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Volume 6, Issue 1, February Issue - 2018, Pages:236-242

Authors: Devadason C*
Abstract: Lysozme, a humoral defence protein, played an important bactericidal activity in Atlantic salmon and halibut. Turbidometric and bactericidal killing assays were used to determine the lysozyme level and serum killing activity respectively. A challenged experiment in native halibut (Hippoglossus hippoglossus) with different doses (103,105, 109) of Aeromonas salmonidae (MT423) has been proved that there is no significant relationship between the lysozyme level and bactericidal killing activity (p>0.005). Lysozyme level in halibut serum was significantly higher than that of Atlantic salmon serum. Lysozyme activity of serum collected from fish during the summer was found to be significantly higher, ranging from 650 to 850 µg.  Halibut serum showed varying level of killing activity (KI) during summer (0.266-0.873) and winter (0.255-1.344) whereas Atlantic salmon had very poor killing activity                    (0.414 -6.867). There was no correlation between the lysozyme activity of the serum and bactericidal activity in the serum of A. salmonicida infected halibut.
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Full Text: 1 Introduction Fin fish and shell fish farming have been commercialized to fulfill the protein requirement of rapidly arising population and is one of the fastest growing food production sectors in the world, but infections caused by bacteria, viruses, and parasites cost the industry billions of dollars in worldwide losses each year (Ugelvik et al. 2017). Salmon louse infection and economic losses reported by Costello, (2009). Lysozyme is considered to play an important role in humoral defence mechanisms in fish (Lindsay, 1986). These lysozyme are associated with innate immunity, which forms the first line of defense against infections, however, its primary role is lysis of the bacterial cell wall and opsonization (Alhazmi et al., 2014). Lysozyme (mucopeptide-N-acetyl muramylhydrolase) is a low molecular weight cationic protein which hydrolyses N-acetylmuramic-b-1-4-acetyly glucosamine linkages in bacterial cell wall (Ossermann &  Lawlor, 1966) particularly sugars moiety of the peptidoglycan of the cell wall of gram (+) ve bacteria causing lysis and seems likely that lysozyme may also play a role in the destruction of some gram (-)ve bacteria (Grinde, 1989). These lysozyme has been detected in blood, mucus and phagocytic cells of fish (Fletcher &White,1973; Studnicka et al., 1986).  Further, its presence in the blood of invertebrates was also reported by Barnes (1974). Cheng et al. (1977) used the tubidometric assay to determine the lysoszyme activity in the hemolymph of Biophalaria glabrate challenged with heat killed Bacillus megaterium. Similarly, Fletcher &White (1973) and Murray & Fletcher (1976) found that lysozyme was consistently present throughout the year in the sera of flat fish, including plaice (Pleuronectes plattessa L.), flounder (Platichthys flesus L) and turbot (Scophthalmus maximus L). Lysozyme levels have been increase to response of microbial attack and physical stress (Ingram, 1980). Mock &Peters (1990) reported reduction in lysozyme concentration in response to stress in rainbow trout. Reddy et al. (2004) and Wang et al. (2005) are employed to the study of lysozyme activity and serum bacterial killing activity (Ourth &Wilson, 1981) and macrophage killing activity (Sharp & Secombes, 1993). Lysozyme isolated from rainbow trout (Oncorhynchus mykiss) serum also have bacteriacidal activity against a range of Gram (-)ve bacterial pathogens of fish, including Vibrio salmonicida, Aeromonas salmonicda and Yersinia ruckeri (Grinde, 1989; Rainger &  Rowley AF, 1993). A. salmonicida, the etiological agent of furnculosis in trout and salmon, has been a worldwide economic threat to intensive culture of the commercial valuable fish for over fifty years (McCarthy & Roberts, 1980). A. salmonida  subsps  salmonicida is gram (-)ve bacteria , non-motile, fermentative rods which produce cytochrome oxidase and catalase. It can be differentiated on the basis of their biochemical properties and the ability to produce a brown water-soluble pigment when culture on media containing tyrosine (Austin &  Austin, 1987). This study was designed to understand the antibacterial properties of lysozyme against Aeromonas salmonicida gram (-)ve virulent bacteria. 