Vitamin B12 produced by Cetobacterium somerae improves host resistance against pathogen infection through strengthening the interactions within gut microbiota.

BACKGROUND: Pathogen infections seriously affect host health, and the use of antibiotics increases the risk of the emergence of drug-resistant bacteria and also increases environmental and health safety risks. Probiotics have received much attention for their excellent ability to prevent pathogen infections. Particularly, explaining mechanism of action of probiotics against pathogen infections is important for more efficient and rational use of probiotics and the maintenance of host health. RESULTS: Here, we describe the impacts of probiotic on host resistance to pathogen infections. Our findings revealed that (I) the protective effect of oral supplementation with B. velezensis against Aeromonas hydrophila infection was dependent on gut microbiota, specially the anaerobic indigenous gut microbe Cetobacterium; (II) Cetobacterium was a sensor of health, especially for fish infected with pathogenic bacteria; (III) the genome resolved the ability of Cetobacterium somerae CS2105-BJ to synthesize vitamin B12 de novo, while in vivo and in vitro metabolism assays also showed the ability of Cetobacterium somerae CS2105-BJ to produce vitamin B12; (IV) the addition of vitamin B12 significantly altered the gut redox status and the gut microbiome structure and function, and then improved the stability of the gut microbial ecological network, and enhanced the gut barrier tight junctions to prevent the pathogen infection. CONCLUSION: Collectively, this study found that the effect of probiotics in enhancing host resistance to pathogen infections depended on function of B12 produced by an anaerobic indigenous gut microbe, Cetobacterium. Furthermore, as a gut microbial regulator, B12 exhibited the ability to strengthen the interactions within gut microbiota and gut barrier tight junctions, thereby improving host resistance against pathogen infection. Video Abstract.

Screening and evaluating honokiol from Magnolia officinalis against Nocardia seriolae infection in largemouth bass (Micropterus Salmoides).

Nocardiosis caused by Nocardia seriolae is a major threat to the aquaculture industry. Given that prolonged therapy administration can lead to a growth of antibiotic resistant strains, new antibacterial agents and alternative strategies are urgently needed. In this study, 80 medicinal plants were selected for antibacterial screening to obtain potent bioactive compounds against N. seriolae infection. The methanolic extracts of Magnolia officinalis exhibited the strongest antibacterial activity against N. seriolae with the minimal inhibitory concentration (MIC) of 12.5 µg/ml. Honokiol and magnolol as the main bioactive components of M. officinalis showed higher activity with the MIC value of 3.12 and 6.25 µg/ml, respectively. Sequentially, the evaluation of antibacterial activity of honokiol in vivo showed that honokiol had good biosafety, and could significantly reduce the bacterial load of nocardia-infected largemouth bass (p < .001). Furthermore, the survival rate of nocardia-infected fish fed with 100 mg/kg honokiol was obviously improved (p < .05). Collectively, these results suggest that medicinal plants represent a promising reservoir for discovering active components against Nocardia, and honokiol has great potential to be developed as therapeutic agents to control nocardiosis in aquaculture.

Ononin: A candidate anti-parasitic drug isolated from Spatholobi caulis against infections of Dactylogyrus intermedius (Monogenea).

Dactylogyrus is a common parasitic pathogen, which causes high mortality of fish when presents in large numbers, resulting in serious economic losses. Herbal medicines contain myriad of bioactive compounds is a valuable reserve for developing safe and effective anti-parasite drugs. Here, we conducted bioassay-guided fractionation to isolate and identify the anti-parasitic constituents from Spatholobi caulis. Among five extraction solvents (petroleum ether, chloroform, ethyl acetate, methanol and water), S. caulis methanolic extract had the highest parasiticide activity in Carassius auratus, and therefore subjected to further separation and purification using multiple chromatography methods. One compound exhibiting the strongest parasiticidal activity was obtained and identified as ononin by analyzing its spectral data (NMR and ESI-MS). The EC50 value of ononin against Dactylogyrus was 0.655 mg/L and showed 100% parasiticide activity with 3.0 mg/L. The 24, 48, 72, 96 h LC50 for goldfish were 4.691 (the 95% CI of 4.526-4.873) mg/L, 4.612 (4.441-4.800) mg/L, 4.472 (4.345-4.607) mg/L, 4.288 (4.155-4.428) mg/L, respectively. The present results discovered for the first time that ononin had potent parasiticidal activity and have the potential to be developed as new anti-parasitic drug for the control of Dactylogyrus.

