Effect of Nigella sativa (Black Cumin Seed) to Enhance the Immunity of Common Carp (Cyprinus carpio) Against Pseudomonas fluorescens

The effect of Nigella sativa (black cumin) seed extracts supplemented diets at 2%, 4% and 6% doses with common diet as control (0%) fed to common carp, Cyprinus carpio against Pseudomonas fluorescens was investigated critically on weeks 1, 2 and 4 as immunomodulator. Immunological factors including bactericidal activity and phagocytic action were examined along with the disease resistance. The outcomes of the research recommend 4% dose of N. sativa oil enriched diet as an effective immune response and disease resistance agent for C. carpio against P. fluorescens although further investigation will facilitate to optimize the precise quantity comparing with the weight of fish body.


Introduction
The common carp is one of the major aquaculture species in the globe Vandeputte [1]. Common carp occur within the temperature range of 3-35°C Froese and Pauly [2]. The optimum water temperature for growth and propagation is 20-25°C. Physically, common carp live in the middle and lower sections of rivers and in areas where the water is shallow and the bottom is mud-spattered FAO [3]. According to Chowdhury et al., [4] Aeromonas sp. and Pseudomonas sp. are very frequent in fish disease particularly in carp and live fishes in Bangladesh. Cultured fish species suffer from Aeromonas sp. and Pseudomonas sp. infections with signs like dermal lesion, scale loss, frayed fins, tail and fin rot and Dropsy Khatun et al., [5]. Several approaches have been made to control these diseases such as disinfection, sanitary prophylaxis and chemotherapy with particular emphasis on antibiotics. Conversely, use of chemicals and antibiotics is often costly and detrimental because it guides the antibiotic and chemical resistance in pathogen as well as reduces consumer preference Harikrishnan et al., [6]. One of the most proficient methods of controlling diseases in fish culture is tightening the defense mechanisms of fish through prophylactic administration immunostimulants from synthetic or natural sources Robertsen, [7].
In recent times, immunostimulants of herbal origin have been shown to have the capacity to boost disease resistance in fish against a number of diseases by improving nonspecific and specific defense mechanisms Harikrishnan et al., [6]. Many herbs have been reported that develop the immunity of fishes, such as Achyranthes aspera Rao et al., [8], Nigella sativa and Quercetin Awad et al., [9], U. dioica Nya and Austin, [10]; Awad and Austin, [11], Viscum album, Urtica dioica and Zingiber officinale Dügenci et al., Common Carp (Cyprinus carpio) Against Pseudomonas fluorescens [12], Eclipta alba Christybapita et al., [13], Astragalus membranaceus and Lonicera japonica Ardo et al., [14], Rheum officinale Xie et al., [15],, Allium sativum, Lupinus perennis, Mangifera indica and Radix astragalin seu Hedysari and Radix angelicae sinensis Jian and Wu, [16] [17] etc. against pathogens. The genus Pseudomonas, of the Pseudomonadaceae family, are motile gram-negative aerobic bacteria, plump-shaped rods with polar flagella which have an key role in pathogenicity Public Health Agency of Canada, [18]. It is eminent that pseudomonads are ubiquitous bacteria in environment. Due to their ability to exploit a wide range of organic compounds, they inhabit a significant ecological arrangement in the carbon cycle Tryfinopoulou et al., [19]. The genus Pseudomonas is prevalent throughout nature and especially characterized by promoted metabolic adaptability and enzymatic organization Franzetti and Scarpellini, [20]. P. fluorescens has ability to form extra cell enzymes such as amylse, protease, chitinase, cellulase and gelatinase Soesanto et al., [21]. Many strains of Pseudomonas fluorescens show potentiality for biological control of phytopathogens especially root pathogens Couillerot et al., [22]. Nigella sativa, known as the black cumin seed, is a spice and food preservative. It comprises many applauded therapeutic properties such as bronchodilatory, hypotensive, antibacterial, antifungal, analgesic, anti-inflammatory Khan, [23], anticancer, anidiabetic, antiradical, immunomodulator, spasmolytic, bronchodilator, hepatoprotective, antihypertensive and renal protective Ramadan, [24], anti-nociceptive, anti-ulcer, anti-histaminic Tembhurne et al., [25]. The present study also intends to evaluate the effect of dietary Black Cumin Seed oil (Nigella sativa) on Common carp, Cyprinus carpio against Pseudomonas fluorescens infection.

