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β-Glucan and triterpenes are two important derivative compounds from traditional medicinal mushroom, such as Ganoderma lucidum and Antrodia cinnamomea. β-glucan and triterpenes are considered to have immunoregulatory properties in disease treatment for long years. The immunoregulatory effects are usually activated through some transcription of pro-inflammatory genes and possess immunomodulatory activity. Difficulty in healing wound now is a common condition that occurred in diabetic patients, and the physiological hyperglycemic status of diabetic patients resulting in the wounds continue to produce an inflammatory response. Thus, we hope to use β-glucan and triterpenes for difficult wound healing that possess immunomodulatory activity on the wound micro-environment and stimulate the positive effects on healing. In this chapter, these two important derivative compounds from traditional medicinal mushroom were examined by diabetic mammal’s wound healing models. In these models, the skin wounds’ microenvironment is expected close to diabetic foot, suffering in hyperglycemic and inflammatory status. The results are clearly presented, with the immunomodulatory effects from mushroom β-glucan and triterpenes that involved in modulating the cell-mediated immune system to cause cellular proliferation and further to introduce healing performance of the chronic inflammation wounds.

In order to simplify the survey of pesticide (including carbamate, organochlorine, organophosphate and synthetic pyrethroid pesticides) residues in fishery products (including bivalve, crustacean, fish and cuttlefish), multiresidue determination methods were developed through a solid phase extraction (SPE) technique. In the present procedures, samples were extracted with acetonitrile except for bivalve and cuttlefish samples. Mixed solvent of water, acetone and acetonitrile was added to bivalve prior to homogenization, whereas water was needed in cuttlefish samples for blending. For both kinds of sample, an additional procedure of salting-out was needed during extraction. Tandem SPE cartridges of C18 and aminopropyl, using acetonitrile as the only solvent, were used to clean up extracts from either method. A total of 91 pesticides in four major pesticide groups were tested in this study Gas chromatography (GC, equipped with electron capture detector and flame photometric detector) and high-performance liquid-chromatography (HPLC) equipped with fluorescence detector were used for analysis. The validation of the method was evaluated for each fishery product using samples spiked with all pesticide standards at three concentration levels. The results indicated percentage of recovery ranged from 60%-120% and coefficients of variation ＜20% for all but 10 of the pesticides analyzed (including 1-naphthol, 3-hydroxy carbofuran, aldicarb sulfoxide, heptachlor, trifuralin, acephate, dichlorvs, methamidophos, monocrotophos and omethoate). Residue extraction techniques described in this report are rapid and suitable for screening of pesticide residues in monitoring programs.

In this study, diverse haemocytes from Pacific white shrimp Litopenaeus vannamei were spread by flow cytometer sorting system. Using the two commonly flow cytometric parameters FSC and SSC, the haemocytes could be divided into three populations. Microscopy observation of L. vannamei haemocytes in anticoagulant buffer revealed three morphologically distinct cell types designated as granular cell, hyaline cell and semigranular cell. Immune genes, which includes prophenoloxidase (proPO), lipopolysaccharide-β-glucan binding protein (LGBP), peroxinectin, crustin, lysozyme, penaeid-3a and transglutaminase (TGase), expressed from different haemocyte were analysed by quantitative real time PCR (qPCR). Results from the mRNA expression was estimated by relative level of each gene to β-actin gene. Finally, the seven genes could be grouped by their dominant expression sites. ProPO, LGBP and peroxinectin were highly expressed in granular cells, while LGBP, crustin, lysozyme and P-3a were highly expressed in semigranular cells and TGase was highly expressed in hyaline cells. In this study, L. vannamei haemocytes were firstly grouped into three different types and the immune related genes expression in grouped haemocytes were estimated.

The present study aims to investigate the effects of mushroom beta glucan (MBG) on wound recovery in partial hepatectomy (PH) in Nile tilapia (Oreochromis niloticus) and in rat skin wound healing examination. Following PH, we focussed on the effects on liver repair ability using in vitro and in vivo tests. In vitro, we examined whether the MBG has an impact on liver cell proliferation, mainly through 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assays and bromodeoxyuridine (BrdU) cell proliferation assay detection method. Results showed that MBG treatment was remarkable in enhancing cell proliferation of hepatocytes and in maintaining the cellular viability. Immunohistochemical staining to analyse Wnt/β-catenin signalling also showed that MBG has the effect of promoting cell proliferation of liver tissues after PH surgery.

