ZAFAR IQBAL., R. MUGHAL and U. SHEIKHDepartment of Zoology, University of the

Punjab,New Campus, Lahore.

A paper read at 31st Pak. Cong. Zoology (International) University of AJK,

Muzaffarabad. 19-21st April, 2011

ABSTRACTSix freshwater fish species, Labeo rohita (Hamilton); Catla

catla (Hamilton); Ctenopharyngodon idella (Valenciennes, 1844); Hypophthalmichthys molitrix (Richardsons, 1845) and Carassius auratus (L.) were studied for fungal infection.

Labeo rohita and C. auratus showed fungal infection with typical cotton wool like appearance at gills, head, fins, skin and bloody spots at the sites of infection. Grossly, the skin color was faded and fins were eroded in infected fishes. The fungi isolated from the infected areas 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 were labeled. The slides were prepared by taking the material from each colony and stained with Trypanblue in Lactophenol. The stained slides 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 only Aspergillus sp was isolated from caudal fin. The probable causes of fungal infection in these fishes are discussed.

IntroductionFungi: Heterotrophs (require living /dead matter for growth/reproduction)Habitat: Freshwater/marine; at cool/ warm temperature

Class Oomycetes (water mould) Members are ubiquitous fungi, normal flora of estuarine ecosystem, have worldwide

distribution. virtually every freshwater is susceptible to infection by at least one fungal species

Family 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 FUNGAL INFECTION IN FISHES

Pathogenic fungi-considered opportunists and attack fish when ;

Stressed Immuno-compromised due to unfavorable environmental

condition or secondary to bacterial / viral infection or when they have lost their mucus protection because of

trauma or excessive handling (Neish,1990; Byl et al, 1993;Quinjou et al,1998)

Some members of family Saproleniace are also primary pathogens that cause disease without predisposing factors

SAPROLEGNIASIS

EUS: ULCERATIVE EPIZOOTIC SYNDROME

(Aphanomyces invadans)

In carps and snakehead, Pakistan (1995-96)/S.E.Asia, Lilly et al, 1998 attack skin/muscles

Others Names

GM: Granulmatous disease

RSD: Red sore disease

UM: Ulcerative disease

A disease caused by Genra; Saprlegnia; Achyla; Dictyuchus in salmonids/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/granulmatous

gill rot acute/localized fungal disease of gills in many freshwater fish/worldwide

AIMS

Isolation of pathogenic Fungi from some imported ornamental fish and culturable fishes

MATERIALS AND METHODS

Isolation of fungusSterilization of sample In 1 % Formaldehyde - 5 min dip or 70% alcohol -5min dipRinse in distilled water

Fish 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/ autoclave 90mins.at15psi)

INOCULATION OF AGAR PLATESFungus from infected fish/sterilized needle/ inoculated agar plate

Incubation of agar plates

one week at room temperature (28° C) before inoculation

5 days at 32-35° C after inoculation

Observations of fungal coloniesPhysical examination and labeling of colonies

Slide preparationFor identification of fungus

STAINING OF SLIDES Stain: Trypanblue in Lactophenol

100ml 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. rohita

Fish Infected organ Fungus

Carassius auratus Gills Aspergillus sp

Mucor sp

Pelvic fins Aspergillus sp

Caudal fins Aspergillus sp

skin Penicillum sp

Labeo 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 pathogenic

Smoked 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 within feed.

Infection results into aflataxicosis- (poisoning leading to cancerous spleen, kidney) produced by aflatoxins,

aflatoxins, elaborate on feed stuff by proliferating Aspergillus mould during storage of oil seed in warm and humaid environment as found in tropical countries

Toxins can be deposit on pellets, if eaten by fish cause acute toxicity- 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, Aspergillus spp, Rhizopus spp, Mucor spp, and

Penicillum spp from 8 edible smoked-dried freshwater fishes; Claris gariepinus; Chrysichthys nigrodigitatus; Sarotheodon galilaeus,Heterotis niloticus, Heterbranchus bidorsalis, Synodontis schall,Synodonts clarias and Clarias angullaris These fish species were rich in protein and dietary minerals such as calcium, sodium, zinc, potassium, phosphorous and iron and with low fungal infestation hence are recommended for human consumption.

Junaid, et al., (2010) isolated 7 fungi from stockfish, in Nigeria, as under; 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 to Pakistan and sold live to hobbyist.

Cause of infection may be the use of contaminated feed in the pet shop

Or the fish might be infected before arriving in Pakistan.The infection of Labeo rohita may also be attributed to the

contaminated kitchen waste fed to fish.Same may be the case with Mucor spp. and Penicillium spp

infection in 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 and humans

CONCLUSIONThe presence of the toxigenic fungi

increase the risk for mycotoxin production. Mycotoxin can pose an important danger to human and animal health because they are toxic to vertebrates and other animals in low concentration. The consumption of these fungi exposes the consumers to the toxic metabolites produced by the fungi.

Public Health awareness is necessary and stressed.

Acknowledgement

Special thanks to Directorate of Research and Development PU Lahore, for providing funds for this Study.

Thanks to Chairman Department of Zoology and Botany PU Lahore for providing facilities.

Many thanks to Mr. Abdul Rehman of Zoology Department for making this presentation.

Thanks