Antagonism of Aeromonas hydrophila by propolis and its effect on the performance of Nile tilapia, Oreochromis niloticus

Fish & Shellfish Immunology 27 (2009) 454–459

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Antagonism of Aeromonas hydrophila by propolis and its effect on the performance of Nile tilapia, Oreochromis niloticus Azza M.M. Abd-El-Rhman* Fish Health and Management Dep, Central Laboratory for Aquaculture Research, Abbassa, Abu-Hammad, Sharkia 44662, Egypt

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 May 2009 Received in revised form 10 June 2009 Accepted 21 June 2009 Available online 27 June 2009

Propolis, a resinous substance collected by Apis mellifera bees from various plant sources and mixed with secreted beeswax, is a multifunctional material used by bees in the construction, maintenance, and protection of their hives. The collected propolis sample, from High Egypt, was dark-green with olive-odor. The minimal inhibition concentration (MIC) of propolis-ethanolic-extract, against Aeromonas hydrophila, was 80 mg Propolis-ethanolic-extract and crude propolis (1%) were added to artificial basal diet with (30% crude protein) to evaluate their efficacy on the fish growth-performance, immunostimulation and resistance to A. hydrophila. Two hundred and twenty-five Oreochromis niloticus (8  0.45 g/fish) were divided into three equal treatments (T) of triplet replicates. The fish of T1 were fed on basal diet (control). The fish of T2 were given the basal diet, containing propolis-ethanolic-extract. The fish of T3 were given the basal diet containing crude propolis for 28 day. The fish were intraperitoneally challenged by A. hydrophila (0.2  107 cells ml1) at the end of the feeding period and kept for 15 more days. The best growth rate and feed conversion ratio were obtained with T2. The increase in the average daily gain, specific growth rate and feed efficiency ratio were highly significances in T2 followed by T3 when compared with the control group. The HCT-level and monocyte-counts were increased (T2). No significant change, in the large lymphocytic-count was found among the three treatments (28–27–28%), while the neutrophil-count was significantly decreased (7%) with T2 and increased (13.11%) with the control. A significant increase in serum lysozyme and serum bactericidal activities was found with T2. The RLP against A. hydrophila was high with T2 and T3. The propolis-ethanolic-extract enhanced the growth, immunity and resistance of O. niloticus against A. hydrophila more than the crude propolis. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Propolis Oreochromis niloticus Aeromonas hydrophila MIC Growth Lyzosyme Serum bactericidal

1. Introduction Propolis is a resinous sticky substance produced by honeybees. The bees collect it from trees, buds, flowers and other botanical sources and mixed with hypo pharyngeal secretions for protection of hives as sealer, draught excluder and embalming substance to cover arises from hive-invaders and against bacterial and fungal infection [1,2]. Propolis has many different biological and pharmacological properties such as antibacterial, antifungal, antiviral, antiprotozoae, local-anesthetic, anti-inflammatory and immunostimulant [3–5]. The most potent microbicidal componant in propolis is flavanone pinocembrin (5,7-dihydroxyflavanone) [6]. Propolis exhibits bacteriostatic activity against different bacterial genera and can be bactericidal at high concentrations [7]. Fish diseases, especially bacterial infections, are a major problem facing the fish farming industry, which is currently growing fast with an * Tel.: þ20 55 3400498. E-mail address: [email protected] 1050-4648/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2009.06.015

annual increase of approximately 12% [8]. The motile Aeromonas group, especially Aeromonas hydrophila, affects a wide variety of freshwater fish species and occasionally marine fish and has been associated with tail and fin rot, hemorrhagic septicemia and epizootic ulcerative syndrome (EUS) [9,10]. Nile tilapia, Oreochromis niloticus (L.) is an important species for freshwater aquaculture, and the improvment of its culture and disease-resistance is a major challenge facing fish culturists. The use of antibiotics, in disease prevention and growth promotion, can bring about the emergence of drug-resistant microorganisms and leave antibiotic residues in the fish and in the environment [11–13]. Moreover, the chemotherapy may kill or inhibit the normal microflora in the digestive tract, which is beneficial to fish [14]. One of the most promising methods for controlling diseases in aquaculture is strengthening the defence mechanisms of fish through prophylactic administration of immunostimulants [15]. Non-specific Immunostimulants have been recently attracting the attention, since they enjoy lower costs and being easily incorporated in the diet, besides having a low impact on the environment [2].

