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Figure 3 . Effect of antibody response to heat killed in (=6 fishes/group) by ELISA. Each value represents the arithmetic mean±SEM (*<0.05) of six fishes.

The number of antibody producing cells in response to SRBC was observed from 7 to 28days of feeding in all experimental diet groups ( Fig. 4 ). Among the experimental diet, a higher number of plaque forming cells showed in group C and it reached the maximum (61±4.4) at 21days of post immunization. The least number of antibody producing cells was observed in control (11±1.5) than group A (38.7±2.33), group B (45±3.4) and group C (47.3±5.9) at 28days of feeding.

Figure 4 . Effect of the number of antibody producing cells against SRBC in (=6 fishes/group) by plaque forming cell assay. Each value represents the arithmetic mean±SEM (*<0.05) of six fishes.

The spleen of C. catla is a reddish-brown, elongated, thick and flattened structure, lying along the intestine and near the pancreas, trunk-kidney. The spaces within the connective tissue framework are filled with splenic pulp, the distinction of which into red pulp and white pulp is difficult in histological preparations. The pulp in general consists of large venous sinuses and their branches, representing the red pulp (RP), white pulp (WP), nodule-like ellipsoids of lymphoid tissue and dark-staining melanomacrophage centres (MMC) ( Fig. 5 ). Heat killed A. hydrophila challenged fish spleen showed loosely packed red and white pulp (arrow) ( Fig. 5a and c ); macrophage aggregation can be viewed in Fig. 5b and collapsed MMC and progression of MMC are observed in Fig. 5d–h , which showed that the challenged fishes get immunized and develop their immunological parameters. Through this histological evidence it is proved that C. catla increased their immune response depending on the C. dactylon extract concentration.

Figure 5 . Light micrographs of spleen of . infected spleen shows loosely packed red and white pulp and collapsed MMC, which shows heavy infection. Arrows: melanomacrophage centres i.e., cluster of macrophage cells. RP : ; WP : ; MMC : ; BV : .

The present study was carried out to investigate whether the oral administration of ethanolic extract of plant C. dactylon mixed diet is capable of protecting C. catla against A. hydrophila . Therefore, the plant extract was used for oral administration with prepared diet in the laboratory and to challenge with A. hydrophila for studying the specific immunity on fish. In Indian major carp, outbreak of motile aeromonad septicemia due to the potential opportunistic pathogen A. hydrophila is an easily infectious bacterium, mostly when fishes are exposed to both physical and environmental stressors.

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