2 Materials and Methods 2.1 Collection of Serum and Mucus from healthy fish  The healthy halibut (n=11) and Atlantic salmon (n=11) were obtained from the Scottish office of Agriculture Environment and fisheries department (SOAEFD) field station, Scotland  and were lightly anaesthetized by immersion in benzocaine (ethyl-p-aminobenzoate). Samples of mucus were scraped from the dorsal surface using a scalpel and store at -200C until use. Blood samples were taken from the caudal vein using a 23G needle and syringe and allowed to clot at room temperature (RT). The blood samples were then centrifuged at 4000g for 15 min, the serum collected and stored at -200C until required for assay. 2.2 Infectivity trial Two year old halibut, weight range from 250g-300g were reared in seawater in 1 m diameter plastic tanks. They were fed daily on commercial dry pellets and appeared to be healthy with no signs of disease. The effect of intraperitonial injection of three doses of A. salmonicida viz., 109, 105, 103 cells/fish was examined. There were four fish per test and control group fish which were injected with sterile phosphate buffer solution (PBS). Each group of fish was maintained in seawater in separate 0.75m diameter circular tanks with continuous aeration. To reduce the risk of cross -contamination, the tanks were covered with polythene sheeting. The temperature of the seawater was monitored throughout the experiment. Prior to the infection of bacteria, the fish in each group were lightly anaesthetized as described above, and a 1 ml sample of blood was taken from the caudal vein using 23G needle and syringe, to enable measurement of pre-infection levels of lysozyme. Each fish was then individually dye-marked using a pan-jet loaded with alcian blue, and injected intraperitonially with 100microlitre of the appropriate dose of A. salmonicida for test fish, or sterile PBS for control fish. Blood samples were taken as described above, after 4 and 8 hrs. After 24hrs the fish were killed and bled using a vacutainer and needle (Greiner). The blood samples were allowed to clot at RT, and were then centrifuged at 3000g for 10 min to collect the serum. Samples were stored at 40C during the trial, and on return to the laboratory, were stored at -200C until required for assay. The serum samples from individual fish in each group were assayed for lysozyme activity and bactericidal activity using the turbidometric assay and serum bactericidal assay Pech (1985). 2.3 Turbidometric Assay This assay was a modification of the method of Parry et al. (1965), which is based on a decrease in optical density (OD) or absorbance due to the lysis of bacterial cells by lysozyme. Before this assay was applied, it was optimized. Micorcoccus lysodeikticus (80mg/ml in 0.05M sodium phosphate buffer, pH 6.2) was added to varying concentration of hen egg white lysozyme as four replicates in a microtitre plate. Sterile PBS added Micorcoccus lysodeikticus was used as a negative control and sterile PBBS alone as a blank. The decrease in OD per minute, measured at 540nm and 370C was recorded using a Dynatech MR 7000 microplate reader. Lysozyme was measured in terms of units, where one unit is equivalent to a decrease in OD of 0.001 per minute (DOD/min =0.001). Lysozyme activity of serum and mucus was determined using this assay. Sterile twofold dilutions of each serum and mucus samples, range from 1:5 to 1:5120, at 100microlitre per well were set up. The Micorcoccus lysodeikticus at a concentration of 0.4mg/ml was dispersed by a Dynatech MR 7000 reagent dispenser at 100microliter per well. The plate was shaken for 20 sec and the OD read at 1 min intervals at 540nm and RT over a 5 min period. 2.4 Culture of Aeromonas salmonicida (MT 423) A. samonicida (MT423) , which is highly virulent , A layer possessing form, was obtained from the SOAEFD, Aberdeen , and cultured on tryptone soya broth (TSB, Oxoid) was seeded from the agar culture, and incubated at 220C for 16 h to reach log phase growth. The bacterial cells were pelleted by centrifugation at 2000g for 10 min at 220C, and washed twice in sterile PBS. The cells were then resuspended in sterile PBS and aggregated bacteria were dispensed by passage through a 26G needle. The bacterial suspension was measured spectrophotometrically and adjusted to give a density of 109 cells/ml in PBS (OD=1.0at 540nm). 