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This study was designed to evaluate the increased Oerochromis niloticus resistance to Aeromonas hydrophila by using crude propolis and its ethanolic-extract as non-specific immunostimulant and to study their effect on growth performance.

2.3.1. Growth performance The average weight-gain (AWG), specific growth rate (SGR), feed conversion ratio (FCR) and feed efficiency ratio (FER) were calculated according to the following equations:

2. Materials and methods

AWGðg=fishÞ ¼ Average final weightðgÞ  Average initial weightðgÞ=experimental periodðdayÞ:

2.1. Crude propolis and its ethanolic-extract Crude propolis sample was collected in summer from Upper Egypt using propolis traps and kept in a dark and dry place until used. Propolis-ethanolic-extract was prepared by adding 30 ml of absolute ethanol to 3 g minced propolis in bottles which were sealed and shaken in darkness for 1 day at room temperature. The extract was then filtered twice and stored in sealed bottles at 4  C until used [2]. 2.1.1. Antibacterial activity Agar-disc-diffusion method was employed for the determination of antibacterial activity of propolis-ethanolic-extract against Aeromonas Hydrophila. A. hydrophila was obtained from The Fish Disease Dep, Central Laboratory for Aquaculture Research in Abbassa. A. hydrophila was isolated from the liver of Oreochromis niloticus, suffering from hemorrhagic septicemia. A suspension of A. hydrophila (0.5 Mac-Farland scales) was spread on solid medium plates. Filter-paper-discs were impregnated with 20, 40 and 60 ml of propolis-ethanolic-extract and another with ethanol only (control). The inoculated plates were incubated at 35  C for 24 h. Diameters of the inhibition zones were measured in millimetres according to [16]. The test was performed in triplicate. 2.1.2. Minimal inhibition concentration (MIC) The minimum inhibitory concentration, of propolis-ethanolicextract, was determined by broth micro dilution method. Propolisethanolic-extract was evaporated at 50  C. Then 0.5 g of the residue was dissolved in 1 ml distilled water at 35  C. Serial dilutions from (1:10) using tryptic soya broth (TSB) was prepared and inoculated by one loopful of A. hydrophila broth (24 h live). The test-tubes were incubated at 35  C for 18–24 h and checked for bacterial growth. A loopful, from each tube, was streaked on propolis free agar medium to check the bacterial growth. The plates were incubated at 35  C for 24 h. 2.2. Feed preparation Commercial basal diet (crude protein 30%) was crushed, and then divided into three parts. The first part was mixed with ethanol (control), second part was mixed with 1% propolis-ethanolicextract and the third part was mixed with ethanol containing crude propolis (1%). The diet was reformed into pellets, spread to dry and stored at 4  C for the feeding experiment [2]. 2.3. Feeding experiment Two hundred and twenty-five (225) Oreochromis niloticus (8  0.45 g/fish) were randomly collected from earth ponds of Abbassa Fish Farms, Abu-Hammad Sharkia, Egypt. The fish were acclimatized for two weeks, and then divided into three equal groups. Each group, in the three replicates contained 25 fish. The first group (T1) was fed on the control diet (diet free from propolisethanolic-extract and crude propolis). The second group (T2) was given the basal diet containing propolis-ethanolic-extract. The third group (T3) was given the basal diet containing the crude propolis. The fish were hand-fed twice daily ad libitum for 28 days. The water of the aquaria was changed daily. The fish were weighed 0, 7, 14 and 28 day from the start of the experiment.

SGRð%=dayÞ ¼ 100ðIn final body weightðgÞ  ln initial body weightðgÞÞ=experimental periodðdayÞ: FCR ¼ Feed intakeðgÞ=weight gainðgÞ: FER ¼ Body weight gainðgÞ=Feed intakeðgÞ: 2.3.2. Organ index The fish were killed by rapid cervical chopping, and the fish were weighed at the end of experiment. The liver and spleen were removed and weighed. Moreover, the hepatosomatic and splenosomatic indices were calculated according to [17].