Ophiopogon japonicus inhibits white spot syndrome virus proliferation in vivo and enhances immune response in Chinese mitten crab Eriocheir sinensis.

White spot syndrome virus (WSSV) is a fatal pathogen threatening global crustacean industry with no commercially available drugs to control. Herbal medicines have been widely used to treat a number of viral infections, which could offer a rich reserve for antiviral drug discovery. Here, we evaluated the inhibition activities of 30 herbal medicines against WSSV in Chinese mitten crab Eriocheir sinensis. A WSSV infection model in E. sinensis was firstly established in order to determine the antiviral effects of the plant extracts and to explore the potential action mechanisms. Results showed that the highest anti-WSSV activity was obtained by the treatment of Ophiopogon japonicus extract (93.03%, 100 mg/kg). O. japonicus treatment decreased viral loads in a dose-dependent manner and significantly improved the survival of WSSV-challenged crabs. O. japonicus reduced the expression of vital genes in viral life cycle in vivo, particularly for the immediate-early stage gene ie1. Further results indicated that O. japonicus could repress the JAK-STAT signaling pathway to block ie1 transcription. Moreover, O. japonicus could modulate certain immune genes such as the myosin, toll-like receptor, crustin, and prophenoloxidase in the interactions between WSSV and crabs. The up-regulated expression of pro-autophagic factors (Gabarap and Atg7) and elevated levels of antioxidant enzymes (SOD, CAT and GSH) suggested that O. japonicus may induce autophagy and attenuate WSSV-induced oxidative stress. Taken together, O. japonicus could inhibit WSSV proliferation and improve the survival of WSSV-challenged crabs. Thus, O. japonicus may have the potential to be developed as a preventive or therapeutic agent against WSSV, and its effective compounds merit further isolation and identification.

Dietary supplementation of salidroside increases immune response and disease resistance of crucian carp (Carassius auratus) against Aeromonas hydrophila.

Some medicinal plants have been known as immunostimulants, and the medicinal plants extract has been used to control the outbreak of the disease in aquaculture for many years. In this study, a total of 270 crucian carp (30 ± 5 g) were randomly distributed in 9 aquaria (55 cm l × 40 cm w × 50 cm h) and divided into three feeding groups including 0 (Control), 50 mg kg-1 (Diet A) and 100 mg kg-1 (Diet B) of salidroside. The expression of immune-related genes (IL-1ß, TNFα, MYD88, CXCL-8, TGF-ß, and IL-11) in the kidney had a significant increase when the crucian carp fed with Diet B for 4 weeks (P < 0.05). Meanwhile, the expression of IL-1ß, TNFα, and CXCL-8 in the spleen was significantly up-regulated when the fish fed with Diet B (P < 0.05). Higher serum alkaline phosphatase (AKP) activity, catalase (CAT) activity, superoxide dismutase (SOD) activity, and complement C3 content were found in the fish which fed with salidroside-supplemented diet. Our results also proved that fish fed with salidroside-supplemented diet for four weeks, especially at a concentration of 100 mg kg-1 diet, improved the protection of crucian carp against A. hydrophila. The amount of A. hydrophila in the kidney and spleen was significantly decreased in salidroside-supplemented diet groups (P < 0.05). In conclusion, the present results demonstrate that the addition of salidroside for four weeks can improve the immune response of crucian carp and increase the protection against the pathogen, especially at the concentration of 100 mg kg-1 diet. The protective effect of the salidroside to the crucian carp could be used as alternatives to antibiotics for controlling fish diseases in aquaculture.

