1. ZAFAR IQBAL., R. MUGHAL and U. SHEIKH
Department 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

2. ABSTRACT
Six 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.

3. Introduction
Fungi: 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

4. 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

5. 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

8. 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

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

10. MATERIALS AND METHODS
Isolation of fungus
Sterilization of sample
In 1 % Formaldehyde - 5 min dip
or 70% alcohol -5min dip
Rinse in distilled water
Fish collection- Pet shop/PU Research fish farm
Observations of fungal infection
Fresh mount preparation/microscopy

11. PREPARATION OF CULTURE MEDIA
Malt 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)

12. INOCULATION OF AGAR PLATES
Fungus 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 colonies
Physical examination and labeling of colonies
Slide preparation
For identification of fungus

13. 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.

14. RESULTS AND DISCUSSION
Fish examined /infected
Labeo 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

26. 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

27. 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).

38. 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.

39. 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

40. CONCLUSION
The 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.

41. 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.

42. Thanks