fungal infection in silver carp, hypophthalmichthys ...

Sci.Int.(Lahore),26(1),261-266,2014

ISSN 1013-5316; CODEN: SINTE 8

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FUNGAL INFECTION IN SILVER CARP, HYPOPHTHALMICHTHYS MOLITRIX (VALENCEINNES) REARED IN EARTHEN POND Zafar Iqbal, Uzma Najam and Saira Saleemi Department of Zoology, University of the Punjab Quaid-E-Azam Campus, Lahore. Pakistan. Corresponding author: [email protected]

ABSTRACT: This study was conducted to investigate fungal infections in a freshwater culturable fish, silver carp, Hypophthalmichthyes molitrix. A total of 35 freshly caught fishes were studied. The body weight (BW) and total length (TL) ranged from 53.55–246.0g 15.96–30.6cm respectively. Different body parts of fish such as; head, eyes, operculum, gills, buccal cavity, skin and all fins were examined for fungal infection. Thirteen fishes showed fungal infection, with clinical signs such as: ruptured skin, eroded scales, damaged fins, cataract in eyes and lesions on skin. Fungi isolated from these tissues of fish were inoculated on three different media, Sabouraud Dextrose agar (SDA), Potato Dextrose agar (PDA) and Malt Dextrose agar (MEA). A total of 223 agar plates were prepared (71 MEA, 57 SDA and 95 PDA). Inoculated agar plates were incubated for 5-6 days at 25-300 C. The fungal colonies appeared on agar plates, which were black, green, grey, white and orange in colors. Slides were prepared by taking material from fungal colonies on agar plates and were stained with 0.05% Trypanblue in lactophenol. Four fungal genera; Aspergillus., Mucor., Penicillium and Rhizopus were isolated and identified. Incidence of Aspergillus sp. was the highest (98.64%) compared to other three genera (0.45%). The infection level on different parts of fish was variable such as: gills 17.93%, buccal cavity 10.72%, eyes 10.24%, operculum 12.10%, head 10.3% skin 16.14%, dorsal fin l1.8%, caudal fin 4.93%, pectoral fin 3.14%, and skin lesions 2.7%. The infection on anterior parts (61.29%) of the fish was significantly higher than the posterior part (38.72%) (χ2=3.39; P=0.05). Pure culture plates produced uniform colonies of same colour, showing the growth of one type of fungus, Aspergillus spp. The fungal infection observed in silver carp probably occurred through the use of unhygienic and contaminated feed given to fish in pond. Fish infection by pathogenic fungi reduces the economic and nutritional value of fish. The present situation points our attention for very good fish health management practices in the fish ponds. Key Words: Silver carp, rearing pond, fungal infection, Aspergillus sp.

Introduction The carp culture is growing fast, especially in the province of Punjab Pakistan, where the numbers of private fish farms have increased to 7829 (area 45650 acres) [1]. Three indigenous carp species, Labeo rohita (Hamilton) Catla catla (Hamilton) Cirrhinus mrigala (Hamilton) and two Chinese carps, grass carp Ctenopharyngodon idella (Valenceinnes) and silver carp, H. molitrix are reared extensively under semi-intensive culture system in Pakistan [2, 3]. The growth of fish farming has also raised the issues of fish diseases. [4, 3] reported four common fish diseases in fish ponds in Punjab; saprolegniasis, lernaeasis, bacterial hemorrhagic septicemia and anoxia. Environmental conditions that arise in the fish farms during rearing of fish, represent a considerable stress that make fish more susceptible to attack by a wide range of pathogens such as; parasites, bacteria, fungi and viruses. Diseases affect growth of fish and result in poor production [5, 6]. The skin of the fish with loss of mucus, or without mucus is unprotected and fungal spores starts developing on it and increase susceptibility by the attack of fungus such as; Saprolegnia sp. [7]. The injuries in fishes which are caused by high stocking density, unsuitable water quality and excess of organic manure may be the result of increased Saprolegnia infection in fish [8]. Some species of genus Saprolegnia causes infection in fishes such as; salmonids [9], carps [10, 3], catfish [11]. [12] reported mortality of L. rohita due to fungal infection. High mortality rate in many species of

