Molecular cloning of a putative agglutination/immobilization antigen located on the surface of a novel agglutination/immobilization serotype of Cryptocaryon irritans

A surface agglutination/immobilization antigen was purified from the novel agglutination/immobilization serotype (serotype G37) of the ciliated protozoan Cryptocaryon irritans, a parasite of seawater fishes. Serum from fish immunized with C. irritans theronts had agglutination/immobilization activity against theronts in vitro. However, fish and rabbit antisera raised against serotype G32 (reported previously) caused little agglutination/immobilization of serotype G37 theronts. Immunological analysis indicated that the 37 kDa theront surface membrane protein may be the agglutination/immobilization antigen of this serotype. The full-length 37 kDa antigen cDNA contained 1171 base pairs, encoding a 331-amino acid protein with hydrophobic N- and C-termini, which are characteristically found in proteins containing a C-terminal glycosylphosphatidylinositol anchor. In addition, the genetically characterized nucleotide sequences of the first internal transcribed spacer region of ribosomal DNA of these 2 serotypes were compared. The internal transcribed spacer rDNA sequence of serotype G32 was identical to that of isolates from Pingtung, Taiwan, and from the USA. On the other hand, the sequences of serotype G37 were not identical to those of any C. irritans isolate.     

卵形鲳鯵对刺激隐核虫的免疫应答和免疫保护研究

用刺激隐核虫(Cryptocaryon irritans)的幼虫对卵形鲳鯵(Trachinotus ovatus)进行腹腔注射和体表感染,然后每隔一周用阻动试验(Immobilization assay)检测免疫鱼的抗血清和皮肤培养液对刺激隐核虫幼虫的阻动效价,在第14周中,分别用亚致死剂量和致死剂量的刺激隐核虫幼虫对免疫鱼攻毒以检测所产生的免疫保护力。实验结果显示:两种免疫方法都能让卵形鲳鯵的血清和皮肤生成阻动刺激隐核虫幼虫的特异性抗体,并能使被免疫鱼获得明显的免疫保护,但是体表感染免疫组的血清和皮肤培养液的阻动效价都要比腹腔注射免疫组高,所获得的免疫保护力也更强。同时还发现,免疫鱼血清和皮肤培养液中的抗体存在明显的差异:两者的最初生成时间、达到峰值的时间、变化规律以及阻动效价等都不一致。因此,我们推测鱼类的系统免疫应答和皮肤黏膜免疫应答有可能是相互独立的,或者是不同步的。鱼类的体液免疫应答,特别是黏膜免疫应答对抵御刺激隐核虫的感染起了重要的作用,采用刺激隐核虫虫体疫苗可能成为预防海水鱼类白点病的一种选择。       

In vitro culture technique for Cryptocaryon irritans, a parasitic ciliate of marine teleosts

A medium for the in vitro culture of Cryptocaryon irritans, which is an obligatorily parasitic ciliate of marine teleosts and causes 'white spot disease', was developed. The medium consisted of a layer of cultured fish cells (FHM), with an agarose gel layer covering the cell layer. The agarose gel contained 0.22% agarose, 10% fetal calf serum, 100 I.U. ml(-1) Penicillin G potassium and 100 microg ml(-1) streptomycin sulphate. Theronts of C. irritans transformed to trophonts and grew to 180 microm in mean length in the medium, although they gradually decreased in number. When trophonts fully developed in medium were transferred into seawater 4 d after inoculation, approximately 70% of them transformed to encysted tomonts and released theronts. When fish were challenged with theronts obtained from in vitro-raised parasites, approximately 40% of the theronts were recovered from fish, indicating comparative infectivity of in vitro-raised theronts to those of in vivo-raised theronts. This is the first report that C. irritans fully developed in vitro and its entire life cycle was completed without a host fish.     

