HISTOPATHOLOGICAL MANIFESTATIONS IN COMMERCIALLY IMPORTANT FISH, CLARIAS BATRACHUS(L.) FOUND INFECTED WITH SAPROLEGNIA DICLINA.

Transcription

1 Page1168 Indo American Journal of Pharmaceutical Research, 2014 ISSN NO: Journal home page: INDO AMERICAN JOURNAL OF PHARMACEUTICAL RESEARCH HISTOPATHOLOGICAL MANIFESTATIONS IN COMMERCIALLY IMPORTANT FISH, CLARIAS BATRACHUS(L.) FOUND INFECTED WITH SAPROLEGNIA DICLINA. Rekha Chauhan, Ashiq Hussian Beigh and Magid Hassan Bhat Department of zoology and applied aquaculture, Barkatullah University, Bhopal. ARTICLE INFO Article history Received 09/02/2014 Available online 03/03/2014 Keywords Saprolegnia Diclina, Clarias Batrachus, Pathogenicity, Histopathology. ABSTRACT Present study was carried out on commercially important fresh water fish (Clarias batrachus).a total number of 36 fishes infected with ulcerative mycoses were collected from Larpur reservoir, Bhopal. Isolation of fungi from different parts of body of fish showed the presence of Saprolegneous fungi ( Saprolegnia diclina) in all the specimens collected. Pathogenicity of isolated fungi was tested on host fish with concentration of 2x10 4 zoospores /ml and all the fishes got infection leading to 100% mortality within ten days. Histopathological studies of infected fishes showed varying degree of destruction in the tissue. Skin showed loss of epidermis, necrotized hypodermis and penetration of hyphae in underlying musculature with fibrillar granuloma. Degenerative liver and kidney cells with fungal spores were found, gills showed degeneration of lamellae. The aim of present work is to find out the parasitic ability S.diclina with its histtopathological studies. Corresponding author Dr. Rekha Chauhan, Department of zoology and applied aquaculture, Barkatullah University, Bhopal. rekhatarun98@gmail.com Please cite this article in press as Dr. Rekha Chauhan, et al. Histopathological Manifestations in Commercially Important Fish, Clarias Batrachus (L.) Found Infected with Saprolegnia diclina. Indo American Journal of Pharm Research.2014:4(02). Copy right 2013 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 Page1169 INTRODUCTION Fungal infection in fishes are very common and most of the fungal infections are caused by water molds of family saprolegniaceae. Saprolegnia infection in fish appears as white cottony patches on body surface leading to destruction of cells of epidermis, dermis and underlying musculature. Saprolegnia diclina have been reported as fish pathogen by [11]. [23] in (Perca fluviatilis L.) and [12] in (Rutilus rutilus L.) reported the given fungi. Later on [21] found this species associated with necrotic lesions of adult Catla catla, Cirrhinus mrigala, Colisa lalia, Labeo rohita, L.calbasu and Puntius sophore. [22] recorded this fungi from eggs of Cyprinus carpio and adults of Colisa lalia. Later on some other workers like [17,18,7 and 2] reported S. diclina from some fresh water fishes. C.batrachus have been reported as host of S.ferax and S.parasitica by[1] and Aspergillus niger by [3].[16] also reported Saprolegnia spp. infection in cat fish (Clarias garipenus).work on pathogenicity of this fungi and histology of C.batrachus due to fungal infection is very rare. Pathogenicity of S.diclina have been reported by [22 and10] [6] reported both pathogenicity and Histological alterations in S.diclina infected tissue of fish. Present piece of work has been designed to find out the pathogenicity of S.diclina isolated from C.batrachus and its histological studies to find out the parasitic ability of given species of fungi. MATERIAL AND METHODS A total number of 36 fishes showing external symptoms like white fungoid patches and ulcerations on body were collected from Larpur reservoir, Bhopal and brought to the laboratory in sterilized polythene bags for further examination. The fishes were kept in aquaria with continuous aeration. The fishes were observed to note external symptoms. To avoid bacterial contamination all the glass wares, instruments and media were sterilized, along with all aseptic conditions, Streptomycine sulphate 100mg/ml were used in media. Innoculation was done in Laminar flow in sterilized conditions. The agar plates were incubated at 16 ±2 C for the growth of cultures. Growth of colony was observed in 24 hours. For full growth of colony, plates were kept for 4-6 days in incubator. Preparation of pure cultures Pure cultures were prepared by picking up small tuft of mycelium and grown on agar media (Potato Dextrose Agar). For the development of zoospores and reproductive structures cultures were prepared on baits. For that pretiplates were filled with ml sterilized tap water, small piece of media with fungal growth were kept in these plates and baited with different baits at temperature 16±2 C. Glycine seeds (Soyabean seeds) was used as baits. Identification of the isolates All pure cultures were examined for colonial growth, morphological features and microscopical characteristics. For identification, slides were prepared from each colony by taking small tuft of mycelium and stained with Lacto-phenol cotton blue. The slides were observed under microscope. Identification of fungi was carried out on the basis of keys of [4,19,8 and 9]. Pathogenicity Tests Ten fishes having average length of 28± 6 cm and average weight of 20± 4 gm were used for innoculation. Concentration of 2x10 4 zoospores were prepared by using haemocytometer and 0.2 ml were injected intra-peritonially. Fishes were observed for ten days. Histological Examination For histological examination infected tissue of skin with muscles, gills, liver and kidney were fixed in aqueous Bouin s fluid for hours. The tissue was then processed routinely and prepared into paraffin blocks. The blocks of the tissues were cut at 4-6 µm thickness and stained with Haematoxylin and Eosin (H-E).Standard histological procedures [15] were followed for histopathological investigations. RESULTS AND DISCUSSION All the collected 36 infected Clarias batrachus showed patches of fungal infection which leads to ulcerative lesions.(fig-1.). Mycological examination of revealed the presence of zoosporic fungi.wet cultures with reproductive structures were developed within six days. On the basis of taxonomic characters it was isolated as Saprolegnia diclina, Humphrey. With non septate hyphae, abundant zoosporangia having the size of µm in length and µm in diameter, encysted zoospores 9-11 µm in diameter, oogonia spherical to oval µm thick with diclinous antheridia (Fig-2,3,4and5).present finding of S.diclina as fish pathogen is supported by reports of [21,22,17,18,7,10 and 1]. Fig-1. Showing fungoid patches on body of Clarias batrachus (L.) which leads to ulcerations.