Ursolic acid from Prunella vulgaris L. efficiently inhibits IHNV infection in vitro and in vivo.

Infectious hematopoietic necrosis virus (IHNV) is a fish viral pathogen that causes severe disease and huge economic losses in the salmonid aquaculture industry. However, anti-IHNV drugs currently are scarce. For the purpose of seeking out anti-IHNV drugs, the anti-IHNV activities of 32 medicinal plants were investigated by using epithelioma papulosum cyprini (EPC) cells. Among these plants, Prunella vulgaris L. (PVL) showed the strongest inhibition on IHNV replication with an inhibitory percentage of 99.3% at the concentration 100 mg/L. Further studies demonstrated that ursolic acid (UA), a major constituent of PVL, also showed a highly effective anti-IHNV activity. The half-maximal inhibitory concentration (IC50) at 72 h of UA on IHNV was 8.0 µM. Besides, UA could significantly decrease cytopathic effect (CPE) and the viral titer induced by IHNV in EPC cells. More importantly, UA also showed a strong anti-IHNV activity in vivo, as indicated by increasing the survival rate of rainbow trout and inhibiting viral gene expression. Intraperitoneal injection of UA increased the relative percentage of survival of rainbow trout by 18.9% and inhibited IHNV glycoprotein mRNA expression by > 90.0% in the spleen at the 1st-day post-infection. Altogether, UA was expected to be a therapeutic agent against IHNV infection in aquaculture.

Synthesis and antiviral activity of a new arctigenin derivative against IHNV in vitro and in vivo.

Viral diseases in aquaculture were challenging because there are few preventative measures and/or treatments. Our previous study indicated that imidazole arctigenin derivatives possessed antiviral activities against infectious hematopoietic necrosis virus (IHNV). Based on the structure-activity relationship in that study, a new imidazole arctigenin derivative, 4-(8-(2-ethylimidazole)octyloxy)-arctigenin (EOA), was designed, synthesized and its anti-IHNV activity was evaluated. By comparing inhibitory concentration at half-maximal activity (IC50), we found that EOA (IC50 = 0.56 mg/L) possessed a higher antiviral activity than those imidazole arctigenin derivatives in our previous study. Besides, EOA could significantly decrease cytopathic effect (CPE) and viral titer induced by IHNV in epithelioma papulosum cyprinid (EPC) cells. In addition, EOA significantly inhibited apoptosis induced by IHNV in EPC cells. Further data verified that EOA inhibited IHNV replication in rainbow trout, with reducing 32.0% mortality of IHNV-infected fish. The results suggested that EOA was more stable with a prolonged inhibitory half-life in the early stage of virus infection (1-4 days). Consistent with above results, EOA repressed IHNV glycoprotein gene expression in virus sensitive tissues (kidney and spleen) in the early stage of virus infection. Moreover, histopathological evaluation showed that tissues from the spleen and kidney of fish infected with IHNV exhibited pathological changes. But there were no lesions in any of the tissues from the control group and EOA-treaten group. In accordance with the histopathological assay, EOA could elicited anti-inflammation response in non-viral infected rainbow trout by down-regulating the expression of cytokine genes (IL-8, IL-12p40, and TNF-α). Altogether, EOA was expected to be a therapeutic agent against IHNV infection in the field of aquaculture.

Isolation of anti-Saprolegnia lignans from Magnolia officinalis and SAR evaluation of honokiol/magnolol analogs.