cultured carps such as H. molitrix, has been associated with Saprolegnia parasitica [13]. According to [14] Aspergillomycosis is the disease of tilapia, Oreochromis sp. caused by Aspergillus spp. [15] reported Fusarium sp., Saprolegnia sp., Penicillium sp. and Mucor sp. in the eggs of Aspencer percicus. Infections caused by Fusarium sp. have increased in recent years in freshwater fish. [16] isolated 16 species of fungi from infected eggs of rainbow trout, O. mykiss in Iran. [17] isolated Saprolegnia sp. from farmed raised fingerlings of L. rhita, C. catla and C. mrigala. [18, 19] reported the occurrence of Saprolegnia sp. and Achyla sp. in C.idella and C. catla. Infection of L. rohita and H. molitrix by two fungal genera Aspergillus and Alternaria has also been reported. [20, 21, 22) have reported incidence of fungal genera such as; Aspergillus, Rhizopus, Mucor, Penicillium, Alternaria and Blastomyces in six ornamental fishes, (C. auratus, its two verities black moor and shubunkin; koi, Cyprinus carpio: platy, Xiphophorus maculates, and guppy, Poecilia reticulate ) imported to Pakistan. The aim of study was to assess the incidence of pathogenic fungi on silver carp reared in earthen ponds at Punjab University Research fish farms Lahore. MATERIALS AND METHODS The total of 35 specimens of silver carp, were obtained from Fish Research Farms, University of the Punjab, Lahore from February to July 2012. The fishes were brought to laboratory in sterile polyethylene bag in aerated pond water and kept in

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glass aquaria with continuous air supply at ambient temperature. Total length (TL) and body weight (BW) of each fish was measured and fungal infection and health status of every specimen was observed. The body of the fish was divided into two parts (Anterior part; comprising, head, eyes, operculum, gills, buccal cavity and posterior part; comprising all the fins, skin, abdomen and rest of the body) to note the infected sites. Chi-square test was applied to compare the infection in anterior and posterior part of the body of the fish [21]. The glassware (containing media and distilled water covered with aluminum foil), vials and test tubes (cotton plugged) were autoclaved at 121°C at 1.054 kg/cm2 for 15 min. Antibiotic streptomycin sulphate 250mg was added to each preparation of media to reduce bacterial contamination. The fish samples were disinfected from surface to prevent secondary contamination with airborne spores by dipping fish in 1% formaldehyde for 1 to 5 min. Then the sample was transferred to 70% alcohol and finally several washings were given in sterilized distilled water. The fungi were isolated from infected organs of fish with sterile needle, inoculated on Malt extract agar (MEA) (Oxoid, UK), Sabouraud dextrose agar (SDA) (Oxoid, UK) and Potato dextrose agar (PDA) (M096-India). Isolation was done in Laminar flow air cabinet to avoid contamination. The agar plates were incubated at 28-30º C and fungal growth was observed after 4-7days. Pure fungal culture was done by picking a small portion of colony with the help of sterilized loop and poured it into distilled water to make spore suspension. Then 0.25ml of spore suspension was taken with sterilized dropper and poured it into agar plate and agitated the spore suspension on the agar plates. The pure culture plates were incubated for 4-5 days and observed it. Slides were prepared by taking material from each colony and stained with 0.05% Trypanblue in Lectophonel. The slides were observed under microscope and photographed. The fungal identification was done with the help of identification keys and literature [23, 24]. RESULTS A total of 35 fishes were examined in this study. The body weight of the fish ranged from 53.55 to 246.0g and total length of fish varied from 15.96 to 30.6cm. Out of 35 fishes, 13 fishes were having fungal infection. The infection was 37.14%. Site of infection was operculum, skin, eyes, pectoral fins and pelvic fins. Skin was infected in six fishes followed by operculum infection in three fish each and eye infection in two fish each. Whereas, pelvic fins and pectoral fins showed infection in one fish each.The clinical signs such as: rupture skin, eroded scales, damaged fins, cataract in eyes and lesions on skin were evident on infected fish (Fig.1). Fungi isolated from various tissues of fish were inoculated on 3 types of agar plates. A total of 223 plates were prepared. Out of which, 71 were of MEA, 57 were of SDA and 95 were of PDA. The tissue wise description of % infection is shown in Table 1. The highest infection 17.93% was seen on gills and lowest infection (1.8%) from dorsal fin. Anterior part of the fish (61.28%) was having