The present study showed that oral mushroom beta-glucan treatment significantly increased IFN-γmRNA expression but significantly reduced COX-2 mRNA expression within the lung. For LLC tumor model, oralGanoderma lucidumorAntrodia camphoratapolysaccharides treatments significantly reduced TGF-βproduction in serum. In addition, IL-12 and IFN-γmRNA expression were significantly increased, but IL-6, IL-10, COX-2, and TGF-βmRNA expression were substantially following oral mushroom polysaccharides treatments. The study highlights the efficacious effect of mushroom polysaccharides for ameliorating the immune suppression in the tumor microenvironment. Increased M1 phenotype of tumor-associated macrophages and attenuated M2 phenotype of tumor-associated macrophages could be achieved by ingesting mushroom polysaccharides.

Background Binding of beta 1,3/1,6 glucan of Ganoderma lucidum (G. lucidum) with the receptor results in a series of signal transfers (signalling cascades), which activates the transcription factors for regulating inflammation. Excess cholesterol intake leads to an increase in the distance between fat cells and capillaries, which may cause hypoxia in the fat tissue of obese mice. This hypoxia induces the death of fat cells, resulting in the inflammation of adipose tissue or an increase in the inflammatory gene expression associated with obesity. Methods The current study examined the immunomodulation effect of G. lucidum beta 1,3/1,6 glucan according to immunoglobulin, poly-Ig receptor expression, Nature Killer cell (NK cell) activity, lymphocytes proliferation and cytokines expression. Results Our present study shows that feeding G. lucidum beta 1,3/1,6 glucan to mice induces IgA or IgG expression in the serum and small intestine washing fluid and enhances poly-Ig receptor expression in the small intestine moreover, the observation of the IL-2 and Nature killer cell activity were exchanged. Conclusions The effect of a high-cholesterol diet in the inflammatory response was observed in heart, liver, kidney, spleen, and colon tissues through histopathological evaluations. The presented evidence demonstrates that the inflammation response in the high-cholesterol diet group was much higher than in the other groups and the beta 1,3/1,6 glucan reduces inflammation in obese mice fed a high-cholesterol diet.

A continuous cell line (KF-101) derived from the caudal fin of the koi carp Cyprinus carpio was established and characterized. The KF-101 cell line multiplied abundantly in Leibovitz's L-15 medium containing 10% foetal bovine serum at 25° C, and was subcultured for >90 passages over a period of 3 years. Immunocytochemistry revealed that the KF-101 cells contain keratin, junction proteins connexin-43 and occludin, and ectodermal stem-cell marker Pax-6, but not vimentin. Furthermore, the KF-101 cells reacted with anti-human DARPP-32 and anti-human GATA-4 antibodies, and the labelling was regulated according to the cell cycle. The labels of the DARPP-32 and GATA-4 antibodies in the KF-101 cells were the suggested phosphatase-1 inhibitor-1 and GATA-3, respectively. In addition, the KF-101 cells were susceptible to koi herpesvirus but were resistant to eel herpesvirus, iridovirus, grouper nodavirus and chum salmon (Oncorhynchus keta) virus. The results indicate that the KF-101 cells are suitable materials for investigating biological and virological development.

