Annals of Microbiology, 58 (3) 527-534 (2008)

Antimicrobial activity of the cyanobacteria Anabaena wisconsinense and Oscillatoria curviceps against pathogens of fish in aquaculture

Mostafa M. EL-SHEEKH1*, Aida M. DAWAH2, Azza M. ABD EL-RAHMAN3, Hamed M. EL-ADEL4, Reham A. ABD EL-HAY2

1Botany Department, Faculty of Science, Tanta University, Tanta; 2Department of Limnology, 3Department of Fish Diseases, Central Laboratory for Aquaculture Research (CLAR), Abbassa, Sharkia; 4Botany Department, Faculty of Science, Benha University, Benha, Egypt

Received 18 February 2008 / Accepted 8 May 2008

Abstract - Cultures of the blue green algae (cyanobacteria) Anabaena wisconsinense and Oscillatoria curviceps were isolated from fish farms and their antimicrobial effects were studied. Solvent extracts of exponential phase algae were screened for antimicrobial activity against different species of Gram positive (Lactobacillus sp. and Bacillus firmus) and Gram negative (Aeromonas hydrophila, Pseudomonas fluorescens and Pseudomonas anguilliseptica) bacteria and the fungi (Aspergillus niger and Saprolegnia parasitica) which were all isolated from diseased fish. Maximum antimicrobial activity was observed with ethanolic extract of O. curviceps against Lactobacillus sp. and Aeromonas hydrophilia, whereas, methanol extracts of A. wisconsinense and O. curviceps had antibacterial effects against B. firmus, A. hydrophila, P. fluorescens, P. anguilliseptica, and the fungi A. niger and S. parasitica. It was also observed that solvent extracts of A. wisconsinense when injected into Oreochromis niloticus which was already injected intraperitoneally with 2 x 106 CFU/ml P. anguilliseptica significantly decreased fish mortality from 50 to 12.5%.

Key words: antimicrobial activity, cyanobacteria, fish diseases.

IntroductIon

Algae are important components of the aquatic ecosystems and are the primary source of food. Algae are also one of the richest sources of bioactive compounds including antibacterial and antifungal compounds. (Borowitzka, 1988a, 1988b, 1995; Volk and Furkert, 2006). There has been an increasing interest in cyanobacteria as a potential source of antifungal and antibacterial substances (Glombitza and Koch, 1989; Schwartz et al., 1990; El-Sheekh et al., 2006). Several studies have already demonstrated that organic solvent extracts of some members of Rhodophyceae, Gelidiella acerosa (Shelat, 1981), Laurencia pinnatifida (Uddin et al., 1991) and cyanobacteria, Nostoc muscorum (DeCano et al., 1990), Chroococcus sp., Oscillatoria sp., Anabaena sp., Synechocystis aquatilis and various microalgae (Katircioglu et al., 2006), Fischerella sp. (Raveh and Carmeli, 2007) possesses antibacterial or/and antifungal activities. The antimicrobial activities are attributed to the ability of cyanobacteria to produce a large number and variety of bioactive allelochemical substances, with a diverse range of biological activities and chemical structures that can affect many biochemical processes within cells. An increasing number of such metabolites are being found to be directed against oxygenic photosynthetic

processes, which, in the microbial world, are unique to algae and cyanobacteria. Such chemicals are likely to be involved in regulating natural populations, and are potentially useful as biochemical tools, and as herbicidal or biocontrol agents (Smith and Doan, 1999). Bacterial infections are considered the major cause of mortality in aquaculture (Grisez and Ollevier, 1995). Because of the growing bacterial resistance against commercial standard and reserve antibiotics, the search for new active substances with antibacterial activity against pathogenic bacteria is of increasing importance (Service 1995; Mundt et al., 2003). In our previous study it was demonstrated that the active principal antimicrobial compound extracted from the cyanobacterium Nostoc muscorum was a phenolic compound (El-Sheekh et al., 2006). The present work is an attempt to demonstrate the antibacterial and antifungal activity of Anabaena wisconsinense and Oscillatoria curviceps. These species were isolated from Abbassa fish farm in Egypt and are the dominant species in the farm. The extracts from these algae were also used in vivo to protect the fish from pathogenic bacterial and fungal infections.