To control the fish fungal pathogen Saprolegnia, the effects of the petroleum ether extracts of Magnolia officinalis were evaluated by a rapeseed (Brassicanapus) microplate method in vitro. By loading on an open silica gel column and eluting with petroleum ether-ethyl acetate-methanol, honokiol (C18H18O2) and magnolol (C18H18O2) were isolated from Magnolia officinalis. Saprolegnia parasitica growth was inhibited significantly when honokiol concentration was >8.0 mg/L, and magnolol concentration was >9.0 mg/L, with EC50 values of 4.38 and 4.92 mg/L, respectively. Six honokiol and magnolol derivatives were designed, synthesized and evaluated for their anti-Saprolegnia activity. According to the results, double bond and hydroxyl played an important role in inhibiting Saprolegnia. Mechanistically, through the scanning electron microscope observation, honokiol and magnolol could cause the Saprolegnia parasitica mycelium tegumental damage including intensive wrinkles and nodular structures. Moreover, compared to traditional drugs kresoxim-methyl (LC50 = 0.66 mg/L) and azoxystrobin (LC50 = 2.71 mg/L), honokiol and magnolol showed a lower detrimental effect on zebrafish, with the LC50 values of 6.00 and 7.28 mg/L at 48 h, respectively. Overall, honokiol and magnolol were promising lead compounds for the development of commercial drugs anti-Saprolegnia.

Synthesis and in vitro activities evaluation of arctigenin derivatives against spring viraemia of carp virus.

Spring viraemia of carp virus (SVCV) is a viral fish pathogen causing high mortality in several carp species and other cultivated fish. However, robust anti-SVCV drugs currently are extremely scarce. For the purpose of seeking out anti-SVCV drugs, here a total of 35 arctigenin derivatives were designed, synthesized and evaluated for their anti-viral activities. By comparing the inhibitory concentration at half-maximal activity (IC50) of the 15 screened candidate drugs (max inhibitory response surpassing 90%) in epithelioma papulosum cyprini (EPC) cells infected with SVCV, 2Q and 6 A were chosen for additional validation studies, with an IC50 of 0.077 µg/mL and 0.095 µg/mL, respectively. Further experiments revealed that 2Q and 6 A could significantly decrease SVCV-induced apoptosis and have a protective effect on cell morphology at 48 and 72 h post-infection. Moreover, the reactive oxygen species (ROS) induced upon SVCV infection could be obviously inhibited by 2Q and 6 A, while SVCV-infected cells were clearly observed. On account of these findings, 2Q and 6 A could have a promising application for the treatment of infection of SVCV and provide a considerable reference for novel antivirals in aquaculture.

Magnolol protects Ctenopharyngodon idella kidney cells from apoptosis induced by grass carp reovirus.

Many natural products from medicinal plants are small molecular weight compounds with enormous structural diversity and show various biological activities. Magnolol is a biphenol compound rich in the stem bark of Magnolia officinalis Rehd et Wils., and is able to suppress viral replication in GCRV-infected grass carp (Ctenopharyngodon idella) kidney (CIK) cells in the previous study. In this study, in vivo studies demonstrated that magnolol was efficient to restrain the replication of GCRV and repair the low level of superoxide dismutase and total antioxidant capacity in serum at the non-toxic concentration in vivo. Furthermore, magnolol inhibited CIK cell apoptosis induced by GCRV and kept the normal cellular morphological structure, reflecting in the protection of CIK cells from cell swelling, the formation of apoptotic bodies, the disappearance of cellular morphology and nuclear fragmentation. Reverse transcript quantitative polymerase chain reaction (RT-qPCR) showed that magnolol facilitated the expression of apoptosis-inhibiting gene bcl-2, while suppressed the expression of apoptosis-promoting gene bax in GCRV-infected cells. Besides, RT-qPCR and enzyme activity assays proved that magnolol suppressed the expression of caspase 3, caspase 8 and caspase 9. Moreover, interactions between magnolol and proteins were predicted by using the STITCH program, which revealed that ten proteins including caspase 3, were involved in the apoptosis pathway, p53 signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway and toll-like receptor signaling pathway. Further assays were performed to test the effect of magnolol on apoptosis pathway, which showed that magnolol dramatically inhibited the activity of caspase 3 rather than those of caspase 8 and caspase 9. Collectively, the present study revealed that magnolol heightened the resistance of grass carp against GCRV infection and refrained GCRV-induced apoptosis, which may be attributed to the direct interaction of magnolol with caspase 3. The present results make a contribution to understanding the mechanisms by which small-molecule drugs possess antiviral activities, and lay a foundation for the development of broad-spectrum antiviral compounds in aquaculture industry.

