CHAPTER 6 IMMUNOMODULATORY EFFECTS OF DIETARY INTAKE OF CHITIN, CHITOSAN AND LEVAMISOLE. AND CONTROL OF AEROMONAS HYOROPHrA INFECTION IN PONDS

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1 CHAPTER 6 IMMUNOMODULATORY EFFECTS OF DIETARY INTAKE OF CHITIN, CHITOSAN AND LEVAMISOLE ON THE IMMUNE SYSTEM OF CYPRINUS CARPIO AND CONTROL OF AEROMONAS HYOROPHrA INFECTION IN PONDS

2 CHAPTER 6 IMMUNOMODULATORY EFFECTS OF DIETARY INTAKE OF CHITIN, CHlTOSAN AND LEVAMISOLE ON THE IMMUNE SYSTEM OF CYPRINUS CARPI0 AND CONTROL OF AEROMONAS HYDROPHILA INFECTION IN PONDS 6.1. Introduction Overcrowding tends to adversely affect the health of cultured fish. Warm water aquaculture in Asia has problems with bacterial diseases such as motile aeromonads septicaemia, furunculosis, columnaris, and edwardsiellosis. Among these, disease caused by Aerornonas hydrophila is most widespread in freshwater fish (Karunasagar et al., 1991). A. hydrophila is associated with disease in carp, eels, milkfish, channel cat fish, tilapia and ayu and produces stress related diseases in salmonids with the common symptoms of ulcerations, exophthalmia, abdominal distension etc (Amin et al., 1985; Miyazaki and Jo, 1985; Rahman et al., 1997). The use of antimicrobials for disease control and growth promotion in animals increases the selective pressure exerted on the microbial world and encourages the natural emergence of bacterial resistance. Vaccination may be the most effective method of controlling fish disease, even though disease caused by bacteria like A, hydrophila has not been controlled by vaccination due to their heterogeneity. However, when applied to hatchery conditions, some immunization techniques are not as effective as they should be. lmunostimulants may represent an alternative and a supplemental treatment to vaccination in the prevention of diseases in aquatic animals.

3 lmmunostimulants and immunomodulators comprise a group of biological and synthetic compounds that enhance the non specific cellular and humoral defense mechanisms in mammals. These substances, such as levamisole, p-glucan, peptidoglycan, chitin, chitosan yeast and vitamin combinations, as well as various products derived from plants and animals are effective in preventing disease (Baulney et al., 1996; Verlhac et al., 1998; Mullero et al., 1998; Kawakami et al., 1998; Estban et al., 2000; Ortuno et al., 2001; Miles et al., 2001; Estban et al., 2001; Villamil, 2003; Dautremepuits et al., 2004). Most of the research on immunostimulants has been focused on the treatment of tumors in human and animals (Nishimura et al., 1984; Azuma and Jolles, 1987). The basis for this approach in tumor therapy is the fact that natural or synthetic immunostimulants activate macrophages, neutrophils, natural killer cells and T-cell mediated immunity, thus increasing the natural ability to destroy tumor cells. lmmunostimulants also have the ability to increase resistance to viral, bacterial and fungal infections (Suzuki et al., 1984; Anderson, 1992; Siwicki and Dunier, 1994; Siwicki et al., 1994). Chitin is a natural polymer found abundantly in the shells of crustaceans, insects and in fungi. Chitin and its deacetylated product chitosan are commercially manufactured from shells of shrimp and crab. Chitosan has many applications in medicine, agriculture and aquaculture. In aquaculture, it is used as an immunostimulant to protect salmonids against bacterial disease (Anderson and Siwicki, 1994; Siwicki et al., 1994), enhancing the respiratory burst and phagocytic activities in gilthead sea bream (Esteban et al., 2000; Ortuno et al., 2000; Esteban et al., 2001; Cuesta et al., 2003), immersion and

