PATHOGENICITY OF MYXOBOLUS INFECTION AND ITS EFFECT ON PROTEIN EXPRESSION IN CATLA CATLA IN CENTRAL GUJARAT REGION

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1 Journal of Cell and Tissue Research Vol. 10(1) (2010) ISSN: (Available online at Original Article PATHOGENICITY OF MYXOBOLUS INFECTION AND ITS EFFECT ON PROTEIN EXPRESSION IN CATLA CATLA IN CENTRAL GUJARAT REGION CHAVDA, D., BHATT, S., SREEPADA, R. A. 1 AND SHETH, A. B.R. Doshi School of Biosciences, Zoology Laboratory, Bakrol Vadtal Road, Satellite Campus, Sardar Patel University, Vallabh Vidyanagar ; 1 National Institute of Oceanography, Dona Paula, Goa E. mail: bhatt.sujata@gmail.com Received: February 23, 2010; Accepted: March 23, 2010 Abstract: Aquaculture of Major Carps (IMCs) has increased in India during the recent years. Myxobolus infection in fish Catla catla was detected during December February 2008 in a pond in central Gujarat (India) during the monitoring of hygienic conditions of carp culture. Infected catla exhibited hemorrhagic condition with necrotic patches on the body, lethargic condition and enlargement of spleen. Myxozoan parasites were detected in gill and muscle. Gills showed the formation of round to elliptical cyst in secondary lamella measuring 37 to 39 µm widths and 41 to 43 µm lengths. Hyperplasia and necrotic changes in epithelia and in connective tissues of gills were observed. In the muscles, the spores were seen along the myoseptal boundaries as well as in the connective tissues along with the destruction and necrosis of myofibril and of connective tissues. The histology of spleen indicated the influence of infection resulting the damage in cell structure and connective tissues. The polyacrylamide gel electrophoresis (SDS-PAGE) of protein from infected fishes showed the newly expressed proteins viz., 68 kda protein in gill and muscles; and 107 kda and 122 kda proteins in spleen. Hematological analysis showed the destruction of blood cells and presence of microscopic organisms. Anthropogenic activities in the pond, feeding behavior of fishes and very close contact of spores with fishes appear to be responsible for myxobolus infection in catla. Key words: Myxobolus infection, Catla catla? INTRODUCTION Fisheries have always played a pivotal role in food and nutrition all over the world. India ranks third among the world freshwater fish producers with Indian major carps viz, Catla catla, Labeo rohita and Cirrhinus mrigala being the most preferred cultured species [1]. The major carps are fast growing species and greatly propagate on an extensive/intensive scale in a polyculture system; and among the major carps, Catla catla is the fastest growing species [2]. The carps are reared in the fertilized tanks; and excessive manuring can lead to water quality deterioration. Several fish diseases like haemorrhagic septicaemia, saprolegniasis, dactylogyrosis, sporozoan and trichodiniasis have been observed in major carps in tanks with heavy organic loading [3]. During the monitoring of hygienic conditions of carp culture ponds of Central Gujarat, myxobolus infection has been detected in catla. 2157