MAterIAls And Methods

Algal analyses.Isolation and culturing of algae. The cyanobacterial strains * Corresponding author. E-mail: mostafaelsheekh@yahoo.com

528 M.M. El-Sheekh et al.

were isolated from fish pond at Abbassa fish farm, Egypt according to Venkataraman (1969). The axenic cultures were obtained using centrifugation cleaning and antibiotic treatment as recommended by Bolch and Blackburn (1996) and finally tested for purity on PYE agar medium (1.5% agar plus 0.2% bacteriological peptone and 0.2% yeast extract). Algae were cultured in 250 ml Erlenmeyer flasks, containing 150 ml sterilised BG11 medium (Rippka et al., 1979). The Erlenmeyer flasks were inoculated with 30 ml of axenic algae. The flasks were aerated with sterile air using 0.2 µm bacterial filter and incubated at 30 °C under continuous illumination provided from cool white fluorescent tubes giving light intensity of 100 µmol·m-2·s-1. This 250 ml Erlenmeyer culture was used for growth measurements and also as inoculums for 20 l glass carboy cultures.

Carboy culture. Algal cultures were also maintained in 20 l glass carboys filled with 19 l of tap water. The water and carboy were disinfected with commercial bleach Clorox (5.25% solution of sodium hypochlorite, NaOCl). For this purpose the bleach was aerated overnight, neutralised with sodium thiosulphate the following day and then aerated overnight once again. Media and algae were added after the second day and maintained for biomass production under continuous illumination (100 µmol·m-2·s-1) and the same conditions as in Erlenmeyer flasks culture.

Determination of algal dry weight. Twenty millilitres of algal suspension from 250 ml Erlenmeyer culture were centrifuged at 805 x g for 10 min. The cell pellet was washed twice with distilled water, transferred to a pre-weighed dry filter paper and dried in an oven at 100 °C overnight to a constant weight (Leganes et al., 1987).

Harvesting, extraction and processing the algal biomass. Algal mass in logarithmic phase of growth was harvested from 20 l glass carboy cultures by filtration using a cheese cloth of cotton or nylon. The algal cells were then dried at 60 °C, ground in an electric coffee mill and the resulting powder was extracted with ethanol, methanol and chloroform 1:15 (p:v) using a soxhlet extractor at 55-60 °C. All samples were refluxed until saturation (24 h) and the respective extracts were dried in a rotavapor. Subsequently, the residual extracts were suspended in the respective solvents to final concentration of 1 mg/20 µl (Lima-Filho et al., 2002).

Isolation and identification of fungi. Aspergillus niger was isolated from fish muscles and the identification was done according to Raper and Fennel (1965). Saprolegnia parasitica was obtained from Fish Diseases Department, Central Lab for Aquaculture Research in Abbassa, Agriculture Research Centre, Egypt, which was isolated previously from diseased Oreochromis niloticus.

Media used for maintaining microorganisms. The media used for primary isolation and identification of bacteria were, Tryptic soya agar medium (TSA, Becton), Tryptic soya broth medium (Becton), and Nutrient agar medium (Difco, 1974), prepared according to Cruickshank et al. (1975). Bacteria were identified based on Analytical Profile Index (API 20E, BioMerieux) a standardised identification system for Enterobacteriaceae and other non-fastidious Gram negative rods. The media used for isolation of fungi were

Oxytetracycline glucose yeast extract agar (Oxoid, 1982) for Aspergillus niger and Sabouroud medium (Larone, 1995) used for culturing of Saprolegnia parasitica.