Magnolol and honokiol from Magnolia officinalis enhanced antiviral immune responses against grass carp reovirus in Ctenopharyngodon idella kidney cells.

Medicinal plants have been widely used for a long history. Exploration of pharmacologically active compounds from medicinal plants present a broad prevalent of application. By examining viral mRNA expression in GCRV-infected Ctenopharyngodon idella kidney (CIK) cells treated with thirty kinds of plant extracts, we identified Magnolia officinalis Rehd et Wils. was able to preferably suppress viral replication. Further studies demonstrated that the main ingredients of magnolia bark, namely, magnolol and honokiol presented protective pharmacological function when treated GCRV-infected CIK cells with a concentration of 2.00 µg/ml and 1.25 µg/ml, respectively. Furthermore, reverse transcript quantitative polymerase chain reaction (RT-qPCR) and western blot showed that both magnolol and honokiol were efficient to restrain the replication of GCRV in CIK cells at non-toxic concentration (2.51 ± 0.51 µg/ml for magnolol, and 3.18 ± 0.61 µg/ml for honokiol). Moreover, it was found that magnolol and honokiol promoted the expression of immune-related genes. Magnolol obviously significantly increased the expression of interferon (IFN) regulatory factor (IRF)7 rather than that of IRF3 in the GCRV-infected cells, leading to the activation of type I IFN (IFN-I). Simultaneously, magnolol drastically facilitated the expression of interleukin (IL)-1ß, but failed to induce the molecules in nuclear factor (NF)-κB pathways. Differently, honokiol strikingly motivated not only the expression of IL-1ß, but also those of tumor necrosis factor α (TNFα) and NF-κB. Interestingly, though honokiol motivated the expression of IFN-ß promoter stimulator 1 (IPS-1), IRF3 and IRF7, it failed to up-regulate the expression of IFN-I, indicating that honokiol enhanced the host innate antiviral response to GCRV infection via NF-κB pathways. Collectively, the present study revealed that magnolol and honokiol facilitated the expression of innate immune-related genes to strengthen the innate immune signaling responses to resist GCRV infection, which contributed to understanding the mechanisms by which small-molecule drugs possessed antiviral activities. In addition, these results lay a foundation for the development of broad-spectrum antiviral compounds in aquaculture industry.

In vitro immunocompetence of two compounds isolated from Polygala tenuifolia and development of resistance against grass carp reovirus (GCRV) and Dactylogyrus intermedius in respective host.

The present study was undertaken to isolate some compounds from methanol extract of Polygala tenuifolia and evaluate their immunostimulatory properties and antiviral activity using grass carp Ctenopharyngodon idella kidney (CIK) cells and GCRV. By applying insecticidal bioassay-guided, chromatography techniques and successive recrystallization, two purified compounds were obtained. The changes of expression of selected immune genes (Mx1, IL-1ß, TNFα, MyD88 and IgM) in C. idella kidney cell lines were evaluated after exposure to these isolated compounds. The results showed that compound 1 and 2 up-regulated to varying degrees of Mx1, IL-1ß, TNFα, and MyD88 in C. idella kidney cells. WST-8 kit assay verified the two compounds has no toxic effects on CIK cell, and furthermore, have in vitro antivirus activity. Especially, that there is keeping 79% cell viability when exposure to compound 2 (100 mg L(-1)). According to in vivo insecticidal assays against Dactylogyrus intermedius, compound 2 exhibited higher efficacy than compound 1, which was found to be 87.2% effective at the concentrations of 5 mg L(-1) and safe to goldfish (Carassius auratus). Besides, the purified compounds were identified by spectral data as: (1) 1,5-Anhydro-D-glucitol and (2) 3,4,5-trimethoxy cinnamic acid. Overall, the results indicate that bath administration of these compounds modulates the immune related genes in C. idella kidney cells and to some extent, eliminate the virus and parasitic infections.