2 The myxosporea are common parasites of fishes; and some of them are reported to be serious pathogens associated with epizootics and causing heavy losses in aquaculture [4]. Myxosporian parasites are known to infect both marine and freshwater fishes all over the world [5-10]. Histopathological analysis of tissues is an important approach for the detection of myxobolasis [11]. Severe damage to the gills has been obseved by histological studies during the myxobolasis of catla, common carp and gible carp [5,12,13]. Skeletal muscles have been reported to be a location for myxosporean infection in fresh water fishes[14]. Myxobolus infecti-on in fishes is also reported to damage spleen [15]. Examination of change in the expression of proteins by SDS-PAGE has proved to be useful in classification and identification of organisms [16]. The SDS-PAGE and Western blot analysis have been used for the identification of proteins of triactinomyxon spores of Myxobolus cerebr-alis in salmonid fishes [17]. In the present investigation, myxozoan infection of catla has been investigated by analyzing the damage caused to gills, muscles, spleen and blood by histopathological analysis; and by looking to the change in the expression of proteins by SDS-PAGE in infected tissues. Till now, very little is known about myxozoan infection in aquaculture units in Gujarat. Therefore, the present study has been undertaken to investigate diseased conditions in IMCs with a view to help fish farmers in identification and investigation of prevailing diseases and to take appropriate measures to prevent heavy losses of carps. MATERIALS AND METHODS J. Cell Tissue Research by drag net and transported to the laboratory in live conditions. Blood samples were collected from the caudal vein in heparinized tubes for hematological studies. Subsequently, the spleen, gills and muscles were excised and stored at -20 C to study the changes in the expression of proteins. Tissues were also processed for histopathological analysis. Histopathological and hematological analysis: The histopathological analysis of gills, spleen and muscles were carried out by standard Haematoxylene-eosin staining of paraffin sectioned tissues. For the ultramicroscopy study, gills and muscles were fixed in 2.5% glutaraldehyde, post fixed in 2% osmium tetra oxide in 0.1 M phosphate buffer (ph 7.2 at 4 C) and embedded in spurr s resin. Semi thin sections were cut (Leica ultracut UCT ultra microtome, All India Institute of Medical Sciences, AIIMS, New Delhi) and stained with toludine blue. For the hematological analysis, blood smear from infected and healthy fishes was stained with Giemsa. The photographs were taken with Axioplan image analyzer (MPM-400 from Carl Zeiss, Germany) at 40X and 100X magnification. SDS PAGE analysis: The gills, muscles and spleen (0.5gm each) from infected and healthy fishes were homogenize in 10 ml of phosphate buffer (ph 7.0), centrifuged at 10,000 rpm (10 min) and supernatant subjected to polyacrylamide gel electrophoresis using 10 % polyacrylamide resolving gel as well as 5% stacking gel. Gels were run using a Dual midi vertical gel Electrophoresis system at 100V constant voltage in 1X buffer (25mM Tris, 25mM glycine, ph 8.3, 0.1% SDS). Proteins were visualized by silver staining kit (Genie, Bangalore) [18]. Sample collection: Catla catla with morphological symptoms of infection and without infection were collected from Napad village pond, with three hac. of water area (Dist. Anand, Gujarat) during December 2007-February The pond was stocked with 10,000 fingerlings of IMCs. Fishes were fed with rice bran and oil cake (1:1). Before stocking, the pond was manured with cow dung. Fishes were collected RESULTS Morphological symptoms: Infected fishes were found to be weak, lethargic, and crowding near the water surface. Gills were pale, covered with a heavy coat of slime. Gill filaments were covered with large number of cysts. Hemorrhage was seen on the body surface as bluish red marks with several necrotic patches (Fig. 1). 2158