In vitro tests for antimicrobial activity of algal extracts.Paper disk assay. Nutrient agar media plates were inoculated with 0.1 ml of 108 CFU/ml broth culture of tested bacterial suspension and immediately sterile paper discs impregnated with 30 µl of the cyanobacterial extracted materials that air dried, were placed over the agar surface. The plates were incubated at 37 °C for 24 h and examined for zones of inhibition. Discs with the appropriate solvents used in extraction were used as control (Bauer et al., 1966).

Hollow well technique. This method was used for antifungal activity. Sterile glass beads were placed in sterile Petri dishes and Czapek-Dox agar medium (Thom and Raper, 1945) was allowed to solidify after being inoculated with definite number (104/ml) of fungal spore suspension. After solidification the glass bead was removed leaving a hole. Two hundred and fifty microlitres algal extract were pipetted in the hole. Then the plates were incubated at 28 °C for 72 h after being inoculated with the fungus and examined for inhibition zones (Attaie et al., 1987). Holes with the appropriate solvents used in extraction were used as control.

In vivo test for antimicrobial activity of algal extracts.Bacterial preparation for experimental injection. Pseudomonas anguilliseptica was cultivated on TSA plates and incubated at 25 °C for 24 h. The bacterial growth was recultivated on TSA plates and incubated at 25 °C for another 24 h. The bacterial cells were collected, resuspended, diluted by saline and measured at wave length 650 nm to give optical density 1.2 (107 CFU/ml).

Bacterial isolation. One hundred and twenty individuals of Oreochromis niloticus (between apparently healthy and with those showing signs of disease) were randomly collected from a fish production pond (Abbassa fish farm, Egypt). Forty of the fish varying in body weight and length were taken for bacterial and fungal isolation. The fish organs were dissected under aseptic conditions and incubated at 25 °C for 24-48 h for isolation and identification of bacteria according to Austin and Austin (1993), Bergey et al. (1984) and API 20E strip system. Lactobacillus sp., Bacillus firmus, Aeromonas hydrophilia, Pseudomonas anguil-liseptica and Pseudomonas fluorescens were isolated from eye, stomach, kidneys, gills and intestine of Oreochromis niloticus, respectively.

Fish infection. Eighty of the apparently healthy fish (70-80 g body weight) were allotted into four equal groups and placed in four (77 x 37 x 48 cm) glass aquaria. These aquaria were supplied with chlorine free natural filtered sterilised water and aerator. The physical and chemical analysis of water was as follows: water temperature 28.8 °C, dissolved oxygen 5.8 mg/l, pH 8.6, ammonium 0.60 mg/l, nitrite 0.032 mg/l, nitrate 0.495 mg/l and total phosphorus 0.779 mg/l. The first group was injected intraperitoneally with 2 x 106 CFU/ml Pseudomonas anguilliseptica and kept for 18 h prior to injection with 0.5 ml algal extract (Nau et al., 1995; Abd El-Rhaman and

Ann. Microbiol., 58 (3), 527-534 (2008) 529

El-Ashram, 2005). The second group was infected with 2 x 106 CFU /ml P. anguilliseptica. The third group was just injected with algal extract and the fourth group was not injected with bacteria or algal extract. All groups were observed for 21 days post-infection.

Statistical analysis. Data were analysed and treatments compared using the one-way ANOVA with 95% confidence limits (p < 0.05).

results

Identification of bacteriaBased on the morphology and biochemical characteristics, the bacterial isolates were tentatively identified as Lactobacillus sp., Bacillus firmus, Aeromonas hydrophila, Pseudomonas fluores-cens and P. anguilliseptica (Table 1).

the growth curve of Anabaena wisconsinense and Oscillatoria curvicepsGrowth of Anabaena wisconsinense from Erlenmeyer flask cul-ture increased exponentially through the first 16 days, there

after, decline phase began and it continued till the 18th day. On the other hand, Oscillatoria curviceps showed an exponential increase through the first 14 days and there after the decline phase began (Fig. 1). Algal cultures were also maintained in 20

FIG. 1 - Growth of Anabaena wisconsinense and Oscillatoria curviceps from 250 ml Erlenmayer flask culture. Vertical bars represent ± standard error of the mean of three replicates.