4 dietary supplements (Kono et al., 1987; Kawakami et al., 1998). However, Shiau and Yu, (1999) reported depressed growth in tilapia after feeding chitin and chitosan. The present study was undertaken to evaluate the comparative efficacy of chitin, chitosan and levamisole on enhancing non-specific immunity as well as their effects on the immune response against A. hydrophila and growth of common carp under field conditions Materials and methods Bacterial strain A. hydrophila strain AH-PU 13 was isolated from diseased fish collected from local fish farm according to Shome and Shome, (1999). The species level identification of the strain was carried out by comparative biochemical tests (Joseph and Carnahan, 1994), and the polymerase chain reaction technique developed by Chilaka, (2001). Subcultures were maintained on Tryptone soy agar slopes (Hi-media, Mumbai, India) at 5OC and routinely tested for pathogenesis (Joseph and Carnahan, 1994) by inoculation into common carp through intramuscular injection (Davis and Hayasaka, 1983). A stock culture in Tryptone soy broth (Himedia, Mumbai, India) was stored at -70 C with 0.85% (w/v) NaCl and 20% (vlv) glycerol to provide stable inocula throughout the study (Chabot and Thunne, 1991 ; Yadav et al., 1992) Fish Fingerlings of common carp (C. carpio) at the size of c 2.2 g were brought from a fish farm in Kurunjipadi, near Pondicherry, India, and acclimated

5 in well water for a week followed by pond water for another week before start of the experiments. Fish were fed with diets prepared in the laboratory throughout the period of study. Five fish from the stock were randomly checked for any disease they are carrying. After ascertaining all the fish are healthy they were stocked into the ponds lmmunostimulant preparation Chitin and chitosan were prepared from shrimp shell waste according to Madhavan and Nair, (1974) with some modification (Gopalakannan et a!., 2000). Levamisole Hydrochloride was purchased from (Sigma, USA) Pond site selection Four ponds with 50 m length, 5.5 m breadth and 0.9 m depth were selected near Pondicherry, to conduct the field study, Initially soil and water quality parameters including temperature, ph, DO2, ammonia, nitrite, alkalinity and hardness were analyzed following the standard methods described by APHA, (1995) and Jackson, (1967). The pond was filled with well water followed by initial addition of organic fertilizers cow dung. For conditioning the pond and to increase the plankton productivity during the initial acclimation period of fish the ponds were fertilized with organic manure. Subsequently no fertilizers were added. The initial phytoplankton and zooplankton population in the four ponds ranged from and cells/l respectively and the plankton population declined during the second week of experimental period. Water quality parameters were monitored in all the four ponds once in a week.

6 Experimental regime In each pond, 125 C. carpio fingerlings (8.1 r 0.68 cm length; 7.32 r 2.2 g weight) were stocked. Fish in pond A were fed with experimental diets incorporated with chitosan (1%) and fishes in ponds B, C and D received separate diets with chitin (I%), levamisole (250 mglkg of feed) and nonsupplemented (controls) diet separately. Length and weight of 10 fish randomly selected from each pond were measured every 15 days for a period of three months using a measuring scale and an analytical electronic balance (to 1 mm and 0.01 g respectively). Blood was collected by the caudal vein puncture and pooled from a random sample of five fish in each experimental pond after anaesthetizing them with MS-222 (Sigma, USA) in every 30 days. The blood (heparinised 150 iu 1 ml) collected from each group was tested for WBC count, Lysozyme activity and Nitroblue tetrazolium assay Feed A pellet feed composed of 40% groundnut oil cake, 33% rice bran, 20% soyabean meal, 5% fish meal and 2% minerals and vitamins mixture (each 250 g minerals-vitamins mixture provide vitamin A - 500,000 iu., vitamin D3-100,000 iu., vitamin B2-0.2 g, vitamin E - 75 units, vitamin K g, calcium pentathonate g, nicotinamide- 0.1 g, vitamin BIZ- 0.6 mg, choline chloride- 15 g, calcium - 75 g, manganese g, iodine- 0.1 g, iron g, zinc g, copper g and cobalt g) was used as control diet. Chitin (10 g), chitosan (10 g) and levamisole (250 mglkg) along with vitamins and minerals