Wild caught ornamental fish: a perspective from the UK ornamental aquatic industry on the sustainability of aquatic organisms and livelihoods

The ornamental aquatic industry involves the global commercial trade of live aquatic organisms such as fish, invertebrates and plants. It comprises a range of businesses including collectors, breeders, exporters, importers and retailers. Together, these form a supply chain through which aquatic organisms pass from their point of origin to the end point e.g., domestic aquaria and ponds. On a worldwide basis, the legal and legitimate ornamental aquatic trade is subject to regulation and monitoring throughout the majority of its supply chain. Approximately 90% of ornamental freshwater fish species traded are captive-bred, but, due to their complex breeding cycles, 90–95% of ornamental marine fish species are wild-caught. The ornamental aquatic industry and consumers therefore have a responsibility to ensure that wild-caught species are sourced sustainably, legally and to good welfare standards. Such good practice should be considered a necessity for the longevity, not only of the ornamental aquatic industry, but of the livelihoods which depend on it and the future of ecosystems dependent on such communities.     

Ornamental fish: a potential source of pathogenic and multidrug-resistant motile Aeromonas spp.

Aeromonas spp. are ubiquitous bacteria that cause diseases in fish and other aquatic animals. They are the natural inhabitants of different aquatic environments, such as freshwater, brackishwater and marinewater. Extrinsic stressors, such as crowding, unhygienic handling, poor water quality, polluted feeding and inadequate nutrition, can predispose fish to Aeromonas infection. In ornamental fish, motile Aeromonas spp. are known as aetiological agents of motile aeromonad infections, which cause significant mortality in fish and economic loss in the ornamental fish industry. The existence of different virulence factors leads to the virulence potential of motile Aeromonas spp. There are several antimicrobials used to treat bacterial infections in ornamental fish. However, the extensive use of antimicrobials in the ornamental fish industry causes multidrug resistance. This article reviewed a multitude of virulence factors that are related to the ornamental fish-borne Aeromonas pathogenicity and the antimicrobial resistance determinants related to the multidrug resistance phenotypes of motile Aeromonas spp. in ornamental fish.     

Fish host range of seven isolates of Cryptocaryon irritans (Ciliophora)

Host specificity of the marine ciliate Cryptocaryon irritans was investigated using seven parasite isolates and six experimental fish species. The low degree of host specificity of C. irritans to teleost fish has been confirmed. Furthermore, five fish species occurring outside the geographical range of this parasite were also susceptible to infection; three of these could be at risk to Cryptocaryon through aquaculture practices.     

Stress and welfare in ornamental fishes: what can be learned from aquaculture?

The ornamental fish trade is estimated to handle up to 1·5 billion fishes. Transportation and handling of fishes imposes a range of stressors that can result in mortality at rates of up to 73%. These rates vary hugely, however, and can be as low as 2%, because they are generally estimated rather than based on experimental work. Given the numbers of ornamental fishes traded, any of the estimated mortality rates potentially incur significant financial losses and serious welfare issues. Industry bodies, such as the Ornamental Aquatic Trade Association (OATA), have established standards and codes of best practice for handling fishes, but little scientific research has been conducted to understand the links between stress, health and welfare in ornamental species. In aquaculture, many of the same stressors occur as those in the ornamental trade, including poor water quality, handling, transportation, confinement, poor social and physical environment and disease and in this sector directed research and some resulting interventions have resulted in improved welfare standards. This review considers the concept of welfare in fishes and evaluates reported rates of mortality in the ornamental trade. It assesses how the stress response can be quantified and used as a welfare indicator in fishes. It then analyses whether lessons from aquaculture can be usefully applied to the ornamental fish industry to improve welfare. Finally, this analysis is used to suggest how future research might be directed to help improve welfare in the ornamental trade.     

Protective immunity in grouper (Epinephelus coioides) following exposure to or injection with Cryptocaryon irritans

The protective immunity of grouper (Epinephelus coioides) against Cryptocaryon irritans was determined after immunisation by surface exposure or intraperitoneal injection. Specific antibody titres of immunised fish serum and skin culture supernatant were determined by enzyme-linked immunosorbent assay and immobilisation assays. Specific antibody can be detected in some immunised fish at Week 1 and in all immunised fish at Week 2, and the peaks were between Weeks 4-6. Specific antibody was still evident in the serum and skin of immunised fish at Week 8, and provided good protection against challenge with C. irritans. These findings indicated that humoral and skin mucosal immunity play important roles in fish against C. irritans infection.     