TABLE 1 - Physical and biochemical characteristics of the isolated bacteria

Character Lactobacillus sp. Bacillus firmus

Pseudomonas fluorescens

Pseudomonas anguilliseptica

Aeromonas hydrophilia

Gram stain +ve +ve -ve -ve -veShape Cocci, bacilli Bacilli Bacilli Bacilli Cocci, bacilliArrangement Single, short chain Single Single Single SingleOxidase + - + + -Catalase + + + + +O/F O/- - O /- - FMotility - - + + +Indol - - - + +V.P. - - . + +M.R. . + . + +H2S - - + - -Gelatin - + + + +Nitrate - + + + +Arginine . + . + +Ornithine . - + - +Lysine . - - - -Production of acid from:Glucose + - + - +Sucrose + D + + +Sorbitol + + + - -Xylose + - + + -Maltose + + + + +Manitol + + D + -Galactose + D + + +Growth on:0% NaCl . + + + +3% NaCl . + + - +5% NaCl . + - - -6.5% NaCl . + - - -7% NaCl . - . - -Growth at 45 ºC . + . - -

O/F: Oxidative/Fermentative, V.P.: Voges- Proskauer reaction, M.R.: methyl red, (.): test was not carried out, D: different result with different replicates, mostly positive.

530 M.M. El-Sheekh et al.

l glass carboys and the resulting biomass (0.5 g/l) was used for extractionand antimicrobial activities investigation.

Antimicrobial activities of Anabaena wisconsinense extractsMethanolic extract of Anabaena wisconsinense inhibited the growth both Gram negative and Gram positive bacteria except Lactobacillus species and the diameter of the zones of inhibi-tion varied from maximum of 50 to a minimum of 30 mm in P.

anguilliseptica and P. fluorescens, respectively. The inhibition zone with Aspergillus niger was 20 mm in diameter in case of methanolic extraction of Anabaena. Chloroform and ethanol extracts of A. wisconsinense were not as effective. Ethanolic extract of A. wisconsinense gave inhibition zones 34, 24 and 12 mm diameter with Aeromonas hydrophilia, B. firmus and P. fluorescens while the zone of inhibition was 26 mm with A. niger. However, Saprolegnia parasitica was not inhibited by all algal extracts (Table 2).

TABLE 2 - The antimicrobial activities of Anabaena wisconsinense extracts against different test organisms

Organisms Algal extract (µl)

Inhibition zone (diameter, mm)

Methanol Chloroform Ethanol

Algal extract Control Algal extract Control Algal extract Control

Bacteria

Aeromonas hydrophilia 30 32.0 ± 2.2b 6.0 ± 0.06c n.d. 0.0 ± 0.0a 34.0 ± 1.4a 6.0 ± 0.29a

Bacillus firmus 30 32.0 ± 1.9b 8.0 ± 0.08b n.d. 0.0 ± 0.0a 24.0 ± 1.4b 8.0 ± 0.34a

Lactobacillus sp. 30 n.d. 4.0 ± 0.70c 16.0 ± 0.98c 0.0 ± 0.0a n.d. 6.0 ± 0.08a

Pseudomonas anguilliseptica 30 50.0 ± 3.5a 10.0 ± 0.33a 20.0 ± 2.03b 0.0 ± 0.0a n.d. 6.0 ± 0.18a

Pseudomonas fluorescens 30 30.0 ± 2.3b 0.0 ± 0.0d 30.0 ± 3.1a 0.0 ± 0.0a 12.0 ± 1.2c 6.0 ± 0.08a

Fungi

Aspergillus niger 250 20.0 ± 1.8a 0.0 ± 0.0a n.d. 0.0 ± 0.0a 26.0 ± 3.1a 0.0 ± 0.0a

Saprolegnia parasitica 250 n.d. 0.0 ± 0.0a n.d. 0.0 ± 0.0a n.d. 0.0 ± 0.0a

Data presented are the mean diameter of ± standard error of inhibition zones of three replicates; values having different script at the same column are significantly (P < 0.05) different. Significance of diameters lower than 10 mm is marginal. n.d.: not detected.

