The Tiger shrimp was administered by OMP intramuscularly (IM) at 10, 20, 30 μg/kg bw. The THC was collected every two days from day 2 until day 14 after imunization. The data showed that after day 2 administered with OMP decreased THCs compared to control (day 0) and increased again at day 4 and day 6. Futhermore THCs decreased again in day 8, after second imunization, and then increased at day 10, day 12 and day 14. This result indicated that administration OMP 20 μg/kg bw stimulated the highest THC in tiger shrimps compare to other doses (Figure 1). This phenomenone was similar to Johansson statement, stated that ß-glucan treatment as immunostimulant could decrease haemocyte at the moment and then increased dramatically (Johansson et al. 2000). (Yeh et al. 2005) reported that administration of Sargassum duplicatum extract could increase haemocyte of Litopenaeus vannamei. Haemocyte is part of cells in haemolymph, it has function to protect infectious disease as an immune response of the invertebrate organism (Van de Braak, 2002).
Total haemocyte count
The total haemocyte count (THC) of tiger shrimp was significantly different with intramuscular administration of OMP and V. harveyi infections (P < 0.01). Greatly improved THC was observed in the treatment of OMP 20 μg with THC value of 317.5 × 104 cells/ml (Figure 2).
The haemocyte circulation system may cause modulation of THC. Indeed, invertebrates (including shrimp) blood circulation system is open while haemocytes are distributed in both the vascular system and tissues. Consequently, an increase of THC values was assumed from either proliferation or movement of cells from tissues into haemolymphs. The concentration of cells varied as function of the development stage of an intermoult cycle of P. japonicus post larvae (Tsing et al. 1989). A decrease of THC may be due to cell lysis or increased movement of cells from haemolymph to tissues (Pipe and Cole, 1995). The decreasing of THC is also presumably due to the altered physiological conditions of the shrimp, as enhanced susceptibility to bacterial infection (Ford et al. 1993).
Differential haemocyte count
The DHC (hyaline, semigranular, granular) of tiger shrimp were given intramuscular administration of OMP and V. harveyi infections, where hyaline, semigranular, and granular were significantly different among five treatments determined by one-way ANOVA (Figure 3). Phagocytosis is believed to be one of the major cellular defence mechanisms in crustaceans. The semigranular haemocytes are the primary cells involved in the phagocytosis of foreign particles in shrimp (Bachère et al. 1995). The similar morphological haemocytes were also reported as haemoblasts in Tapes philippinarum (Matozzo et al. 2008) and Saccostrea glomerata (Aladaileh et al. 2007b). This cells were identified with different functions which do not play a role in defensive responses like phagocytosis or encapsulation, and they lack the common intracellular enzyme systems associated with host defense, unlike hyaline and granular (Aladaileh et al. 2007b).
Granular haemocytes are also capable of phagocytosis of foreign material but with less frequency than the smaller ones (Hose and Martin, 1989). Granular cells have been proven to play a significant role in the shrimp defence system because of their antibacterial activity (Chisholm and Smith, 1995). Granular played a role in phagocytosis, which may be correlated by their capacity for intracellular killing. Moreover, several studies showed that granular contain high levels of acid phosphatase and phenol oxidase enzymatic activities, as well as the ability to form superoxides and peroxides (Xue et al. 2000). The other haemocytes are the hyaline cells. They are also considered as phagocytes and superoxide anions production (Söderhäll and Cerenius, 1992).
The results of the study showed that treatment significantly increased the percentages of hyaline and decreased the percentages of semigranular and granular cells in the haemolymph. It was presumed that the young are generally known as the hyaline cells and that those cells gave rise to two haemocytic developmental series, i.e. the large- and small-granular cell line. Granular cells of the large-granular cell line mature and accumulate in the connective tissue, and many cells of the small-granular cell line were located in the lymphoid organ (Van de braak, 2002). Decreases in the percentages of semigranular and granular cells were compensated for by a proportional increase in the percentages of hyaline population. In the present study, haemocytes may display recruitment of immature cells from haematopoietic tissues (Hine, 1999). (Aladaileh et al. 2007a) predicted that the increased percentages of particular haemocyte types were due to induced cellular proliferation, recruitment of cells from non-circulating compartments of the haemolymph, or rapid cellular differentiation in response to antigenic challenge.