Organ-somatic index ¼ ½organ weightðgÞ=body weightðgÞ100:

2.4. Blood and serum sampling At the end of the feeding experiment fish were anaesthetized by immersing the fish in water containing 0.1 ppm tricaine methane sulphonate (MS-222). Blood-samples were collected from the caudal vein of fish, by using needles previously rinsed in heparin for the evaluation of hematocrit [18] and differential white blood count [19]. For serum separation, 0.5 ml blood-samples were withdrawn from the caudal vein; of other collected fish into blood Eppendorf tubes without anticoagulant in syringe. The Eppendorf tubes, containing the blood samples were centrifuged at 3000 g for 15 min and the supernatant serum was collected. The serum was stored at 20  C in screw capped glass vials until used for serum bactericidal activity and lysozyme activity studies. 2.4.1. Lysozyme activity The lysozyme activity was measured using photoelectric colorimeter with attachment for turbidity measurement. A series of dilution was prepared by diluting the standard lysozyme from henegg-white (Fluka, Switzerland) and mixed with Micrococcus lysodeikticus (ATCC No. 1698 Sigma) suspension for establishing the calibration curve. Ten ml of standard solution or serum were added to 200 ml of micrococcus suspension (35 mg of Micrococcus dry powder/95 ml of 1/15 M phosphate buffer þ 5.0 ml of NaCl solution). The changes in the extinction were measured at 546 nm by measuring the extinction immediately after adding the solution which contained the lysozyme (start of the reaction) and after a 20 min incubation of the preparation under investigation at 40  C (end of the reaction). The lysozyme content is determined on the basis of the calibration curve and the extinction measured [19]. 2.4.2. Serum bactericidal activity (SBT) The SBT integrates both pharmacokinetic and pharmacodynamic properties in a single set of determinations that examines the ability of the fish’s serum. Bacterial culture, from A. hydrophila, was centrifuged and the pellet was washed and suspended in phosphate buffer saline. The optical density of the suspension was adjusted to 0.5 at 546 nm. This suspension was serially diluted (1:10) with PBS five times. Serum bactericidal activity was determined by incubating 2 ml of this diluted bacterial suspension with

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25

2.08 T2

T3

20

b 15

a

10 a b

b

a

a

c

b

b

HSI

a

2.06

a

HSI (%)

Mean wieght (g)

T1

a

2.04 2.02 2

b

a

1.98

5

1.96 T1

0 0

7

14

T2

T3

Treatments

28

period feeding in day Fig. 1. Mean weight of Oreochromis niloticus fed for 28 day with commercial basal diet supplemented with crude propolis (T3) and its ethanolic-extract (T2) and commercial basal diet free from crude propolis and its ethanolic-extract (control, T1). Means in the column with different superscripts were significantly different (P < 0.05).

20 ml of serum in a micro-vial for 1 h at 37  C. In the bacterial control group, phosphate buffer saline replaced the serum. After incubation, the number of viable bacteria was determined by counting the colonies after culturing on TSA plates for 24 h at 37  C.

Fig. 3. Effect of crude propolis (T3) and its ethanolic-extract (T2) fed for 28 day on hepato-somatic index (HSI) of Oreochromis niloticus with no significant variation among treatments and the control group (T1).

2.6. Statistical analysis Statistical analysis was performed using the one way analysis of variance (ANOVA). It was performed with SPSS statistical software (version 10.0, SPSS). The data were subjected for test of homogeneity of variances and Duncan post-hoc test. Data were considered significantly different when P < 0.05.

2.5. Challenge test 3. Results After the 4 weeks of the feeding experiment, 30 fish from each group were divided into three subgroups (30 fish distributed in 3 aquaria, 10 in each). The fish was challenged intraperitoneally with 0.2 ml 107 cells of 24 h cultures of live A. hydrophila. The challenged fish were kept under observation for 15 days. The dead fish were used for bacterial re-isolation. The mortalities were recorded and the relative level of protection (RLP) among the challenged fish was determined according the following question:

The collected propolis sample was dark-green and sticky with olive-odor at room temperature. The employed MIC dose of propolis-ethanolic-extract 80 mg, for antibacterial evaluation, showed no inhibition-zone against Aeromonas hydrophila 102 cells ml1 of TSB by agar-diffusion method.

RLP ¼ 1  ½percentage of treated mortality=

3.2. Growth performance

percentage of control mortality  100:

The highest growth rate was obtained with propolis-ethanolicextract (T2), followed by the crude propolis (T3), while the lowest one was obtained with the control (T1) (P < 0.05) 28 day from the start of feeding. The growth rate was significant increased (T2), and insignificantly (T3), 7 and 14 day from start of feeding, when compared with T1 (Fig. 1). The average daily gains (ADG), specific growth rate (SGR) and feed efficiency ratio (FER) were highly significant with T2 followed by T3 and the lowest with T1 (Fig. 2). On the other hand, the best feed conversion ratio (FCR) was obtained with T2 (0.8), while the highest one (2.0) was obtained with T1 (Fig. 2).