Anthelmintic activity of saikosaponins a and d from radix bupleuri against Dactylogyrus spp. infecting goldfish.

Disease caused by the parasitic helminths Dactylogyrus spp. results in significant economic damage to the aquaculture industry. Treatment using common chemicals (e.g. formalin) is usually dissatisfactory due to environmental problems, risk of residues, toxicity to fish, and the possibility of anthelmintic resistance. The search for an alternative drug is thus becoming more urgent. This study was designed to evaluate in vivo the anthelmintic efficacy of total saponin (TS), saikosaponin a (SSa), and saikosaponin d (SSd) from radix bupleuri (i.e. the dried root of Bupleurum sp.) based on our previous screening works, with the aim of determining which has commercial potential. Results showed that median effective concentration (EC50) values for TS, SSa, and SSd were 2.01, 1.46, and 0.74 mg l⁻¹, respectively. The acute toxicities against goldfish Carassius auratus for TS, SSa, and SSd were also determined, with median lethal concentration (LC50) of 8.99, 11.20, and 1.54 mg l-1, respectively. The resulting therapeutic indices (TIs) indicated that SSa (TI = 7.67) is a potential therapeutic agent for treating Dactylogyrus infection.

Screening Medicinal Plants for Use against Dactylogyrus intermedius (Monogenea) Infection in Goldfish.

Abstract Methanol extracts of 24 traditional medicinal plants with potential anthelmintic activity against Dactylogyrus intermedius (Monogenea) in Goldfish Carassius auratus were investigated. Abrus cantoniensis, Citrus medica, Dioscorea collettii, and Polygonum multiflorum exhibited 100% activity and were selected for further evaluation by applying five solvents (petroleum ether, chloroform, ethyl acetate, methanol, and water) for the extraction of the samples, followed by an in vivo bioassay. Among the plants tested, water, methanol, and ethyl-acetate extracts of P. multiflorum showed the highest efficacies; EC50 values (median concentration that results in 50% of its maximal effect) were 1.9, 5.4, and 9.1 mg/L, respectively, and extracts showed 100% efficacy against Dactylogyrus intermedius at 100, 12.5, and 25 mg/L. This was followed by ethyl-acetate, chloroform, and methanol extracts of Dioscorea collettii, which demonstrated 100% efficacy at 80, 80, and 120 mg/L and had EC50 values of 19.7, 27.1, and 37.8 mg/L, respectively, after 48 h of exposure. Chloroform and ethyl-acetate extracts of C. medica, which exhibited 100% efficacy against Dactylogyrus intermedius at 100 and 125 mg/L, revealed similar activity and had EC50 values of 58.7 and 51.3 mg/L, respectively. The ethyl-acetate and methanol extracts of A. cantoniensis exhibited the lowest activity and had EC50 values of 279.4 and 64.3 mg/L. Acute toxicities of these active extracts were investigated on Goldfish for 48 h. The findings indicated that extracts of the four plants can be developed as a preferred natural antiparasitic for the control of D. intermedius. Received June 15, 2013; accepted February 11, 2014.

Aquatic environmental safety assessment and inhibition mechanism of chemicals for targeting Microcystis aeruginosa.