7 were incorporated separately before pelletization in a steam cooked diet. Fish were fed at the rate of 3% of the body weight of animals in each pond Challenge Fish fed with chitin, chitosan, levamisole and the non-supplemented diet were challenged with A. hydrophila on day 45 and 90. On day 45, 40 fish in each group were netted out and were given intramuscular injections of A. hydrophila and then reared in separate ponds. On the goth day the remaining fish in the ponds were netted out and injected with A. hydrophila. A challenge study was performed by injecting 100 pl of 12 h grown culture of A. hydrophila at a concentration of X lo6 cfulml. Total count was determined using Neubauer hemocytometer and total viable count was confirmed by spread plate method. Mortality was recorded daily up to 15 days and relative percentage survival was calculated following methods of Amend, (1981) Results Growth The initial weight of the fish was g in all groups. Growth of the fish fed with supplemented diet was weighed significantly more (~~0.05) after the third week of feeding (30th day of study) (Figure 3 and Plate VII). The positive effect of chitosan and levamisole on growth was clearly observed in all the groups except for chitin fed and control fish. The maximum growth was observed in chitosan fed fish ( g) (p<0.001) followed by levamisole ( c 8.4 g) (p<0.001) and decreased growth was observed in chitin fed fish

8 Time (Days) /+Control Chitin -4- Chitosan Levamisole Figure 3. Weight of common carp fed chitin (I%), chitosan (I%), levamisole (250 mg I kg of feed) and a nonsupplemented control diet for 90 days and weighed every 15 days. Each value (mean + SD) is the average performance of 20 fish/ treatment for a period of 90 days. * (P < 0.05), *** (P < 0.001), indicate a significant difference between fish fed commercially prepared immunostimulants and those fed the control diet. Data represented as mean + SD.

9 Plate VII. The effect of immunostimulant on common carp, C. carpio growth in the field study.

10 (43.54 t 4.7g) (p0.05) compared with non-supplemented control fish ( g) WBC count The WBC count was found to be significantly high on the 6oth day ( ~10~) (p <0.01) and goth day (3.32 t 0.29 X lo4) in chitin treated fish and there was little difference in the WBC count in other treatments (Table 9) NBT assay Studies on neutrophil activity clearly showed the enhancing effect of dietary supplements on neutrophil respiratory burst activity as evidenced from the increased NBT reduction (Figure 4). The neutrophil activity was enhanced in all the treatments. The highest significant NBT reduction was observed on the 30th day in chitosan fed fish (1.I 28 t 0.081) (p <0.001) followed by chitin (0.639 k 0.032) (p<0.001), levamisole ( ) (p<0.001) and control groups ( ). In all the treatments, a gradual significant increase of NBT activity (p<0.001) was observed which reached a maximum on the 6oth day. The NBT activity started to decrease after the 601h day and in the chitin treated experiment, the activity was less than the control fish Lysozyme activity Lysozyme activity started to increase after the feeding of supplemented feeds. The maximum activity was observed on the 3oth day chitosan fed fishes ( iu) (p<0.001) followed by chitin (2005 +: 156 iu) (pi0.001),

11 Table 9. WBC count (lo4 mm3) of common carp fed chitin (I%), chitosan (I%), levamisole (250 mgl kg of feed) and a nonsupplemented control diet for 90 days and weighed every 15 days. Each value (mean 2 SD) is the average performance of five fish1 treatment *(P<0.05), significant difference between fish fed commercially prepared immunostimulants and those fed the control diet. Data represented as mean 2 SD. Duration (Daysf Control Chitin Chitosan Levamisole

12 I [ Time (Days) control Chitin Chitosan Levamisole 1 Figure 4. NBT reduction of common carp fed chitin (I%), chitosan (I%), levamisole (250 mg I kg of feed) and a nonsupplemented control diet for 90 days and weighed every 15 days. Each value (mean + SD) is the average performance of five fish I treatment for a period of 90 days ** (P < 0.01), *** (P < 0.001), indicate a significant difference between fish fed commercially prepared immunostimulants and those fed the control diet. Data represented as mean + SD.