TABLE 3 - The antimicrobial activities of Oscillatoria curviceps extracts against different test organisms

Organisms Algal extract (µl)

Inhibition zone (diameter, mm)

Methanol Chloroform Ethanol

Algal extract Control Algal extract Control Algal extract Control

Bacteria

Aeromonas hydrophilia 30 n.d. 6.0 ± 0.6c n.d. 0.0 ± 0.0a 30.0 ± 2.70a 6.0 ± 0.1a

Bacillus firmus 30 n.d. 8.0 ± 0.8b n.d. 0.0 ± 0.0a 14.0 ± 1.7c 8.0 ± 0.3a

Lactobacillus sp. 30 34.0 ± 2.21a 4.0 ± 0.4c n.d. 0.0 ± 0.00a 30.0 ± 2.9a 6.0 ± 0.30a

Pseudomonas anguilliseptica 30 16.0 ± 1.5b 10.0 ± 1.1a 14.0 ± 1.4a 0.0 ± 0.0a 20.0 ± 1.6b 6.0 ± 0.3a

Pseudomonas fluorescens 30 n.d. 0.0 ± 0.00d 10.0 ± 1.20b 0.0 ± 0.0a n.d. 6.0 ± 0.2a

Fungi

Aspergillus niger 250 20.0 ± 1.9a 0.0 ± 0.0a n.d. 0.0 ± 0.0a 28.0 ± 2.0a 0.0 ± 0.0a

Saprolegnia parasitica 250 n.d. 0.0 ± 0.0a 49.0 ± 4.3a 0.0 ± 0.0a n.d. 0.0 ± 0.0a

All experiments are carried out in triplicate. Data presented are the mean diameter of ± standard error of inhibition zones of three replicates; values having different script at the same column are significantly (P < 0.05) different. Significance of diameters lower than 10 mm is marginal. n.d.: not detected.

Ann. Microbiol., 58 (3), 527-534 (2008) 531

Antimicrobial activities of Oscillatoria curviceps extractsMethanol, chloroform and ethanol extracts of O. curviceps caused growth inhibition of Lactobacillus sp., P. anguilliseptica and A. hydrophila, respectively. The inhibition zone varied according on the type of extract from organism to organism. The extract of Oscillatoria exerted no effect on Saprolegnia parasitica (Table 3). Chloroform extract of O. curviceps had inhibition zones 14, 10, 10, and 6 mm in diameter with P. anguilliseptica, P. fluorescens, A. hydrophila and Lactobacillus sp., respectively (Table 3). The highest growth inhibition was attained with chloroform extract of O. curviceps against S. parasitica.

experimental studies of Pseudomonas anguillisepticaInjection of P. anguilliseptica into O. niloticus caused 50% mor-tality indicating that Pseudomonas anguilliseptica is pathogenic to O. niloticus at 2 x 106 CFU/ml (Table 4). The infection of O. niloticus showed clinical signs and post-mortem lesions (Fig. 2). External gross lesions included multiple areas of haemorrhage on the fins and the body as well as scale loss. Internal gross lesions were multiple foci of haemorrhage in the liver, intestine and kid-neys necrotic foci in the liver. These lesions are consistent with bacterial septicaemia.

efficiency of Anabaena wisconsinense methanolic extract as a protection against Pseudomonas anguilliseptica infected Oreochromis niloticusThe intrapretonial inoculation of 2 x 106 CFU/ml of P. anguil-liseptica caused mortality 50% among O. niloticus, while the treated O. niloticus with methanolic extract of A. wisconsinense had mortality of 12.5%. The other two groups did not show any mortalities or clinical signs (Table 5).