Cyanobacteria are a diverse group of Gram-negative bacteria that produce an array of secondary compounds with selective bioactivity against vertebrates, invertebrates, fungi, bacteria and cell lines. Recently the main methods of controlling cyanobacteria are using chemicals, medicinal plants and microorganism but fewer involved the safety research in hydrophytic ecosystems. In search of an environmentally safe compound, 53 chemicals were screened against the developed heavy cyanobacteria bloom Microcystis aeruginosa using coexistence culture system assay. The results of the coexistence assay showed that 9 chemicals inhibited M. aeruginosa effectively at 20 mg L(-1) after 7 days of exposure. Among them dimethomorph, propineb, and paraquat were identified that they are safe for Chlorella vulgaris, Scenedesmus obliquus, Carassius auratus (Goldfish) and Bacillus subtilis within half maximal effective concentration (EC50) values 5.2, 4.2 and 0.06 mg L(-1) after 7 days, respectively. Paraquat as the positive control observed to be more efficient than the other compounds with the inhibitory rate (IR) of 92% at 0.5 mg L(-1). For the potential inhibition mechanism, the chemicals could destroy the cell ultrastructure in different speed. The safety assay proved dimethomorph, propineb and paraquat as harmless formulations or products having potential value in M. aeruginosa controlling, with the advantage of its cell morphology degrading ability.

Developmental toxicity in rare minnow (Gobiocypris rarus) embryos exposed to Cu, Zn and Cd.

Using rare minnow (Gobiocypris rarus) embryos as experimental model, the developmental toxicity of Cu, Zn and Cd were investigated following exposure to 0.001-1.000mg/L for 72h, and the toxicity effects were evaluated by larval malformation rate, heart rate, pericardial area, spontaneous movements, tail length, enzyme activities and biomarker genes. Our results revealed that increased malformation rate provide a gradual dose-response relationship, and the most pronounced morphological alteration was heart and body malformations. Values of 72h EC50 with their 95 percent confidence intervals on G. rarus embryos were 0.103 (0.072-0.149)mg/L for Cu, 0.531 (0.330-1.060)mg/L for Zn, 0.219 (0.147-0.351)mg/L for Cd. Enzyme activities can be regard as a type of low-dose stimulation and high-dose inhibition. Stress and metabolism-related genes (hsp70, cyp1a and mt) were significantly up-regulated, development-related genes (wnt8a, vezf1 and mstn) were significantly down-regulated after the treatment by Cu, Zn and Cd. Overall, the present study points out Cu, Zn and Cd are highly toxic to G. rarus embryos. The information presented in this study will be helpful in fully understanding the toxicity induced by Cu, Zn and Cd in fish embryos.

Effect of intestinal autochthonous probiotics isolated from the gut of sea cucumber (Apostichopus japonicus) on immune response and growth of A. japonicus.

The study isolated 224 bacteria from the intestine of Apostichopus japonicus, then selected and identified three of the bacteria (HS1, HS7, and HS10) which demonstrated amylase, lipase, and protease production capacity as candidate probiotics for sea cucumbers. The three potential probiotics showed no pathogenicity both in hemolytic assays on sheep blood agar plates and after immersing sea cucumbers in a suspension of the bacteria. To reveal the effects of these three potential probiotics on the innate immunity of sea cucumbers, total coelomocyte counts, respiratory burst activity, superoxide dismutase activity, lysozyme activity, acid phosphatase activity, and phagocytic activity by coelomocytes were examined after feeding with four different diets for up to 28 days. Also the specific growth rate and survival rate were investigated after a 60-day feeding trial. Sea cucumbers were fed with 4 diets: one control, three diets supplemented with 1 × 10(9) cell g(-1) of HS1, HS7, and HS10 for 28-60 days. Results showed that sea cucumbers fed diets containing HS1, HS7, and HS10 had led to an enhanced cellular and humoral immune response, notably higher total coelomocytes counts, respiratory burst activity, lysozyme activity, acid phosphatase activity, and phagocytic activity, as recorded during the four weeks of probiotics administration. On the other hand, the survival rate among dietary treatments ranged from 90.71 to 97.97% with significant improvement (P < 0.05) compared to that of the control; and the growth rate observed in the sea cucumbers fed HS1 and HS7 showed sharp increases after 60 days feeding. The present study confirmed the potential beneficial effects of Pseudoalteromonas elyakovii HS1, Shewanella japonica HS7, and Vibrio tasmaniensis HS10 as dietary probiotics in A. japonicus.

Screening of plant extracts for anthelmintic activity against Dactylogyrus intermedius (Monogenea) in goldfish (Carassius auratus).