13 levamisole (1864 rl: 112 iu) (p<0.001) and control ( iu) fishes. Thereafter the activity decreased in all the treatment but remained significantly higher than in the control (Figure 5) (p <0.01) Challenge study Table 10 shows the relative percentage survival (RPS) of groups challenged with the homologous virulent strain of A. hydrophila. Fish fed with chitin, chitosan and levamisole showed low mortality and significantly increased the RPS (p <0.001) Discussion The results of the present study clearly show that dietary chitosan and levamisole supplementation enhances the growth of common carp, where as, chitin supplementation depressed the growth below that of fish fed with a control diet. This contradicts the reports of Kono et al. (1987) and Shiau and Yu, (1999). Feeding of supplemented diet containing 10% chitin, chitosan or cellulose did not affect the growth of red sea bream, Japanese eel and yellow tail (Kono et al., 1987). On the contrary, Shiau and Yu, (1999) observed depressed growth in tilapia after feeding chitin and chitosan at the 2, 5, and 10% level. They also speculated that the depressed growth in tilapia may be due to interference of chitosan and chitin in the absorption of nutrients. In the present study, we fed the fish with chitin (loh), chitosan (1%) and levamisole (250 mg/kg) supplemented diets which is lower than the dosages or levels used by the previous authors. Hence, the chitosan may play a crucial role in

14 Time (Days) Figure 5. Lysozyme activity of common carp fed chitin (I%), chitosan (1%), levamisole (250 mg I kg of feed) and a nonsupplemented control diet for 90 days and weighed every 15 days. Each value (mean + SD) is the average performance of five fish 1 treatment for a period of 90 days *** (P < 0.001), indicates a significant difference between fish fed commercially prepared immunostimulants and those fed the control diet. Data represented as mean -+ SD.

15 Table 10. Mortality of immunostimulants fed and non supplemented common carp in 45th and goth day of challenge with A. hydrophila infection. Group No, of No. of Survival Relative challenged mortalities Percentage fish Survival I 45th ~ a y Chitin 40 9 (22.5)* Chitosan 40 3 (7.5)* Levamisole 40 5 (12.5)" Control (37.5)" 62.5 ' goth Day Chitin (54.4)** Chitosan (27.1)"" Levamisole (36.7)** Control (87) "' 13 * P<0.01; ** P<0.001; ns: not significant "Fish were challenged by intramuscular injection with the A. hydrophila strain. "Relative percent of survival = 1 - [% mortality in the immunostimulants fed group/% mortality in the control group] X 100. RPS values over 50 indicate positive effect of the immunostimulants (Amend, 1981).

16 enhancing the digestion and absorption of nutrients at lower levels. However, fish fed with chitin showed depressed growth after 30 days of feeding. This may have been due to the fish developing tolerance to chitin. A significant increase of the WBC count was not observed in all the treatments except for chitin fed fish. On the 6oth and goth day, fish fed with chitin supplemented diet showed a higher WBC count. This could be due to the continuous feeding of chitin that may have induced stress in fish. Similarly, stress due to viral or bacterial infection, overcrowding, changes in temperature and exposure to sub lethal concentration of copper has been shown to elevate the WBC level in the blood (Harikrishnan et al., 2003; Haney et al., 1992; Hossain and Sheriff, 1995; Wedemeyer et al., 1983). The NBT assay is a quick inexpensive test focusing on the ability of phagocytes to reduce the dye by the production of oxygen radicals. In animals (in vivo) the oxygen radicals are aimed at the destruction of bacterial invaders. The ability of macrophages to kill pathogenic microbes is probably one of the most important mechanisms of protection against disease among fishes. The higher optical density in the NBT assay was observed in all the treatments. Similarly, in another study the injection of Ocimum sanctum L (20 pg) extract into Tilapia mossambicus produced a higher neutrophil activity (Logambal et al., 2000). The supplemented feeds also enhanced the lysozyme activity in all the treatments. 0' Neill, (1981) and Snarski, (1982) have reported that heavy metal pollution affects the lysozyme levels causing alteration in immunomodulatory function in fish and there is a increase in the lysozyme concentration in the fish