Sci.Int.(Lahore),26(1),261-266,2014

significantly higher infection (χ2=3.39; P=0.05) compared to posterior part (38.72%). Fungal colonies of Black, grey, green, orange and white colour was observed. The most prevalent of fungal isolates belong to Aspergillus sp. (98.64%) and few were of Rhizopus sp., ( 0.45%) Penicillium sp. (0.45%) and Mucor sp. (0.45%) (Fig. 2, 3). One hundred pure culture plates were prepared from the original culture plates from each organ. Pure culture of the fungi produced same colour colonies, which were more uniform in outlook. There was uniform growth spreading all over the petri plate. It indicated the establishment of one genus of fungus, but probably one or more species. Only Aspergillus sp. grew in pure culture (Fig.4, 5, 6). DISCUSSION Fungal infections and its isolates were studied in H.molitrix. Fungi were isolated from different body parts of the fish. Isolated fungi were identified as Aspergillus spp., Rhizopus sp., Mucor sp., and Penicillium sp. The most prevalent of fungal isolates were of Aspergillus spp. followed by the other three. The gills with 17.93% infection were the most affected area. Dorsal fin showed the lowest infection (1.8%). Infection on vital organs like gills and eyes seems to be more serious and fatal. Gill infection result in damage of secondary lamellae and may cause respiratory stress and impairment to fish, and eyes may be become blind due to presence of fungal hyphae [10, 19]. Anterior part of the fish was having significantly higher infection than posterior part of the fish. This is in contrast to [19] who reported that posterior part of the fish had significantly higher infection than anterior part. Higher infection on anterior part of the silver carp may be associated with the fact that these parts interacts with the spores first during swimming and feeding activities in the pond. Infection of C. catla and C.idella by Saprolegnia sp. and Achyla sp. was observed by [18] who suggested it to be associated with factors such as injuries caused by high stocking density, unsuitable water quality and excess of organic manure added in the ponds. According to [23] .fungi have wide range of infection, depending on the management of farm and environment. [18] observed fungal infections in four species of carps i.e., C. auratus, H. molitrix, L. rohita and C. idella and isolated five fungal species, Aspergillus sp., Penicillium sp., Alternaria sp., Blastomyces sp. and Rhizopus sp. [25] described that Aspergillus niger lead to internal and external infection in fish. [26] reported that some species of Aspergillus produce a group of mycotoxins called aflatoxins which is major cause of spoilage of grains and other foods. Aspergillus spp. are presumably infectious and spread in fishes through the use of contaminated fish feed. This view has also been supported by [27]. [14] reported a number of Aspergillus species such as A. flavus, A. japonicus, and A. terreus that are involved in the infection of African fish, especially the tilapia. These species presumably cause infection via entry into the fish through contaminated feed. [28] isolated 7 fungal species from stockfish in Nigeria and these included A. flavus, A. fumigatus, A. niger, Trihophyton verrucosum, Rhizopus

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Mucor and Penicillum sp. and among these Mucor sp. showed the highest occurrence. [16] isolated Penicillium expansum, Penicillium citrinium; Aspergillus terruse, Aspergillus clivatus; Alternaria sp. and 11 other fungal species from infected eggs of rainbow trout. Systemic infections cause intense granuloma formation in salmonids [29] in goldfish and tilapia [30]. Fungi isolated in the present study are comparable to the fungi isolated by [31, 32, 23, 16, 19, 20, 21, 22]. However, [32] has characterized Aspergillus spp., Penicillium spp. and Rhizopus spp. as normal

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mycoflora and these species may be considered as opportunistic pathogens as many of these genera possess virulence factors which enable them to cause disease especially under favorable conditions. This study indicates that although most fungi isolated from fishes are considered as normal mycoflora, but they still cause infections in fishes. It is suggested that proper health management practices must be adapted while rearing fish, so that chances of fungal infection can be minimized

.Table. 1. Tissue wise infection in Hypophthalmichthys molitrix S. No. 1 2 3 4 5 6 7 8 9 10

Tissue Gills Skin Buccal cavity Eye Operculum Head Caudal fin Pectoral fin Skin lesions Dorsal fin Total

No. of plates 40 36 35

% infection 17.93 16.14 10.72

34 27 23 11 7 6 4 223

10.24 12.10 10.30 4.93 3.14 2.70 1.80

Fig.1 A silver carp showing fungal infection from middle of the body to caudal peduncle area (F.28)

A.

B.

Fig. 2 A. Fungal colony from Head on PDA (F.13), B. Aspergillus sp. isolated from colony A (Sporangium with hyphae, 100X)


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A.

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B.

Fig. 3 A. Fungal colony from Head on MEA (F.19, B. Penicillium sp. isolated from colony A

A.

B.

Fig. 4 A. Pure culture of fungal colony from head on PDA (F.17) B. Aspergillus sp. isolated from colony A (Sporangium with hyphae, 100X)

A.

B.

C.

Fig. 5 A. Pure culture of fungal colony from head on SDA (F.21) B and C. Aspergillus spp. isolated from colony A (Sporangium with hyphae, 100X)

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A.

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B.

Fig. 6 A. Pure culture of fungal colony from B.C on SDA (F.22) B. Aspergillus flavus isolated from colony A (Sporangium with hyphae, 100X)

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