3 Page1170 Fig-2. Wet culture of fully developed S.diclina on Glycine seed (Soyabean seed prepared from isolate of Clarias batrachus. Fig-3. Mature zoosporangia of S.diclina with zoospores Fig-4. Zoosporangia realesing spores and scattered encysted zoospores. Fig-5. Spherical oogonia of S.diclina with diclinous antheridia. Results of pathogenicity tests showed,the isolated species of fungi was highly pathogenic to C.batrachus causing 100% mortality with in ten days of experimental period.(fig-6). Symptoms of artificially infected fish were similar to those of naturally infected fishes. Present findings are supported by the reports of [11 and 6] who reported 100% mortality in Nile tilapia due S.diclina. Severly infected twelve fishes were dissected for examination of gills,kidney and liver.all these organs were found infected with hyphal layers. Histological examination of skin showed complete loss of epidermis and extensive edema in the hypodermal layer from which fungal hyphae are radiating, degenerative cells of muscular layer, lost their original appearance with the presence of number of fibrillar granulomas with fungal hyphae.encysted zoospores were observed in muscle layer. Gills showed severe infection with necrotized lamellae having fungal spores. Degenerative liver cells were found with fungal hyphae and spores between hepatic tissues and epitheloid cells which aggeregate to form granulomas. Kidney showed severe degenerative changes in glomerular tissue and hyphal threads were observed between interstitial cells and cells start aggregating, spores were found between cells. These findings are justified by reports of [11,5,13,14 and 6].(Fig-7-12)

