsulation, forming capsules. A spectrophotometric nitroblue tetrazolium (NBT) reduction assay was used to demonstrate the production of superoxide anions (O2(-)) by shrimp haemocytes. All the Vibrio spp. were able to induce superoxide anions (O2(-)) during phagocytosis. Live Vibrio sp. induced O2(-) production in haemocytes in a dose-dependent manner. Significant activity was detected with a 40:1 bacteria to haemocyte ratio (P < 0.05). NBT reduction assay for measuring the post-phagocytic killing mechanism in shrimp haemocytes might be a valuable tool for monitoring shrimp health and immunological studies.