3 Histopathological changes in gills, muscles and spleen: Myxobolus parasites were detected inside and outside the cyst in secondary lamellae of gills (Figs. 2a to 2d). The cysts were spherical to elliptical in shape measuring 41 to 43µm in length and 37 to 39µm in width, each containing 27 to 29 parasites and surrounded by flattened epithelial cells. Some of the cysts were also infiltrated by epithelial cells and macrophages. Parasites were oval with one pointed end, with one or two nuclei and measuring 5 to 7 µm in length and 3 to 4 µm in width. Necrotic changes and hyperplasia were extensively observed in the gill filaments. Semi thin sections also revealed the presence of the myxozoan parasites (Fig. 2e), which were as well detected in the muscle sections (Figs. 3a and 3b). Marked enlargement and destruction of myofibrils as well as necrosis of the surrounding tissues were visible. Parasites could be seen along myoseptal boundaries and in connective tissues. Semi thin sections of muscle showed the necrosis of muscle fibers and the presence of parasites (Fig.3c). In infected fishes, enlargement of the spleen was associated with the loss of cell structure and connective tissues (Figs. 4a and 4b). Hematological analysis: In the blood smear, large numbers of enucleated and ruptured RBCs as well as damaged leukocytes were evident (Fig. 5). SDS PAGE analysis: The protein profile of gills, muscle and spleen tissues from infected and healthy fishes are shown in figures 6a, 6b and 6c. In the infected gills and muscles, a newly expressed protein with molecular weight 68 kda was detected (Fig. 6a Lane 1 and Fig. 6b Lane 6). In the spleen two new proteins with high molecular weight, 107 kda and 122 kda were detected (Fig. 6c Lane 1). DISCUSSION In the present investigation, myxobolus were seen in the cyst and in connective tissues with the ruptured cysts in the secondary lamellae of gills. As the gill lamellar surface was covered by ruptured cysts with necrotic tissues, the Chavda et al normal respiratory function of the gill appeared to be impaired. Such a condition has been observed by several workers. Sanaullah and Ahmed [5] reported the presence of myxobolus cyst in different locations of secondary lamellae with hyperplastic gill epithelial cells. In Indonesia, myxobolus infection in common carp is reported to result in lesions, inflammation, congestion and hyperplasia in the gills [6]. Three myxobolus sp. were identified in pond cultured common carp in gills [11,14]. Myxobolus longisporus have been detected in the gill lesions of Cyprinus carpio from China [19]. Presently observed very high number of plasmodia in the gills seem to be responsible for necrosis of the gill filaments and for the disruption of the respiratory function. The visible pinkish coloration of cysts could be due to the accumulation of blood by rupture of blood capillaries and sinuses in the gills. Occasionally the infiltration of the cyst with connective tissue and macrophages demonstrates the formation of granulation tissue in the healing process. In common carp, during the Myxobolus koi Kudo infection, granulation in gill tissue was observed as host tissue reaction in the healing process [6]. Interestingly, the infection is detected only in catla and not in rohu or mrigala. The exact reason of infection only in catla and not in other carps cultured in the same pond is not understood clearly, though feeding behavior of the fishes and possibly very close contact of the spores with the fishes appear to be responsible for this infection. Moreover in this pond organic manure is being added before the stocking of the fishes and the pond is also used as a multipurpose village pond. Heavy organic loading and different anthropogenic activities in the pond are likely to be responsible for this infection. Muscles also exhibited the destruction of myofibrils, necrosis of connective tissues and presence of spores in infected fishes (Fig. 4). Longshaw et al. [20] reported myxozoan infection in muscles, gills and kidney in Cyprinids in UK. In muscles, spores were seen in the coelozoic stage. The damage caused to muscles is also seen as necrotic patches and

4 J. Cell Tissue Research Fig. 1: Arrow showing the hemorrhage with necrotic patches in infected Catla catla. 10 µ 50 µ 50 µ 50 µ 50 µ Figs. 2a to 2e showing secondary lamellae and presence of myxobolus parasite in the infected gills. Cyst with parasites, hyperplasia, necrotic changes, infiltration of cyst with different cells and parasites coming out of the cyst seen clearly (Fig.2e). C-Cyst; M-Myxobolus; Ep-Epithelial sheet; N-Necrosis; In-Infiltration in cyst; Mg-Migration of parasites outside the cyst (Fig. 2a scale bare=10µm; Fig. 2 b, c, d and e scale bare=50 µm) 50 µ 50 µ 50 µ Figs. 3a, 3b and 3c muscle section showing presence of pathogen and necrosis of muscle fibers and connective tissue. M-Myxobolus; C-Connective tissue; F-Muscle fibers. (Fig. 3a, b and c scale bar =50µm) 2160

5 Chavda et al. 50 µ 10 µ Fig.4. Necrosis and loss of cellularity as well as loss of fibrillar connective tissues (a) as compare to control (b) (Fig. 4a and 4b scale bar = 50 µm and 10 µm respectivily). 50 µ 50 µ Fig. 5 Blood smear of infected fishes showing presence of damaged RBCs and damaged leukocytes (a and b). 5a-Damaged RBCs ; 5b-Damaged WBCs (Fig. a and b scale bar is=50µ) hemorrhage on the body surface, Enlargement of spleen together with the loss of cell structure and damage to the fibrilar connective tissues further indicates the response of spleen to invading organisms. The spleen is reported to be an integral component of the lymph reticular system playing a crucial role in generating a response to invading pathogens. A severe infection of Myxobolous pangasii in the spleen of Pangasius hypophthalmus was reported by Molnar et al. [15]. Myxobolus infection in Catla catla exhibited change in the expression of proteins in gills, 2161 muscles and spleen. Looking at the presence of parasites in the gills and muscles, expression of new protein of molecular weight 68 kda appear to be the protein of parasite but further confirmation is required. The SDS-PAGE and Western blot analysis of spores of Myxobolus cerebralis have exhibited the expressions of protein bands with molecular weight 130 kda and 60 kda as well as four other protein bands with molecular weight 7 kda to 45 kda [17]. In the spleen from the infected fishes, expressions of two new proteins with high molecular weight 107 kda and 122 kda possibly indicate the response to invading pathogens. During the

