ZAFAR IQBAL., R. MUGHAL and U. SHEIKHDepartment of Zoology, University of the Punjab,New Campus, Lahore.A paper read at 31stPak. Cong. Zoology(International) University of AJK, Muzaffarabad.19-21stApril, 2011
ABSTRACTSix freshwater fish species, Labeo rohita (Hamilton); Catla catla(Hamilton); Ctenopharyngodon idella (Valenciennes, 1844);Hypophthalmichthys molitrix (Richardsons, 1845) and Carassiusauratus (L.) were studied for fungal infection.Labeo rohita and C. auratus showed fungal infection with typicalcotton wool like appearance at gills, head, fins, skin and bloody spotsat the sites of infection. Grossly, the skin color was faded and finswere eroded in infected fishes. The fungi isolated from the infectedareas of the fish, were cultured on Maltose extract agar medium.The isolated fungi were incubated for a week at room temperature(28º C) and the growth of fungi was observed.The control plates showed no growth. Different fungal colonies werelabeled. The slides were prepared by taking the material from eachcolony and stained with Trypanblue in Lactophenol. The stainedslides were observed under microscope and photographed.Aspergillus spp; Mucor sp and Penicillium spp were isolated from gills,caudal fin, pelvic fins and skin of C. auratus. In L.rohita onlyAspergillus sp was isolated from caudal fin. The probable causes offungal infection in these fishes are discussed.
IntroductionFungi: Heterotrophs (require living /dead matter for growth/reproduction)Habitat: Freshwater/marine; at cool/ warm temperatureClass Oomycetes (water mould) Members are ubiquitous fungi, normal flora of estuarine ecosystem, have worldwidedistribution. virtually every freshwater is susceptible to infection by at least one fungalspeciesFamily Saprolegniace have members, frequently attributed to cause fungal infection in fishes Fungi attack fish/fish eggs and cause serious diseases.Fungal infection in fish may be Superficial: skin, fins, eyes, gills, Head,eggs Deep: musculature, Brain Infection rout: Skin to musculature; inside to outside through skin –rare Epizootics; Mostly facilitated by poor environmental conditions malnutrition other primary diseases
PREDISPOSING FACTORS FOR FUNGALINFECTION IN FISHESPathogenic fungi-considered opportunists and attack fishwhen ; Stressed Immuno-compromised due to unfavorable environmentalcondition or secondary to bacterial / viral infection or when they have lost their mucus protection because oftrauma or excessive handling (Neish,1990; Byl et al,1993;Quinjou et al,1998) Some members of family Saproleniace are also primarypathogens that cause disease without predisposing factors
SAPROLEGNIASISEUS: ULCERATIVE EPIZOOTIC SYNDROME(Aphanomyces invadans)In carps and snakehead, Pakistan (1995-96)/S.E.Asia, Lilly et al, 1998 attack skin/musclesOthers Names GM: Granulmatous disease RSD: Red sore disease UM: Ulcerative diseaseA disease caused by Genra; Saprlegnia; Achyla; Dictyuchus insalmonids/cyprinids/other fishes/worldwide‘Fish Fungus disease (in ornamental fish; Post, 1987) infect,skin/muscles
BRANCHIOMYCOSIS(Branchiomyces Sanguinis)Icthyophonus disease Ichthyosporidiosis:(Icthyophonous hoferi)Freshwater/saltwater/worldwide.Skin rough/granulmatousgill rot acute/localized fungal disease of gills in manyfreshwater fish/worldwide
AIMSIsolation of pathogenic Fungifrom some imported ornamentalfish and culturable fishes
MATERIALS AND METHODSIsolation of fungusSterilization of sampleIn 1 % Formaldehyde - 5 min dipor 70% alcohol -5min dipRinse in distilled waterFish collection- Pet shop/PU Research fish farmObservations of fungal infectionFresh mount preparation/microscopy
PREPARATION OF CULTURE MEDIAMalt Extract Agar (Oxid, UK) Composition (formula per liter) Malt Extract 12.75g Dextrin 02.75g Glycerol 02.35g Peptone 0.78g Agar 1.50g 2% maltose extract agar medium (2g.MEA+4g.Agar+200ml d/water+flask, double sealed/ autoclave90mins.at15psi)
INOCULATION OF AGAR PLATESFungus from infected fish/sterilized needle/ inoculated agar plateIncubation of agar plates one week at room temperature (28° C) beforeinoculation5 days at 32-35° C after inoculationObservations of fungal coloniesPhysical examination and labeling of coloniesSlide preparationFor identification of fungus
STAINING OF SLIDESStain: Trypanblue in Lactophenol100ml stain (composition) Lactic acid 33ml Phenol 33ml Glycerin 33ml Trypanblue powder 0.05g (Mixture placed in mechanical stirrer over night)Microscopy / Photography (Digipro Labomed USA) At FHM lab. Zoology Dept. and FBR Lab. Botany Dept PU. Lahore Identification of fungus - confirmed by Dr. Abdul Nasir of Botany Dept.PU, Lahore.