With the aim of finding natural anthelmintic agents against Dactylogyrus intermedius (Monogenea) in goldfish (Carassius auratus), 26 plants were screened for antiparasitic properties using in vivo anthelmintic efficacy assay. The results showed that Caesalpinia sappan, Lysima chiachristinae, Cuscuta chinensis, Artemisia argyi, and Eupatorium fortunei were found to have 100% anthelmintic efficacy at 125, 150, 225, 300, and 500 mg L(-1) after 48 h of exposure. Crude extract of the five plants were further partitioned with petroleum ether, chloroform, ethyl acetate, methanol, and water to obtain anthelmintically active fractions with various polarity. Among these fractions tested, the ethyl acetate extract of L. chiachristinae was found to be the most effective with a 50% effective concentration (EC50) value of 5.1 mg/L after 48 h of exposure. This was followed by ethyl acetate extract of C. chinensis (48 h-EC50 = 8.5 mg L(-1)), chloroform extracts of C. sappan (48 h-EC50 = 15.6 mg L(-1)), methanol extract of C. chinensis (48 h-EC50 = 15.9 mg L(-1)), and chloroform and petroleum ether extract of L. chiachristinae (EC50 values of 17.2 and 21.1 mg/L, respectively), suggesting that these plants, as well as the active fractions, provide potential sources of botanic drugs for the control of D. intermedius in aquaculture.

In vivo anthelmintic effect of flavonol rhamnosides from Dryopteris crassirhizoma against Dactylogyrus intermedius in goldfish (Carassius auratus).

The purpose of the present study was to assess the anthelmintic property of plant-derived polyphenolic compounds extracted and isolated from Dryopteris crassirhizoma against Dactylogyrus intermedius in goldfish. The active ethyl acetate extract was loaded on an open silica gel column and eluted with chloroform-methanol. According to (1)H-nuclear magnetic resonance (NMR), (13)C-NMR, and mass spectral data, the structures of three purified compounds were identified as protocatechuic acid, sutchuenoside A, and kaempferitrin. Among these compounds, sutchuenoside A and kaempferitrin were observed to be effective with median effective concentration (EC50) of 3.01 and 2.71 mg L(-1), respectively. The alterations in the tegument of the parasites treated with isolated compound were examined using scanning electron microscopes. Ultrastructural micrographs revealed shrinkage of body surface, dense tegumental folds, and disheveled protuberances. The structural deformities in the treated parasites were indicative of an efficient anthelmintic activity of the isolated compound kaempferitrin. In addition, the 48-h median lethal concentration for sutchuenoside A and kaempferitrin against goldfish were 12.03- and 11.98-fold higher than corresponding EC50. The present results showed that ethyl acetate extract of D. crassirhizoma may be considered as a potent source, and sutchuenoside A and kaempferitrin as new natural parasitic agents against D. intermedius.

Anthelmintic efficacy of Santalum album (Santalaceae) against monogenean infections in goldfish.

Dactylogyrus spp. and Gyrodactylus spp. are helminth ectoparasites that are a significant threat to the aquaculture industry. Existing treatments could cause the threats of anthelmintic resistance, risk of residues, environmental contamination, and toxicity to fish. Importantly, there is no report on a treatment against these two parasites. This study explored the possibility of using the extracts of Santalum album to treat Dactylogyrus sp. and Gyrodactylus sp. infections in goldfish. Results showed that among the four extracts (chloroform, methanol, ethyl acetate, and water) of S. album, the chloroform extract is the most effective and 40 mg/L is a safe and the lowest effective dosage. In addition, we found that Gyrodactylus elegans is more sensitive than Dactylogyrus intermedius when exposed to the extract of the medicinal plant. Finally, it is substantiated that bath treatment with long duration and multiple administrations could eliminate a greater proportion of monogenean infections. These findings show the potential for the development of effective and safe therapy to treat Dactylogyrus sp. and Gyrodactylus sp. infections of fishes.