4 3.5

T1

T2

T3

a

3

b

2.5

3.1. Antibacterial activity and MIC

2

a 1.5

c

0

SSI

a c

b

Average daily gain

b

SGR

FCR

a

0.1

c

b

0.12

FER

Growth parameters at the end of feeding experimental Fig. 2. Effect of crude propolis and its ethanolic-extract on growth parameters (average daily gain, specific growth rate (SGR), food conversion ratio (FCR), and feed efficiency ratio (FER)) of Oreochromis niloticus fed for 28 day. T1 commercial basal diet free from crude propolis and its ethanolic-extract, T2 fish group fed with commercial basal diet containing propolis-ethanolic-extract, T3 fish group fed with commercial basal diet containing crude propolis. Means in the column with different superscripts were significantly different (P < 0.05).

SSI %

c 0.5

SSI

0.14

a

1

0.08

a a

0.06 0.04 0.02 0 T1

T2

T3

Treatments Fig. 4. Effect of crude propolis (T3) and its ethanolic-extract (T2) fed for 28 day on spleno-somatic index (SSI) of Oreochromis niloticus with no significant variation among treatments and the control group (T1).

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T1

T2

a

50

b c

40

(%)

80

T3

30 a a a 20 10

a c

b

a

a b

ab

Bacterial count (CFU/µl)

60

b

b

Small Large Monocytes lymphocytes lymphocytes

70

a

60 50 40 b

30 20 c

10 0

0 HCV

457

T1

Neutrophils

T2

T3

Treatments

Differential white blood-count and hematocrit-value Fig. 5. Effect of crude propolis (T3) and its ethanolic-extract (T2) fed for 28 day compared to control group (T1) on the differential white blood-count and hematocritvalue of Oreochromis niloticus. Means in the column with different superscripts were significantly different (P < 0.05).

Fig. 7. Effect of crude propolis (T3) and its ethanolic-extract (T2) feeding for 28 day on serum bactericidal activity against Aeromonas hydrophila of Nile tilapia compared to the control group (T1). Means in the rows with different superscripts were significantly different (P < 0.05).

3.3. Organ index

number of the bacterial colonies, in T2 and T3, was significantly lower than the control group (Fig. 7).

The hepatosomatic (Fig. 3) and splenosomatic (Fig. 4) indices were not increased significantly in T2 and T3.

3.7. Challenge test

3.4. Hematocrit-value and differential white blood cell-count The HCT-level was significantly increased with T2 (21%), when compared with the control (9.5%) as shown in Fig. 5. The small lymphocytic-count was significantly increased with T2 than T3, when compared with the control (Fig. 5). The monocytic-count was increased with T2 only (11%) without any significant change with T1 and T3 (7%). The large lymphocytes were not significantly changed in the three treatments (28–27–28%), while the neutrophil-count was significantly decreased (7%) with T2 and increased (13.11%) with the control (Fig. 5). 3.5. Lysozyme activity The serum lysozyme activity was significantly increased with T2 followed by T3, when compared with the control group (Fig. 6). 3.6. Serum bactericidal activity (SBT) The serum bactericidal activity against A. hydrophila, was significantly higher with T2 and T3, than the control group. The

The fish-mortalities kept increasing in time until the 6th day post-challenge (PC) (Fig. 8). T2 and T3 exhibited enhanced immunity against the challenging with A. hydrophila infection (42 and 45% mortality respectively) when compared with T1 (85% mortality). The RLP, against A. hydrophila, was highly significantly increased in fish of T2 (50.59%) when compared with T1 (0.0%). 4. Discussion Propolis is a very complex mixture of balsam and resin (50%), wax (30%), essential oils (10%), pollen (5%) and of various other substances (5%) like sugars and vitamins [20,21]. The collected propolis sample was dark-green with olive-odour. [2] reported that propolis varies in colour according to its botanical source and declared that the most common propolis colour is dark-brown. The agar-diffusion-method showed that 60 mg propolis-ethanolic-extract had no activity against Aeromonas hydrophila. On the other hand [16,22] found inhibition zones for propolis-ethanolicextract against Staphylococcus aureus which ranged from 0 to14 mm diameters and 8–8.8 mm against Escherichia coli [23].

90

2.5 a 2 b 1.5

c

1 0.5

Cumulitive mortality, %

Lysozyme content (µg/ml)

3

80 70 60 50 40 30 20

0

0 T1

T2

T3

Treatments Fig. 6. Effect of crude propolis (T3) and its ethanolic-extract (T2) on serum lysozyme content after 28 day feeding compared to the control group (T1). Means in the rows with different superscripts were significantly different (P < 0.05).