RESULTS AND DISCUSSIONFish examined /infectedLabeo rohita /infected (rahu) Catla catla (thala) Ctenopharyngodon idella (grass carp) Hypothalmichthys molitrix (silver carp)Carassius auratus /infected (gold fish)Clinical signs and symptoms Cotton wool like appearance and Bloody spots on infected area
Table1. Fungal infection/ mycosis in C. auratus and L.rohitaFish Infected organ FungusCarassius auratus Gills Aspergillus spMucor spPelvic fins Aspergillus spCaudal fins Aspergillus spskin Penicillum spLabeo rohita Caudal fin Aspergillus sp
Aspergillomycosis; principally in African fishes, tilapia (Olufemi,1985)Infection is caused through contaminated feed. A. flavus, A.japanicus, A. terreus more pathogenicSmoked dried fishes contaminated (Bukola, 2006) with A. flavus, A.teres, A. funigats,A. niger, Mucor sp, Penicillium italicum, P.viridatus(I,2 had highest rate of infection) in atlantic cod, tuna, bonga,ribbon fish, stark and many other fish spp.Aspencer percicus eggs infected with Penicillim spp, Mucor spp,Saprolegnia spp. 7-22 % mortality (Jalilpoor et al, 2006).Aspergillus invasion is by both spore and hyphae growing in withinfeed.Infection results into aflataxicosis- (poisoning leading to cancerousspleen, kidney) produced by aflatoxins,aflatoxins, elaborate on feed stuff by proliferating Aspergillus mouldduring storage of oil seed in warm and humaid environment asfound in tropical countriesToxins can be deposit on pellets, if eaten by fish cause acutetoxicity- haemorrages syndrome, lead to mass mortality,carcinomas, hepatomata eg. In salmonids (Haller & Roberts,1980;Olufemi, 1985,1986a).
Fayioye et al., (2008) isolated four fungi, Fusarium spp, Aspergillusspp, Rhizopus spp, Mucor spp, andPenicillum spp from 8 edible smoked-dried freshwater fishes; Clarisgariepinus; Chrysichthys nigrodigitatus; Sarotheodongalilaeus,Heterotis niloticus, Heterbranchus bidorsalis, Synodontisschall,Synodonts clarias and Clarias angullaris These fish species wererich in protein and dietary minerals such as calcium, sodium, zinc,potassium, phosphorous and iron and with low fungal infestationhence are recommended for human consumption.Junaid, et al., (2010) isolated 7 fungi from stockfish, in Nigeria, asunder; Aspergillus flavus, Trihophyton verrucosum, Aspergillus niger,Aspergillus fuigatus, Rhizopus spp, Mucor spp, and Penicillum spp.Mucor spp had higher occurrence.Goldfish is tropical fish, transported live from South East Asia toPakistan and sold live to hobbyist.
Cause of infection may be the use of contaminated feed in the petshopOr the fish might be infected before arriving in Pakistan.The infection of Labeo rohita may also be attributed to thecontaminated kitchen waste fed to fish.Same may be the case with Mucor spp. and Penicillium spp infectionin these two fishes.Detection of Aspergillus spp. Mucor spp and Penicillium spp from C.auratus and L. rohita may be health hazards for the animals andhumans
CONCLUSIONThe presence of the toxigenic fungi increase therisk for mycotoxin production. Mycotoxin canpose an important danger to human and animalhealth because they are toxic to vertebrates andother animals in low concentration. Theconsumption of these fungi exposes theconsumers to the toxic metabolites produced bythe fungi.Public Health awareness is necessary andstressed.
AcknowledgementSpecial thanks to Directorate of Research andDevelopment PU Lahore, for providing funds for thisStudy.Thanks to Chairman Department of Zoology andBotany PU Lahore for providing facilities.Many thanks to Mr. Abdul Rehman of ZoologyDepartment for making this presentation.