The monocotylid monogenean Dendromonocotyle pipinna infects the dorsal skin of the blotched fantail ray, Taeniurops meyeni, and is problematic for Hualien Farglory Ocean Park, an aquarium in Taiwan. Over the last 2 years, eight rays have died due to heavy infections with this parasite. In this study, we found that the epidermis of T. meyeni with attached D. pipinna was not markedly thinner but contained decreased numbers of mucous cells and numerous vacuoles. We examined the effects of temperature (from 16 to 30 degrees C in 2 degrees C increments), salinity (from 10 to 50 per thousand in 5 per thousand increments), desiccation (from 1 to 10 min) and sodium hypochlorite (from 5 to 20 ppm in 5 ppm increments) treatment on the embryonation period and hatching success of D. pipinna eggs, with the goal of disinfecting equipment used in aquaria. Temperature strongly influenced embryonation period: eggs first hatched 4 days after being laid at 30 degrees C and 16 days after being laid at 16 degrees C. However, hatching rate was not significantly influenced by incubation temperature, since the final hatching rates under the incubation temperatures tested herein were not significantly different from one another. Hyposalinity had a lethal effect on D. pipinna eggs, completely preventing the hatching of eggs cultured at 10 and 15 per thousand salinity. Hypersalinity was only partially effective, with a hatching rate close to 7% at 50 per thousand salinity. Desiccation was effective at preventing hatching, and the effectiveness increased with increasing treatment duration. The hatching rate of D. pipinna eggs was significantly decreased when incubated under desiccating conditions for even 1 min. Furthermore, a complete inhibition of hatching was achieved by desiccating eggs for 10 min. Sodium hypochlorite treatment completely prevented hatching at concentrations higher than 10 ppm after 18 h of exposure, but a concentration of 5 ppm was ineffective at preventing hatching even after 24 h of treatment. We therefore propose effective combinations of sodium hypochlorite and exposure time as a means to sterilize tanks and equipment containing D. pipinna eggs.

The fisheries products in this study comprise fish, bivalve, crustacean and cephalopod collected from different types of markets in Taiwan between the years 2001 and 2003. A total of 91 pesticide residues belonging to four major pesticide groups were tested and analyzed. The test results show that 65.40% of fish, 93.55% of shellfish, 84.92% of crustacean and 98.33% of cephalopod samples contain no detectable residues. There are only two kinds (organochlorine and organophosphate) of totally six pesticides (DDTs, dieldrin, chlorpyrifos, fenitrotion, fenthion and prothion) that have been detected from the fisheries products in this study. For there were pesticides present in the fish products, consumption of fisheries product there was no zero risk. But the exposure of consumer did not exceed the acceptable daily intakes (ADI). For male, there was the highest risk in exposure to dieldrin, which the percentage of ADI was 93.56%. This study also suggests that a yearly monitoring program for organophosphate pesticide residues in fish is necessity.

The total haemocyte count (THC), differential haemocyte count (DHC), respiratory bursts, and phenoloxidase (PO) activity were determined in the Pacific white shrimp Litopenaeus vannamei which administrated by feeding with diets containing mushroom beta glucan (MBG) at 0.05% and 0.1%. Results showed that shrimp fed a diet containing 0.05% MGB had significantly increased THC and semi-granular cells ratio at 28 days. Intracellular superoxide anion (O2-) production were significantly increased at 14 days which shrimp fed a diet containing 0.05% MBG and PO activity were significantly increased at 14 days which shrimp fed a diet containing 0.1% MGB but equal with control at 28 days. In previously study, semi-granular cells were found as the primary immune activator with their high levels of granules, enzymes and proteins. In this study, the effectively enhance immune responses and compare to high induced semi-granular cells in Pacific white shrimps concluded that MBG is an effective and powerful immunostimulants for shrimp. For application in aquaculture, MBG administration through dietary administration is also a convenient and useful practical technique in the future.

Recently studies performed on mushroom isolated polysaccharides demonstrated thatβ-(1,3)-glucan may affect the balance of Th1/Th2 cell response. Using ovalbumin (OVA) as a hypersensitivity inducer, we evaluated the ability of mushroom beta-glucan (MBG) in modulating Th1/Th2 cell responses in B6 mice. As compared to the control group, administration of MBG resulted in an increase of phagocytic activities, Th1 cytokine productions, immunoglobulins including IgG2A and IgA, and a significant expression of the splenic surface markers including CD3, CD4, CD8, and F4/80. In contrast, administration of MBG has significantly suppressed IgE and IgG1 levels and Th2 cytokines including IL-4, IL-5, and IL-6. Histopathological observation of MBG-treated followed by OVA-treated mice showed less filtration of eosinophil in pulmonary tissue sections. Our data suggested that administration of MBG treatments alters the natural course of the IgE-mediated hypersensitivities. In this investigation, we realize the mushroom beta glucan alter the Th2 response toward the Th1 in the allergic, resulting in a reduction in IgE productions which played a substantive role in reducing the severity of IgE-mediated hypersensitivity.