T1

10 1

2

3

4

5

6

7

8

9

T2

T3

10 11 12 13 14 15

Time (day) Fig. 8. Fifteen day cumulative mortality % of Nile tilapia fed commercial basal diet free from crude propolis and its ethanolic-extract (T1), commercial basal diet supplement with 1% ethanolic-extract of propolis (T2) and commercial basal diet supplemented with 1% crude propolis (T3) for 28 day and challenged intrapretonially with pathogenic A. hydrophila.

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The minimal inhibition concentration (MIC) of propolis-ethanolic-extract against A. hydrophila was 80 mg. Moreover [24], found that 100 and 300 ml of Bulgarian propolis-ethanolic-extract inhibited the growth of Helicobacter pylori strains by the disc-diffusionmethod. The gram-positive bacteria were more susceptible to propolis-ethanolic-extract, at low concentration than the gramnegative. [25–30] mentioned that both the Gram-positive and Gram-negative bacteria were sensitive to propolis but the most sensitive one was the Gram-negative, so until now the effective propolis-ethanolic-extract against different bacteria is unsettled. Flavonoids have been considered to be the main biologically active components in propolis [31,32]. The antibacterial activity of propolis varied according to the dosage extraction-solvents, different geographic locations and climate and besides season of collection. However, all the samples of the different geographic locations exhibited a significant antibacterial activity [16,28,30,33–35]. [36] found that the Egyptian propolis contains phenolic acid esters (72.7%); phenolic acids (1.1%); aliphatic acids (2.4%); dihydrochalcones (6.5%); Chalcones (1.7%); flavanones (1.9%); flavones (4.6%) and tetrahydrofuran derivatives (0.7%). It was clear that phenolic acid esters are present in a major quantity (72.7%). Egyptian propolis is characterized by the presence of unusual esters of caffeic acid with C12- C16 fatty alcohols, mainly saturated. Flavonoid aglycones and especially flavanones are typical components of poplar propolis. [36] mentioned that the antibacterial activity of Egyptian propolis due to 2,6-bis-(pentanyloxy)-4-pentanylphenethanol, total flavone [37], caffic acid [38] or polyphenol [39]. Propolis-ethanolic-extract and crude propolis evoked the highest growth rate, average daily gain, specific growth rate and feed efficiency ratio among O. niloticus. The best FCR was obtained with propolis-ethanolic-extract, but [2] found little effect of propolis on gilthead seabream growth, fed for up to 6 weeks. Also they found that the specific growth rate was not significantly affected by the dietary intake of propolis at 0.1 and 10 g propolis kg_1 diet. Propolis contains certain vitamin (B1, B, C, E) and essential minerals (iron, aluminum, manganese) and silicon which improve the digestive cofactors and enzymatic activity. These properties could contribute to improving the digestion and nutrient absorption with a subsequent increase in the fish-weight. The hepatosomatic and splenosomatic indices were not significantly increased in T3 and T2, when compared with T1. The organosomatic indices are indicators of health (hepatosomatic index and splenosomatic index) [40] and [17]. The HCT-level was significantly increased (T2), when compared with (T1). The elevated hematocrit-value could explain the efficiency of the used propolis on the health of the fish status. The reduced hematocrit may indicate that, the fish are not eating well (anaemic) or suffering bacterial infections [17,41,42]. [43] reported that the HCT-values were significantly higher in the group fed on diet supplemented with probiotic, when compared with the control group. The differential leucocytic-count is an indicator of heath in fish [17]. The current study showed insignificant change in the count of the large lymphocytes and monocytes among the experimental groups. On the other hand, the neutrophil-count was significantly decreased T2 and increased in T1. [2] reported that water and ethanolic-extracts of propolis increased the percentage of phagocytes (monocyte-macrophages and acidophilic granulocytes) of gilthead seabream. The monocytes in the circulating blood are precursors of tissue macrophages. Propolis enhanced the macrophage-functions and lymphocyte proliferation as well as resistance to several pathogens and tumors in several mammalian species [44–49]. The neutrophil-count was significantly increased in blood of rainbow trout with infected bacterial [50] or intoxicated with heavy metal [51].