4 Page1171 Fig-7. S.diclina Infected skin of C.batrachus Showed loss of epidermis and necrotized hypodermis with fungal hyphae. Fig-8. Degenerative cells of Hypodermis and underlying musculature of artificially inoculated C.batrachus with S,diclina,having encysted zoospores in musculature. Fig-9. Complete necrotisation of muscles with fibrillar granulomas having fungal hyphae. Fig-10. Degenerated gill lamellae of C.batrachus due to S.diclina infection. Fig-11. Showing aggregated liver cells of C.batrachus which showed the formation of granulomas. Fig-12. Necrotised and aggeregated tissue of kidney of C.batrachus with spores. CONCLUSION It was observed that Saprolegnia diclina ia pathogenic fungi for Clarias batrachus. It was also observed that the growth and parasitic ability is very rapid causing infection and mortality at very fast rate.experimental infection trials showed that no prior infection in fish is required for the growth of S.diclina. Histopathological findings showed that it s a virulent fungi not only infect skin but also penetrate up to internal organs. Further studies are required to find out the control measures of S.diclina infections in C.batrachus. ACKNOWLEDGEMENTS Author is paying sincere thanks to Department of Science and Technology, New Delhi for providing funds and Head of the Department of Zoology and Applied Aquaculture for providing facilities for completion of present work. REFERENCES 1. Chauhan R., Study on certain fungal diseases in culturable and non-culturable species of fishes of Upper lake, Bhopal, J. Chem. Bio. Phy. Sci. 2012; B,Vol- 2,No Chauhan R and Qureshi TA., Fungal infection of fishes. Published by Lap LambartAcademic Publishing, Germany,2012;pp Chauhan R.,lone SA and Beigh AH., Pathogenecity of three species of Aspergillus (a.fumigatus, a.niger & a.sydowii on some fresh water fishes.life science leaflets, vol 48. Pp Coker WC., The saprelegniaceae with notes on other watermolds. Univ. North Carolina Press, Chapel Hill, 1923; N.C., U.S.A. 201p. 5. Hatai K and Egusa S., Studies on the pathogenic fungus of mycotic granulomatosis-i. Isolation and Network of Aquaculture Centres in Asia-Pacific,Bankok.1973; 73 p. 6. Hussian M M A., Hussian W H and Mohhamad M A.,Pathogenicity of Achlya proliferoids and Saprolegnia diclina(saprolegniaceae) associated with saprolegniasis outbreaks in cultured Nile Tilapia(Oreochromis niloticus).world j. of fish and marine sci.2013; 5(2), Khulbe RD., Studies on aquatic phycomycetes of Nani Tal in relation to Taxonomy,Physiology,Ecology and Pathology.D.Sc Thesis, 1985; Kumaun University, Nani Tal,1985 pp.335

5 Page Khulbe RD., The parasitic watermolds. Publ. Almora. Book Depot, Almora,1993; pp KhulbeRD., A manual of aquatic fungi (Chytridiomycetes & Oomycetes). Daya Publishing Housing House, Delhi,2001; pp Leno EM.,Lilian LPV and Jones EBG.,Saprolegnia diclina isolated from pond cultured red drum (Sciaenops ocelatus)min Hong Kong.Mycological research, 1999; 103 pp Mckay DL., Saprolegnia diclina Humphrey as parasite of Salmonid ( Oncorhynchus kisutch) PhD thesis,1967,university of British Columbia.pp Nolard-Tintigner N.Deux epidemies de saprolegniose des poisons par Saprolegnia ferax(gruith) et par Saprolegnia diclina(humphrey).1970;annales de Parasitologie (Paris): Qureshi T A., Chauhan R and Mastan SA Histopathology of mycoses in certain fishes of Bhopal. Indian J. Anim. Hlth.2001; 40 (1): Refai MK, Laila A, Mohamed M K and Shimaa SMS., The assessment of Mycotic settlement of freshwater fishes in Egypt. J. Amer. Sci.,2010; 6(11), Roberts R J., Fish Pathology. 3 rd Ed., W. B Sauders, U. K.2001, pp Robert M D., Wise D J and Jeffery S T., Saprolegniasis (Winter fungus) and Branchiomycosis of commercially cultured channel cat fish.2003;srac online mstate.edu/dept/srac. 17. Sati S C., Aquatic fungi of Kumaun in relation to fish infection. Ph.D.Thesis, Kumaun University,Nani Tal, 1981; pp Sati S C and Khulbe R D., A host range of Saprolegnia diclina Humphrey on certain coldwater fishes of India.Proc.Nat.Acad.Sci.India, 1983;53(8):1V Seymour RL., The genus Saprolegnia.Nova HedwigiaZ.Kryptogamenkd,1970;19, Siddique MMR.,Bashar MA, Hussain MA and Kibria ASM., Fungal Disease of FreshwaterFishes in Natore District of Bangladesh. J. Bangla. Agri. Uni.,2009; Shrivastava G C and Shrivastava R C.,A note on the destruction of eggs of Cyprinus carpio var.communis by the members of Saprolegniaceae. Cur. Sci. & Cult, 1976a ;42: Shrivastava G C and Shrivastava R C.,Ability of Saprolegniaceous fungus to parasitize Colisa fasciatus Bl;1977a,Geobios 4: Willoughby L G.,Mycological aspect of diseases of young perch in Windmere.1970.Jour. Fish Biol.2: Submit your next manuscript to IAJPR and take advantage of: Access Online first Double blind peer review policy No space constraints Rapid publication International recognition Submit your manuscript at: editorinchief@iajpr.com