6 J. Cell Tissue Research Fig. 6a SDS PAGE analysis of infected and control gills. Lane 1 and 2 loaded with infected gill protein which is showing 68 kda proteins; Lane 4 loaded with gill protein from healthy fish and lane 3 loaded with molecular weight marker. Fig. 6b protein profile of infected and healthy muscles. Lane 1 and 2 loaded with muscle protein from healthy fish; lane 4, 5 and 6 loaded with muscle protein from infected fish showing 68 kda newly expressed protein. 2162

7 Chavda et al. Fig. 6c Protein profile of spleen from infected and healthy fishes. Lane 1 and 3 loaded with spleen protein from infected and healthy fishes respectively. Lane 2 loaded with molecular weight marker. Arrow showing expression of new proteins 107 and 122 kda in the infected spleen. infection, damage to the RBCs and leukocytes has also been detected. Myxobolus infection might have allowed secondary infection of microorganisms. Even some planktonic organisms including algal cells were also detected in blood smear (photographs not shown). They may have entered the blood either through necrotic gills or through necrotic patches of the body surface. Protozoan infection and subsequent infection with microorganisms seem to be responsible for the damage to the RBCs and WBCs. To control the protozoan infection in fish stock, improvement in water quality by chlorine treatment has been reported to be effective in controlling the infection in the next season. Apart from chlorine treatment, 1% formaldehyde, 2% Lysol and ethanol treatment were also reported to be effective in controlling the spores [21]. The present findings may help fish farmers in adopting better management of hygienic conditions of the carp ponds and selecting appropriate treatment to control infection. ACKNOWLEDGEMENTS The first author received meritorious fellowship from UGC New Delhi. We are thankful for the same. We also gratefully acknowledge the cooperation from the office of Assistant Director of Fisheries, Government of Gujarat, Anand, as well as the fish farmer Shree Munavarkhan Chandrasingh Rathod. REFERENCES [1] FAO, Aquaculture production. Yearbook of Foods and Agriculture Organization of United Nation, Rome, Italy (2003). [2] Jhingran, V.G. and Pullin, R.S.V.: ICLARM Studies and Reviews. 11, Manila, Philippines., pp. 191 (1988). [3] Toor, H.S., Sehgal, H.S., Schdau R.S.: Aqua., 35: (1983). [4] Lom, J. and Dykova, I.: Myxosporidia (Phylum Myxozoa). Vol. 26. Elsevier Pub. Amsterdam. pp (1992). [5] Sanaullah, M., Ahmed, A.T.A.: J. Fish. Dis., 3: (1980). [6] Rukyani, A.: Asian Fisheries Sci., 3: (1990). [7] Fomena, A., Lekeufack, G.B. and Tang, II C.: J. Biol. Sci., 7(7): (2007). [8] Grankoto, A., Pampoulie, C., Marques, A. and Sakiti, G.N.: J. Fish Biol., 58: (2001). [9] Zhang, J.Y., Wang, J.G., Wu, Y. S., Li, M., Li, A.H. and Gong, X.L..: J. Fish. Dis., 29: 1-7 (2006). [10] Kent, M.L., Andree, K.B., Bartholomew, J.L., El-Matbouli, M., Desser, S.S., Devlin, R.H., Feist, S.W., Hedrick, R.P., Hoffman, R.W., Khattra, J., Hallett, S.L., Lester, R.J.G., Longshaw, M., Palenzeula, O., Siddall, M.E. 2163

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