Studies on haemolytic response of crustacean haemolymph against pathogenic bacteria on blood agar

Abstract

Nonspecific immune responses play very important role in crustacean immune system defending from external pathogens. Haemocytes particularly involve in vital physiological functions such as repair of wounds, transportation of food materials, coagulation of haemolymph, phagocytosis, nodule formation, encapsulation of bacteria and activation of prophenol oxidase system. It is essential to determine the role of haemocytes when pathogenic bacteria get encountered to an animal. Haemolysis of blood cells can serve as an evidence for the better understanding of crustacean immune responses. In this study, haemolymph collected aseptically form Litopenaeus vannamei and Scylla serrata was used for preparing blood agar plates to study haemolytic activity against aquatic and human pathogens. Haemolymph (5 per cent) was supplemented to the basal medium for preparing blood agar plates with the addition of rose bengal dye. The following type cultures are used for the study viz., Edwardsiella tarda, Aeromonas hydrophila, Enterobacter cloacae, Enterobacter cloacae, Enterobacter cloacae, Staphylococcus aureus, Vibrio cholerae and Salmonella paratyphii. The overnight grown cultures was harvested, the optical density (600 nm) adjusted to 0.5 and was added to the freshly prepared plates in duplicate. The plates were incubated at 280C for 48 h. Results obtained from haemolytic activity of crustacean haemolyph were compared with healthy human blood. A clear zone of haemolysis was observed for all the aquatic bacterial pathogens on both shrimp and crab blood agar plates, where no hemolysis was observed for human pathogens. In case of human blood agar, all the isolates produced clear zone of haemolysis. This result shows that specificity of host-pathogen relation towards the crustacean haemocytes and is most accurate method of haemolysis determination. In conclusion, haemolytic assay employing crustacean haemolymph can be used to evaluate the mechanism of host specificity for invasion by the aquatic pathogens.