The purification and characterization of an extracellular protease produced by pathogenic luminous Vibrio harveyi strain 820514, originally isolated from diseased tiger prawn (Penaeus monodon), was presented in this paper. The purification steps included ammonium sulfate precipitation, with columns of hydrophobic interaction chromatography and anion exchange on fast protein liquid chromatography. The protease is an alkaline cysteine protease, heat labile, inhibited by iodoacetamide, iodoacetic acid, N-ethylmaleimide, p-chloromercuribenzoate, and p-chloromercuribenzene-sulfonic acid, and showed maximal activities at pH 8 and 50 degrees C, having a molecular mass of 38 kDa as estimated by SDS-PAGE and gel filtration column. In addition, the protease was also completely inhibited by CuCl2 and HgCl2, but not or only partially inhibited by other inhibitors tested. Furthermore, 2-mercaptoethanol was the most effective reducing agent in the activation of the enzyme. The present protease is the first cysteine protease found in Vibrio species.

Reactive oxygen species (ROS) may act as signaling molecules in the physiology responses and the present study aims to investigate the effect of extracellular hydrogen peroxide on macrophages cellular response. The results obtained in the present study showed that the extracellular hydrogen peroxide affectively alter the membrane potential of the cell membrane and ion exchange channels in the cell membrane through intracellular NAD turnover that may lead to an intracellular calcium ion concentration alteration and subsequently induce the downstream signal activation.

This study attempts to describe the effects of innate immunity responses and field application of mushroom beta-glucan mixture (MBG) in cultured orange-sported grouper, Epinephelus coioides. Chemical analysis for MBG showed that the mixture contains 34.06% of macro-molecular polymers with bio-active linkage such as 3-; 3,4- and 4,6-glucopyranosyl and 6-linked galactopyranosyl residues. Study performed on the innate immunity showed that oral ingestion of MBG at 1.0 g and 2.0 g per kilogram of feed levels may significantly enhance the lysozyme activity, alternative complement activity, phagocytic activity and respiration burst of the experimental groupers. Observation on the experimental challenge of pathogen showed that uses of MBG at 0.1% and 0.2% levels in feed might significantly enhance the protection of grouper against Vibrio alginolyticus. Field trials performed on short and long-term culture showed that feeding of diet containing 0.1% or 0.2% of MBG may significantly enhance the survival of cultured groupers up to 16% when compared with those obtained from controls.

The effect of dietary cholesterol on the growth and survival of juvenile tiger shrimp ( Penaeus monodon ) was tested using semi-purified diets. Twenty-four shrimp per treatment, with an initial mean weight of 268.2 mg, were individually housed in black nylon mesh pots in 40-liter aquaria containing 30 ppt seawater. Diets containing 0, 0.2, 0.4, 0.5, 0.8, and 1.0% supplemental cholesterol were evaluated. The duration of the experiment was 63 days. Shrimp fed diets containing 0% and 1% cholesterol had significantly lower weight gain and poorer survival than those fed the other diets. There was no significant difference in weight gain of P. monodon fed diets containing 0.2–0.8% cholesterol. A diet containing 1% cholesterol had an adverse or toxic effect on P. monodon growth.

The purpose of this paper was to purify and amplify the DNA fragment of Penaeus monodon -type baculovirus (MBV). Using 30-50% caesium chloride gradients, MBV virions and occlusion bodies with density parameters of 1.28-1.29 and 1.32-1.33 g/ml, respectively, were purified. Two oligonucleotide primers have been successfully designed and utilized for the amplification of a DNA fragment of MBV. After 35 amplification cycles of the MBV DNA fragment, a large amount of amplified product with an approximate molecular weight of 600 bp was obtained. This is the first successfully published work on the amplification of MBV using the polymerase chain reaction (PCR). Using the same primers, DNA extracted from MBV noninfected P. monodon, P. japonicus, and P. orientalis had a negative PCR response. However, a positive PCR response was obtained from DNA extracted from MBV-infected postlarval P. monodon. DIG-dot blot hybridization technique using PCR product obtained from the present study as a probe further confirmed that the product is originated from a portion of MBV polyhedrin gene. It is also suggested that PCR product may be beneficial for an accurate and early diagnosis of MBV infection in larval shrimp.