Fish Collection and Acclimatization
One hundred twenty (120) pieces of C. carpio, Common carp (average weight 30±5 g, length 16±2 cm) were collected from Madhumati Matshyo Utpadon Kendro, Chanchra, Jessore, Bangladesh. The collected fishes were taken into the laboratory of Fisheries & Marine Bioscience in Jessore University of Science & Technology, Jessore in November, 2014 and acclimatized for three weeks in rectangular shaped indoor aquarium (100L) with continuous airstone aeration. At the beginning the study, each aquarium was disinfected with chlorinated water. The fishes were fed with commercial supplementary feed (without herbal extract) at the rate of 5% of their body weight twice a day at 09.00 h and 15.00 h while adapting in the experimental environment. The system was subjected to 12 h light: 12 h darkness. Water quality parameters were taken under measurement approach during the entire experiment.

N. sativa Supplemented Diet Formulation
N. sativa seed was collected from local market of Jessore, Bangladesh. Black cumin seed oil was extracted by milling of the seeds from Begum Mill, Puraton Kosba, Jessore. The oil (100 g) was mixed with 1000 ml of 85% ethanol in a 2000 ml conical flask kept for 7 days at room temperature and agitated twice a day following Cooper and Gunn [26] to ensure complete absorption. The extracts were filtered through Whatman No. 1 filter paper and the filtrate was dried under reduced pressure. The residues obtained after evaporation were stored in sterilized screw cap glass container, externally disinfected by flaming on spirit lamp and stored at −20°C until use. The black cumin seed oil extract was mixed at 2%, 4% and 6% with supplementary commercial fish feed in three treatment groups respectively. Control group was fed with the supplementary fish feed (without oil extract) twice a day as the treatment groups were. The experimental basal diets were in pellet form and dried in sun for 06 hours to avoid any microbial contamination.

Culture of Pseudomonas fluorescens
The bacterial strain was cultured in Brain Heart Infusion (BHI) Agar and Nutrient Agar media at the rate of 0.61 g and 0.25 g respectively in 50 ml distilled water. The bacterial strain was gone through serial dilution and put into media in different dilution factors. Then the petri plates were kept in the incubator for 24 hours in 30°C to 120°C. But the bacteria growth was prominent in 37°C. During each experiment freshly cultured bacteria was used to acquire errorless result.

Experimental Design
C. carpio fishes were divided into four groups each containing 30 fishes (Treatment 1, 2, 3 and control) under two feeding regimes (test group and control group). All treatments were assigned randomly in the aquaria and each aquarium represented one observation. Each treatment group was divided into three replicate groups consisting of 10 fishes. Fishes were fed ad labitum two times a day diets enriched with N. sativa extracts at 0% (control), 2%, 4% and 6%. Uneaten food was removed by a siphon after 1 h of feeding to avoid the reduced quality and opaqueness of the water. The study was carried out for 30 days and experimental diets were fed for 7 days, 7 days and 16 days with 1%, 3% and 5% body weight correspondingly. Partial water exchange was done thrice a week to maintain the water quality.

Serum Collection
Firstly blood samples were collected from caudal vein of individual fish with syringe of 1 ml only once to avoid the influence on the assays due to multiple bleeding and handling stress. The collected blood samples were then transferred to Eppendrop, which was containing Ethylenediaminetetraacetic acid (EDTA) solution as anticoagulant to avoid blood clotting. The blood was then centrifuged at 5000 rpm for 10 minutes at 4°C. The serum was separated in the appendrop with the blood cells. The serum was obtained by centrifugation and was stored in sterile serum tubes at -20°C for further use.