17 blood following infection or injection of foreign materials of pathogens (Studnika and Siwicki, 1986; Siwicki and Studnicka, 1987). The serum lysozyme concentration was significantly increased by lower nisin dose ( pg / fish) in turbot Scophthalmus maximus (Villamil et al., 2003). Similarly, many authors reported that administration of P-glucans enhances lysozyme activity in atlantic salmon (Salmo solar, L) and turbot (S. maximus) (Engstad et al., 1992; Jorgensen et al., 1993; Santarem et al., 1997; Paulsen et al., 2001; Baulny et al., 1996). Lysozyme is a cationic enzyme that breaks P - l,4-glycosidic bonds between N-acetylmuramic acid and N-acetyl glucosamine in the peptidoglycan of bacterial cell walls. This action is known to attack mainly Gram positive bacteria as well as some Gram negative bacteria in conjunction with complement (Alexander and Ingram, 1992). Robertson et al. (1994) showed an increased protection against fish bacterial infection correlated to an increment in serum lysozyme levels, phagocytic activity and bactericidal activity of head kidney leukocytes. In this study after challenge with A. hydrophila, the relative percentage survival of fish fed with chitosan and levamisole supplemented feed was higher on the 45th and goth day. This might be due to the enhancement of the non specific immune system of the fish by chitosan and levamisole. There is strong experimental evidence that feeding of glucan can modify the activity of some components of the innate immune system and increase the resistance against disease in several fish species (Galeotti, 1998; Robertsen, 1999; Sakai, 1999). However, the lower RPS in the chitin fed group could be due to suppression of

18 the non-specific immune system of fish (evidence from the table 2) as they might have developed tolerance to chitin administration. Selvaraj et al. (2005) studied the enhancement of IgM production and IL-1 P mrna expression by stimulation of carp head kidney macrophages with the application of P-glucan. Similar observation was observed by Fujiki and Yano, (1997) with the application of sodium alginate and scleroglucan. In continuation with the present study, Singh, (2006) examined the effect of chitin, chitosan and levamisole on IgM and IL-1 I3 mrna expression in common carp. Administration of chitin, chitosan and levamisole through diet and intramuscular injection significantly enhanced the serum IgM level as well as stimulates higher expression of IL-1 P mrna in carps than in control fish. This research supports the finding of the present study, that the chitin, chitosan and levamisole not only enhance the growth but also protected the fish by enhancing the innate immune system. The mechanisms of immunostimulants funtions were studied by many others (Sakai, 1999; Tassakka and Sakai, 2005). In a recent study, Tassakka and Sakai, (2005) discussed the role of Toll like receptors (TLR) in stimulation of innate immune response and cytokines production. Most of the TLR are present on the cell membrane and some on endosome membrane of immune cells. These TLR receptors signals once the immunostimulants localize with the TLR through the adapter protein MyD88 which then activates IR AR-TRAFG TAR pathway (Tassakka and Sakai, 2005) and then the innate immune system. TLR receptors were also identified in Zeebra fish and buffer fish. TLR receptors were found to be co-localize with the specific immunostimulants. For example,

19 CpG-ODN has a specific TLR 9 receptors found in the fish on B cells which activates the innate immune system. Similarly, there may be specific TLR for chitin, chitosan and levamisole present on the cell membrane of immune cells. Further research is needed to find out the receptor which is responsible for stimulation of immune system in carp. Based on the above investigation it is evident that chitosan (1%) and levamisole (250 mg/kg of diet) certainly enhances the non-specific immunity of G. carpio in ponds. Thus chitosan and levamisole act as immunostimulants which appear to improve the immune status and growth of fish C. carpio in fish farms. It is hoped that this base line information will be of potential use in fish farming.