The present study defines a culture system for heart tissue of hard clam,Meretrix lusoria (Roding). The tissue explants were cultured in six synthetic media supplemented with 10% fetal bovine serum and grown at 28° C. All of the media used in the present study support good cell migration and L15 medium gave the best result. Monolayer cell sheets of heart tissue established in 2X L15 growth medium consist of cardiac myocytes, epithelial-like, and fibroblastlike cells. Mitoses were observed in the culture system. To trigger the proliferation of cells, pronase, collagenase, insulin, fibroblast growth factor (FGF), and epidermal growth factor (EGF) were used individually or combined in the L15 growth medium. The results showed pronase and collagenase at a concentration level of 100 μg/ml promoted cell proliferation. In contrast, insulin, FGF, and EGF gave no positive result. To date, cell cultures derived from the hard clam have been subcultured 6 times. Morphologic characterizations for the cells were also performed.

The present paper describes a herpes-like virus, tentatively designated as EHVF (Eel Herpesvirus in Formosa), isolated from diseased Japanese eels (Anguilla japonica Temminck & Schlegel) in Taiwan.This virus had DNA as its nucleic acid, sensitive to ether and chloroform, and resistant to freezing and thawing treatments. Enveloped mature viral particle measured 200 nm in diameter with nucleocapsid of 120 nm.The virus successfully replicated in CCF, CCT, TO-2, BGK, DK-1 and EO-2 cell lines at temperatures from 15°C to 33°C (optimal temperature 24°C). When these cells were infected with EHVF, syncytium formations were observed in CCF, CCT, TO-2, BGK, EK-1 and EO-2 cell lines. However, HeLa, Vero, L, AF, AK, BF-2, CCG, CCO, CF, CHH-1, CHSE-214, EPC, FHM, FS, JF, LF, MH and RTG-2 cell lines were refractory to the multiplication of this virus.No mortalities were observed among eels injected with EHVF, but the virus was reisolated from the kidneys and livers of infected eels 60 days after injection. On the other hand, carp injected with EHVF showed 37% mortality during 60 days and the virus was reisolated from all the dead fish and 84% of the survivors.

In the present study, the ultrastructural changes of BPK cells, a fibroblast-like cell line, derived from the kidney of juvenile black porgy Acanthopagrus schlegeli, under heat shock treatment are described.The BPK cells were maintained in L-15 medium supplemented with 10% fetal calf serum and 0.15 M NaCl at 28|C2. The heating was carried out in precalibrated water baths. Monolayers of cells, grown on coverslips in parafilm-sealed petri dishes were submerged under water for 30 min at 40|C treatments. Cells were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer supplemented with 6.6% sucrose, postfixed in 1% OsO4 and flat embedded in Spurr’s resin. Silver section were cut parallel to the substratum, stained with uranyl acetate and Reynold’s lead citrate, and examined in a Hitachi H-600 electron microscope at 75 KV.

1. 1. Eyestalk unablated and unilaterally ablated Penaeus monodon juveniles had survival rates after 5 months of 75–72.5 and 67.5–60%, respectively. 2. 2. Unilaterally ablated shrimps had significantly higher (P < 0.05) growth rate than unablated shrimps. 3. 3. Eyestalk-ablatement resulted in a decrease in the haemolymph sodium concentration and an increase in the potassium and calcium concentration of shrimps. 4. 4. The osmolarity of haemolymph and total protein concentration of unablated shrimps were demonstrated to be higher than those of unilaterally ablated shrimps. 5. 5. The eyestalk-ablated shrimps possess higher total ATPase and Na+,K+-ATPase activities in the gill than those of unablated shrimps.

The enzyme-linked immunosorbent assay (ELISA) was employed for the sero-diagnosis of Angiostrongylus cantonensis infections in rats and man. Metabolic and ‘purified’ antigens of adult or juvenile A. cantonensis were evaluated by ELISA and compared against Toxocara cant's antigen for sensitivity and specificity. Sera and cerebrospinal fluid (CSF) from both rats and man gave higher ELISA values against antigens of A. cantonensis than those obtained from negative control samples. The results showed that cross reaction occurred between T. canis and A. cantonensis . Of four A. cantonensis antigens tested, a Sephacryl S-300 “purified” fraction was shown to be superior in the ELISA to determine angiostrongyliasis.