Bactericidal Activity (Specific Immune Response Assay)
P. fluorescens was used to determine the efficacy of both supplements to neutralize the bacterial infection. Stock solution of experimental bacterial strain was prepared to continue the advance study. Broth culture of the bacteria was prepared from the stock. A media was ready to use and 25µl, 50µl and 75µl serum was spread over the nutrient agar and BHI agar media with micro pipette. The bacterial broths were streaked smoothly on the serum after they were mixed in the same amount as the serum to check the bacterial load on the plates even after treating with treatment group. Then the plates were kept in the incubator for 24 h at 37°C. Bacterial load was measured to find out the bactericidal activity of experimental fish's serum.

Mucus Collection and Bacteria Culture (Nonspecific Immune Response assay)
Mucus was collected by scarping the body surface of fishes with a scalpel from four groups (0%, 2%, 4% and 6%) and collected mucus was kept in four eppendorps separately. Same like as serum and bacteria culture, four culture plates for each group were prepared as followed by disc diffusion method. 25µl mucus from each group was mixed with same volume of three different diluted bacterial solutions (10 -3 , 10 -4 and 10 -5 ) and finally all plates were placed in an incubator at 37°C for 24 hours. After 24 hours all plates were observed.

Challenge Test
The susceptibility of fish fed with N. sativa oil extract to a bacterial challenge was examined in vivo and P. fluorescens was used as challenge strain. Herbal extract was challenged 4 weeks after the start of treatments. P. fluorescens strains were prepared from maintaining the serial dilution which LD 50 : 10 -6 CFU ML -1 was used. Fish from each group were injected intraperitoneally with 2.9 x 10 -6 cfu ml -1 live P. fluorescens. Each dilution trial was conducted in triplicates and mortality was recorded daily for 7 days post challenge. The survival rates significantly increased when the dietary N. sativa level was 4%. The cumulative mortality was calculated by following Amend [27] and Relative Percentage of Survival (RPS) was calculated as follows: Percentage of Survival RPS 1 % of Mortality in treated group % of Mortality in control group 100

Statistical Analysis
Values for each parameter measured were expressed as the arithmetic mean ±standard deviation (SD). Consequences of herbal diets consumption on hematological and immunological parameters were tested using one-way ANOVA and the mean values were compared by using Duncan's multiple range tests at 5% level of significance.

Results and Discussion
The global market for herbal drug is estimated as US $62 billion and is likely to develop to US $5 trillion by the year 2050. The worldwide pharmaceutical trade was valued US $550 billion in 2004 and has boosted to US $900 billion in 2008 Maggon, [28]. Black cumin seed (N. sativa) is an herbaceous plant and their oil are used by many nations for diverse curative functions. Recently, numerous researches have been conducted designating the considerable role of black cumin seed in increasing immunity and upholding good health status of fish El-Kadi et al., [29]; Hedaya, [30]; Mandour et al., [31]; Abdel-Ghaffar et al., [32]; Taha et al., [33].

Non-specific Immune Response (Mucus)
2% experimental diet showed much more resistance against P. fluorescens than control diet fed fishes. But the significant (p < 0.05) less amount of bacterial load was exhibited in 4% ( Fig. 1) N. sativa mixed feed. The number of bacterial colony again boosted in 6% trial fed in 1 st , 2 nd and 4 th weeks consequently.

Serum Bactericidal Activity (Specific)
The lowest number of bacterial colonies indicated the efficiency of immune cells in serum to kill the pathogen. With N. sativa the lowest number of colonies, 3 × 10 -5 developing on BHI occurred with the 4% dose (Fig. 2), with highly significant differences to the control, (p < 0.05). The results of the present study revealed a significant increase in the bactericidal activity in day 14 with 4% N. sativa seed oil supplemented diet compared to the control group. The result was similar to the serum bactericidal activity found in kelp grouper, Epinephelus bruneus against Vibrio harveyi by Harikrishnan et al. [34]. Aeromonas hydrophila growth was inhibited by N. sativa (3%) oil and Quercetin (1%) fed rainbow trout, Oncorhynchus mykiss serum Awad et al., [9]. Dip treatment of C. carpio with aqueous leaf extract of Azadirachta indica significantly increased serum protein levels and protected the fish from A. hydrophila infection Harikrishnan et al., [35]. The bath administration of Quil A. saponin with Yersinia ruckeri enhanced the in vitro bactericidal activities in rainbow trout Grayson et al., [36]. The phagocytic activity did not significantly enhance with 2.0% and 6.0% enriched diet on first week against P. fluorescens. Phagocytic activity of 2% enriched with more pace than previous week in week 4. However with 4.0% doses the activity significantly increased on weeks 4 as compared with the control (Fig. 3).