The lysozyme activity is an important indicator of the immune defense of both invertebrates and vertebrates. The lysozyme is a fish defense element, which causes hydrolysis of the N-acetylmuramic acid and N-acetylglucosamine which are constituents of the peptidoglycan layer of bacterial cell wall and activation of the complement system and phagocytes by acting as an opsonin [52,53]. In this study propolis-ethanolic-extract and crude propolis significantly increased the serum lysozyme activity, so it stimulated the immune response in Nile tilapia. The increased lysozyme activity has been reported after supplementing the fish-feed, with non-specific immunostimulants as a mixture of propolis and herba epimedii extracts [54], probiotic [55], or injection of b-glucan [56]. The crude propolis and its ethanolic-extract significantly increased the serum bactericidal activity, against A. hydrophila. These results are triggered by an increased lysozyme-activity and monocytic-count which are precursors of macrophages [52]. It is important to estimate the relative level of protection in the treated fish to determine the efficacy of an immunostimulant. The crude propolis and its ethanolic-extract reduced the A. hydrophilainduced mortality when compared with the control group. These results indicate that the crude propolis and its ethanolic-extract activated the immune system of the Nile tilapia. Similarly, decreased mortalities, on challenge with A. hydrophila, were reported in vaccinated Carassius auratus gibelio and injected with propolis water extract [57], Chinese sucker fed on propolis and Herba epimedii extracts [54], O. mossambicus, fed on Eclipta alba leaf aqueous extract [58] and Cyprinus carpio fed on Chinese herbs (Astragalus radix and Ganoderma lucidum) [59]. It could be concluded that the ethanolic-extract of propolis was more effective than the crude propolis in protecting fish against infection. Moreover, it improved the fish performance. References [1] Kosalec I, Bakmaz M, Pepeljnjak S. Analysis of propolis from the continental and Adriati regions of Croatia. Acta Pharmaceutica. 2003;53:275–85. [2] Cuesta A, Rodrı A, Esteban MA, Meseguer J. In vivo effects of propolis, a honeybee product, on gilthead seabream innate immune responses. Fish and Shellfish Immunology 2005;18:71–80. [3] Miyake T, Shibamoto T. Antioxidative activities of natural compounds found in plants. Journal of Agricultural and Food Chemistry 1997;45:1819–22. [4] Santos FA, Bastos EMA, Uzeda M, Carvalho MAR, Farias LM, Moreira ESA, et al. Antibacterial activity of Brazilian propolis and fractions against oral anaerobic bacteria. Journal of Ethnopharmacology 2002;80:1–7. [5] Sforcin JM. Propolis and the immune system: a review. Journal of Ethnopharmacology 2007;113:1–14. [6] Houghton PJ, Woldemariam TZ, Davey W, Baser A, Lau C. Quantitation of the pinocembrin content of propolis by densitometry and high performance liquid chromatography. Phytochemical Analysis 1995;6:207–10. [7] Drago l, Mombelli B, De Vecchi E, Fassina MC, Tocalli L, Gismondo MR. In-vitro antimicrobial activity of propolis dry extract. Journal of Chemotherapy 2000;12:390–5. [8] FAO (Food and Agriculture Organization). Fish Statistics. Rome, Italy: FAO; 2004. [9] Larsen JL, Jensen NJ. An Aeromonas species implicated in ulcer-disease of the cod (Gadus morhua). Nordisk Veterinaer Medicin 1977;29:199–211. [10] Lu CP. Pathogenic Aeromonas hydrophila and the fish diseases caused by it. Journal of Fish China 1992;16:282–8. [11] Weston DP. Environmental considerations in the use of antibacterial drugs in aquaculture. In: Baird D, Beveridge MVM, Kelly LA, Muir JF, editors. Aquaculture and water resource management. Oxford: Blackwell; 1996. p. 140–65. [12] Esiobu N, Armenta L, Ike J. Antibiotic resistance in soil and water environments. International Journal of Environmental Health Research 2002;12: 133–44. [13] FAO/WHO/OIE June 13–16, 2006 Expert consultation on antimicrobial use in aquaculture and antimicrobial resistance. Seoul: Republic of South Korea; 2006. [14] Sugita H, Miyajima C, Deguchi Y. The vitamin B12-producing ability of the intestinal microflora of freshwater fish. Aquaculture 1991;92:267–76. [15] Robertsen B. Modulation of non-specific defense of fish by structurally conserved microbial polymers. Fish and Shellfish Immunology 1999;9:269–90. [16] Gonsales GZ, Oris RO, Fernandes JRA, Rodrigues P, Funari SRC. Antibacterial activity of propolis collected in different regions of Brazil. Venomous Animals and Toxins Including Tropical Diseases 2006;12:276–84.

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