dIscussIon

In the present study five different bacterial species name-ly Lactobacillus sp., Bacillus firmus, Aeromonas hydrophila, Pseudomonas fluorescens, Pseudomonas anguilliseptica and the fungus Aspergillus niger were isolated from O. niloticus and identified tentatively based on their phenotypic characteristics (Table 1). The present observation also confirms the earlier observations of the presence of Lactobacillus sp. (Austin and Austin, 1993; Abd El-Rhaman, 2003), Bacillus firmus (Lavrova and Mikhnovskaia, 1978; Austin, 1983), P. fluorescens (Abd El-Rhaman et al., 2002), P. anguillisceptica (Ellis et al., 1983) in fish. Cipriano (2001), Rehulka, (2002) and Düğncl and Candan (2003) reported that A. hydrophila is highly pathogenic bacteria to both cultured and wild fishes. These results agree with that of this investigation where A. hydrophila was isolated from kidney of naturally infected Oreochromis. The present observation also confirms the earlier observations of the presence of A. niger in fish (Olufemi et al., 1983). However, some of the isolates do dif-fer from the reported species such as its phenotypic characters (Rueger and Koploy, 1980; Guffanti et al., 1986). The study also demonstrated that extracts of A. wisconsinense inhibited growth of Lactobacillus sp., B. firmus, A. hydrophila, P. fluorescens, Pseudomonas anguilliseptica and A. niger. The methanolic extract showed the highest antagonism against A. hydrophila, P. fluorescens, P. anguilliseptica and A. niger. Oscillatoria curviceps extracts had high antimicrobial activity against Lactobacillus sp., A. hydrophila, Pseudomonas anguilliseptica, A. niger and S. parasitica.

FIG. 2 - Oreochromis niloticus experimentally infected with Pseudomonas anguilliseptica. Note hemorrhages on the skin and fins (A), congestion of internal organs (B) and necrosis in the liver (C).

TABLE 4 - Mortality of Oreochromis niloticus injected intraperitoneally with Pseudomonas anguilliseptica and non-treated during 3 weeks incubation period

Time Pseudomonas anguilliseptica(2 x 106 CFU/ml)

Control(0.2 ml sterile saline)

Fish survival (average No.)

Mortality (%)

Fish survival (average No.)

Mortality (%)

0.0 day 20 0.0 20 0.0

1st day 18 10 ± 2.1 20 0.0

2nd day 15 25 ± 3.0 20 0.0

3rd day 13 35 ± 1.4 20 0.0

4th day 12 40 ± 0.4 20 0.0

1st week 10 50 ± 4.3 20 0.0

2nd week 10 50 ± 4.3 20 0.0

3rd week 10 50 ± 4.3 20 0.0

Pseudomonas anguilliseptica was reisolated from the freshly dead Oreochromis niloticus which injected intrapretonially with 0.2 ml of saline. Data presented are the mean ± standard error of the mean of 4 replicates.