Phagocytic Activity
The highest phagocytic activity was exhibited in week 4 fed with 4% N. sativa oil supplemented ration in the current research. Chinese mixed herbal extracts from Rheum officinale, Andrographis paniculata, Isatis indigotica, and Lonicera japonica containing diets increased phagocytosis of the white blood cells in on crucian carp, Carassius auratus gibelio Chen et al., [37]. In common carp C. carpio and large yellow croaker Papillaria crocea fed a ration containing a mixture of Astragalus membranaceus (root and stem), Polygonatum multiflorum, Isatis tinctoria and Glycyrrhiza glabra containing a mixture diets with 0.5 and 1% observed that significantly increased phagocytic activity Yuan et al., [38]. Nile tilapia, Oreochromis niloticus fed a mixture of Astragalus and Lonicera extracts supplementation diets enhanced the phagocytic activity of blood phagocytes and increased survival rate against Aeromonas hydrophila Ardo et al., [14]. A similar result was reported in carp C. carpio fed a diet combating with Astragalus and Ganoderma Yin et al., [39].

Disease Resistance (Challenge Test)
The cumulative mortality was 40%, 20% and 30% in fish fed with 2.0%, 4.0% and 6.0% supplementation diet for 30 days against P. fluorescens. 4.0% dose diet showed highest relative percentage survivability (85.7%) compare to other dose diet in the experiment ( Table 1). The mortality increased to 20% with 6.0% dose diet. The highest mortality of 70% was observed in fish fed with 0% (control) dose diet (Fig. 4) and the survival rate was highest in 4% dose (Fig. 5).   The challenge test revealed that, maximum mortality was exhibited in control group whereas lowest amount of mortality of fish was experienced in the treatment group fed 4% black cumin seed where the cumulative mortality was 20%. The next highest mortality was in 6% fed fish group from 24 days to 30 days and the mortality was 30%. Dietary incorporation of Urtica dioica at 5% showed significantly higher relative percentage survival (up to 95%) against A. hydrophila Ngugi et al., [40]. Similar mortality was noted by Mohamad and Abasali [41] in C. carpio against A. hydrophila injection. In the challenge test of Yilmaz et al., [42], a significant increase (p<0.05) in the survival rate of Streptococcus iniae infected tilapia fry fed the 84%, 72% and 61% diets supplemented with Cuminum cyminum, while the survival rates of tilapia fry fed the 45% diets did not significantly change in survival rate compared that of tilapia fry fed the 43% diet. Ngugi et al., [40] showed that administration of stinging nettle (U. dioica) at 5% on Victoria Labeo (Labeo victorianus) reduced mortality significantly. The same results were achieved by Harikrishnan et al. [6] for Pueraria thunbegiana supplementation in kelp grouper, Epinephelus bruneus and Harikrishnan et al. [34] for Solanum nigrum supplementation in tiger shrimp, Penaeus monodon against the same pathogenic bacteria Vibrio harveyi. Harikrishnan et al., [43] also reported that, cumulative mortality was 50% and 40% in 0.1% and 1.0% Prunella vulgaris against Uronema marinum infection in Paralichthys olivaceus.

Conclusion
This study demonstrated that N. sativa extract act as immunostimulant and has a positive effect of increasing disease resistance, dominatingly with 4% dose via enhancing the immune response. The dynamic principles of N. sativa have growth promoting capacity and perform as appetizer, prompt the immune system, act as broad spectrum antimicrobial characteristics which will be of immense use in common carp and other carp species culture industry. The present study opens up new approach of research to assess the most effective dose under culture conditions, trialing with purified extract of N. sativa, degree and duration of the resistance offered, administrative regime for different age group of fish and time of application to ensure improved harvest in culture ponds. Further studies are needed to consider the potential of this plant in other fish and pathogen for disease control strategies.