Total plasma protein
The total plasma protein (TPP) or haemolymph protein activity of black tiger shrimp was significantly different with intramuscular administration of OMP and V. harveyi infections (P < 0.01) (Figure 4). A Great improved TPP was observed in treatment of OMP 20 μg with TPP value of 16.56 mg/ml (Figure 4).
The TPP has been observed in previous studies on shrimp. The changing of TPP composition in the blood was affected with animal size, sex, nutritional state, environmental factors (such as temperature, salinity) and the moult cycle but this was inversely related to haemolymph volume (Chen and Cheng, 1993) and a decrease of TPP during infection as well as during repeated haemolymph sampling (Ford et al. 1993). In this study, TPP increased with treatment of OMP-10 and 20 μg/kg bw but decreased during infection of bacteria and treatment OMP-30 μg/kg bw. Those were similar to (Chen et al. 1994), the TPP increased with treatment of 10 and 20 ppt and decreased with treatment of 30 ppt.
Superoxide dismutase and protease enzyme activity
The humoral parameters are SOD and protease activities of tiger shrimp. The SOD and protease activities were significantly different with intramuscular administration of OMP and V. harveyi infections (P < 0.05) (Figure 5). Greatly improved SOD activity was observed in the treatment of OMP 10 μg with SOD value of 139,09 unit /mg protein and protease activity of OMP 20 μg with protease activity value of 130,17 unit/mg protein (Figure 5).
Concern to haemocytes function, superoxide anion produces toxic oxygen metabolites. Production of toxic oxygen metabolites, such as protease and reactive oxygen species (ROS) activation anion superoxide/O2
-, hydrogen peroxide/H2O2, ion hydroxide/OH- and oxygen/O2 (Campa-Cordova et al. 2002) is believed to be mediated through phagocytosis, encapsulation, aggregate nodulation, melanocytes and cytotoxicity which destroy invasive pathogens (Rodriguez and Moullac, 2000), thus providing an explanation for the combined suppression of phagocytosis and superoxide anion (Anderson et al. 1992).
The levels of protease activity were enhanced, caused by intramuscular administration of OMP and V. harveyi infections in tiger shrimp. The protease enzyme has a role to play as a lysosomal enzyme and prophenoloxidase (proPO) activator to become a phenol oxidase (PO) enzyme (Van de Braak, 2002). During that process, it was followed by phenol oxidation to become quinone that is antibacterial molecules (Smith et al. 2003). (Yeh et al. 2005) said that immunostimulants could enhance Phenol oxidase enzyme activity. Similar study in Biomphalaria glabrata reported that serine protease enzyme activity enhancement was followed by phenol oxidase enzyme activity enhancement (Bahgat et al. 2002).
On the other hand, an alteration in the levels of superoxide anion caused by contaminant exposure has been well studied in invertebrates (Wootton et al. 2003). A study on bivalves reported that bivalves’ superoxide generation is generally enhanced with low concentrations of contaminant exposure, but inhibited at higher concentrations (Dyrynda et al. 2000). In the present study, intramuscular administration of OMP and V. harveyi infections presented significant differences from control to produce protease and superoxide anion.
Mortality
Initial mortality tiger shrimp occurred on the next day after challenge. The highest cumulative mortality (46%) was observed in tiger shrimp with the control infection (OMP-free infection) (Figure 6). While shrimp with various OMP injection had reduced mortality until of 6-13%. Similar results had been reported in previous studies on shrimp when using immunostimulant (beta glucan) (Huang et al. 2006). In this study, treatment of OMP-20 μg/kg bw by intramuscular injection showed the most efficient and desirable influence on resistance of tiger shrimp to V. harveyi. However, treatments of OMP-10 μg/kg bw and OMP-30 μg/kg bw reduced of mortality of tiger shrimp but they were not more efficient than treatment of OMP-20 μg/kg bw. It probably concluded that the beneficial effect of OMP of disease resistance of tiger shrimp is dose-dependent and OMP-20 μg/kg bw is an optimal dose after two times of boosters of OMP for 14 days against V. harveyi infection for 24 hours.