532 M.M. El-Sheekh et al.

These results are in agreement with the results obtained by Oufdou et al., (2001) who examined the inhibitory effect of methanolic extract of Pseudanabaena sp. isolated from waste-water stabilisation ponds at Marrakech. Ostensvik et al. (1998) examined aqueous and methanolic extracts from five selected cyanobacteria (Tychonema bourrellyi, Aphanizomenon flos-aquae, Cylindrospermopsis raciborskii, Anabaena lemmermanni, and Microcystis aeruginosa) for antibacterial properties. Methanolic extracts exhibited more pronounced zones of growth inhibition than aqueous extracts. In the Escherichia coli bioassay, methanolic extracts from two cyanobacteria, Tychonema bourrellyi, and Aphanizomenon flos-aquae, showed inhibitory effects. However, Katircioglu et al. (2006) found that acetone and ether extracts of Chroococcus sp. and Oscillatoria sp. had antimicrobial activity against Pseudomonas aeruginosa. While, ethanol extract from Anabaena had antibacterial activity against Bacillus megaterium and B. thuringiensis. However, chloroform extract of Chroococcus sp., Oscillatoria sp., Synechocystis aquatilis, and Chlorella vulgaris did not show any antimicrobial activity against tested microorganisms. Our results confirmed that chloroform extract of O. curviceps gave the highest activity against S. parasitica. Amer (2002) and El-Sheekh et al. (2006) examined the production of antibiotic substance from two species of cyanobacteria Anabaena laxa and Nostoc muscorum. They found that methanolic extract of Anabaena laxa had the highest antimicrobial activity against B. subtilis, E. coli, A. niger and Penicillium sp. Nostoc muscorum showed high biological activity against Bacillus subtilis, B. cereus, E. coli, Salmonella typhy and A. niger. Cyanobacteria produce a variety of bioactive metabolites that may have allelochemical functions in the natural environment, such as in the prevention of fouling by colonising organisms. Cyanobacterial metabolites have the potential for use as antibacterial, antialgal, antifungal and antimacrofouling properties (Dahms et al., 2006). The cyclic peptides laxaphycins are responsible for the antifungal and cytotoxic activity of crude ethanolic extracts from the cultured blue-green alga Anabaena laxa (Frankmölle et al., 1992). The results showed that P. anguilliseptica was pathogenic at 2 x 106 CFU/ml in O. niloticus and gave mortality 50%. The experimental infection of O. niloticus showed clinical signs as haemorrhages all over the body and fins and scale loss. The post-mortem findings indicated septicaemia with haemorrhages in the liver, intestine and kidneys and

necrotic foci in the liver. Our results are in agreement with the results obtained by Stewart et al. (1983) who recorded that P. anguilliseptica caused mass mortalities among pond cultured European eel. Gross lesions reported included petechial haemorrhages in the skin and pale livers with hemorrhagic foci. Successful control of bacterial diseases of cultured fish is both a major management and economic problem for the aquaculture industry (Plumb, 1999). But recently, antimicrobials from algae and cyanobacteria are receiving attention as a candidate to improve the fish health and the prevention of bacterial diseases for environment friendly aquaculture. The O. niloticus which injected experimentally by intrapretonial inoculation with P. anguilliseptica and treated by 0.5 ml of A. wisconsinense methanolic extract showed mortality 12.5%, while experimental injection of P. anguilliseptica without treatment gave mortality 50%. This means that the algal extracts was active in vitro and in vivo. Austin et al. (1992) indicated that extracts derived from Tetraselmis suecica inhibited Aeromonas hydrophila, A. salmonicida, Vibrio anguillarum and V. salmonicida in vitro. The proper management is essential to success of aquaculture operations, while the inadequate management is the principle factor in triggering disease outbreaks (Austin and Austin, 1993; Plumb, 1999). Investigations are required to further determine the mode(s) of action of cyanobacterial antimicrobial compounds and to investigate the possible chemical composition of active materials.

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TABLE 5 - Efficiency of Anabaena wisconsinense methanolic extract against the pathogenic Pseudomonas anguilliseptica injected into Oreochromis niloticus

Pseudomonas anguilliseptica

Methanol extract of Anabaena

Bacteria +Algal extract Control

Examined fish (No.) 20 20 20 20

Dose 2 x 106 CFU/ml 0.5 ml 2 x 106 CFU/ml + 0.5 ml 0.2 ml saline

Route of injection IP IP IP IP

Mortality rate (%) 50 ± 4.3a* 0.0c 12.5 ± 1.4b 0.0c

IP: Intraperitoneal inoculation. * Data presented are the mean ± standard error of 4 replicates; values having different script at the same row are significantly different (P < 0.05).

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