2.5 Serum Bactericidal Assay This assay utilizes the reduction of the tetrazolium dye MTT by bacterial dehydrogenases as an indicator of bacterial viability. This reduction is directly proportional to the number of viable bacteria present. Measurement of a decrease in MTT reduction after incubation with serum is an indication of serum killing. Each serum sample to be tested diluted as 1:10 in sterile PBS and heat inactivated samples of each sera (500C for 1hr) in duplicates at a volume of 50 microliter were added respective wells in the microtitre plate. Once serum samples were loaded, the microtitre plate was exposed to UV radiation for 1 hr to sterilize the serum and reduce the risk of bacterial contamination. Following this, 100 microliter of the bacterial suspension was added to each test well using the Dynatech MR7000 reagent dispenser and the plate was shaken for 20s. A 100 microliter replicates of 1:10 serum diluted with PBS and 100microliter replicates of bacteria suspension were used as controls. A 150microlitre replicates of sterile PBS was used as blank. For each assay, two identical plates were prepared, plate A 0hr and plate B for 1 hr were incubated at 180C. After the set incubation time, the microtitre plate was read at 630nm on a Dynatech MR 7000 microtitre plate reader to measure the background absorbance. Immediately, 10microlitre of MTT (5mg/ml in dH2O ) was added to each well, the plate was shaken, and after exactly 15 min , OD  was recorded. The reduction in OD after 1 hr is an indication of the killing potential of the serum (Pech, 1985). The killing index (KI) of each serum was calculated from the equation KI=MTT reduction after 1hMTT reduction after 0hr Where KI is less than1.0, the serum show the killing activity 2.6 Statistical Analysis Means and standard errors of the means were calculated for each data set and compared using the Student’s t test. A one-way analysis of variance was used to analyze the data for degree differences between population data for lysozyme activity. Significance was measured at a P<0.05 level. 3 Results The lysozyme and bactericidal activities of the serum of Atlantic salmon and halibut showed no significant correlation between both fish (P>0.01). The lysozyme activity of serum of non-immunized salmon showed variation in serum lysozyme activity between the individuals measured. Of the fish examined (n-11), seven showed low lysozyme activity from 80µg to 128 µg whereas halibut serum collected from the fish during winter showed between 84-276µg. In comparison, the lysozyme activity of serum collected from fish during the summer was found to be significantly higher in all samples measured (n=11) , ranging from 650 to 850 µg( Table 1) Of the salmon serum tested, only four serum samples showed bactericidal activity (KI<1.0), ranging from 0.90 to 0.40, and the excessively high index may have been due to experimental variation of error within the assay. Halibut serum collected in winter showed bactericidal activity, with killing index of around 0.60 to 0.25 and three samples did not exhibit serum killing. In comparison, serum collected in summer showed bactericidal activity of varying levels, with killing index of from 0.09 to 0.25 (Table 2). At pre-injection level, all fish showed high levels of lysozyme activity of around 750 µg, the control fish showed remained constant 4 hr after injection with sterile PBS and decreased significantly after 2h around 350µg (Table3). The lysozyme activity of serum of the fish infected at a dose of 103 cells/fish showed significant increases in lysozyme activity, after   Table 1 Lysozyme level (µg) and the serum bactericidal activity (KI) in halibut ±serum collected during winter and summer.  
Winter Season Summer Season Number Lysozyme (µg) KIndex (KI) Lysozyme (µg) K. Index (KI) 1 91±05 0.507 748±07 0.722 2 276±04 1.344** 732±03 0.658 3 276±.02 0.552 756±05 0.357 4 268±00 1.166** 648±02 0.573 5 126±10 0.403 728±04 0.873 6 119±11 0.336 640±06 0.266 7 244±01 0.627 756±12 0.426 8 84±04 1.152** 736±15 0.717 9 142±06 0.365 704±13 0.293 10 119±07 0.357 732±19 0.329 11 144±12 0.255 652±22 0.656            
Value presented mean± SD, ** indicates the non-killing activity of serum, K<1 -Killing activity of serum   Table 2 Lysozyme level (Microgram) and serum bactericidal activity (KI) of naïve Atlantic salmon serum  

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