黄斑蓝子鱼皮肤黏液对刺激隐核虫及一些病原菌的抑杀作用

黄斑蓝子鱼(Siganus oramin)具有天然抗刺激隐核虫(Cryptocaryon irritans)能力, 并从其血清中分离纯化了一种抗虫蛋白。以黄斑蓝子鱼皮肤黏液为对象, 研究了其对刺激隐核虫(C. irritans)、多子小瓜虫(Ichthyophthirius multifiliis)、布氏锥虫(Trypanosoma brucei brucei)及一些病原菌的杀灭和抑制效果。结果显示: 黄斑蓝子鱼黏液对刺激隐核虫、多子小瓜虫和布氏锥虫均具有明显的杀虫效果, 黏液蛋白对3种寄生虫的最低杀寄生虫浓度(Minimum Parasiticidal Concentration, MPC)分别为4.0、5.0和3.0 mg/mL。显微镜观察发现, 刺激隐核虫和多子小瓜虫的幼虫在经过黄斑蓝子鱼黏液作用后, 均出现纤毛脱落、虫体肿胀、外膜破裂和内容物泄漏等现象。黏液抑菌活性实验结果表明, 除大肠杆菌(Escherichia coli)外, 黄斑蓝子鱼皮肤黏液对金黄色葡萄球菌(Staphylococcus aureus)、霍乱弧菌(Vibrio cholerae)、海豚链球菌(Streptococcus iniae)、温和气单胞菌(Aeromonas sobria)、溶藻弧菌(Vibrio alginolyticus)、副溶血弧菌(Vibrio Parahaemolyticus)、星形诺卡氏菌(Nocadia asteroides)和美人鱼发光杆菌杀鱼亚种(Photobacterium damselae subsp.)皆有明显的抑菌效果。在最低抑菌浓度(Minimum Inhibitory Concentration, MIC)测定中, 黄斑蓝子鱼黏液对金黄色葡萄球菌、霍乱弧菌、海豚链球菌、溶藻弧菌、温和气单胞菌和副溶血弧菌的MIC 分别为0.16、0.16、0.08、0.63、0.63和0.63 mg/mL。黄斑蓝子鱼皮肤黏液对小麦赤霉菌(Gibberella saubinetii)和黑曲霉菌(Aspergillus niger)没有明显抑菌作用。       

A new strain of Cryptocaryon irritans from the cultured olive flounder Paralichthys olivaceus

An obligate parasite, Cryptocaryon irritans, which is responsible for the white spot disease of marine fish is known to develop in the temperature regime over 19 degrees C. Recently, however, we found white spot disease of olive flounder Paralichthys olivaceus during winter at water temperatures ranging between 12 and 16 degrees C in Korea. In the present study we isolated a C. irritans-like ciliate from the affected fish and investigated its reproductive characters to compare the newly found ciliate with typical C. irritans. The newly found ciliate had an additional process in the reproductive stage, characterized by a budding before palintomic division, and it showed a higher ability to carry out tomitogenesis at a low temperature (16 degrees C) than at a high temperature (24 degrees C). Nevertheless, the present ciliates still had much in common with typical C. irritans with respect to clinical, histopathological, and morphological characters, suggesting that it is a new strain of C. irritans, adapted to lower water temperature.     

Significant longevity-extending effects of EGCG on Caenorhabditis elegans under stress

Epigallocatechin gallate (EGCG), a main active ingredient of green tea, is believed to be beneficial in association with anticarcinogenesis, antiobesity, and blood pressure reduction. Here we report that EGCG extended Caenorhabditis elegans longevity under stress. Under heat stress (35°C), EGCG improved the mean longevity by 13.1% at 0.1 μg/ml, 8.0% at 1.0 μg/ml, and 11.8% at 10.0 μg/ml. Under oxidative stress, EGCG could improve the mean longevity of C. elegans by 172.9% at 0.1 μg/ml, 177.7% at 1.0 μg/ml, and 88.5% at 10.0 μg/ml. However, EGCG could not extend the life span of C. elegans under normal culture conditions. Further studies demonstrated that the significant longevity-extending effects of EGCG on C. elegans could be attributed to its in vitro and in vivo free radical-scavenging effects and its up-regulating effects on stress-resistance-related proteins, including superoxide dismutase-3 (SOD-3) and heat shock protein-16.2 (HSP-16.2), in transgenic C. elegans with SOD-3∷green fluorescent protein (GFP) and HSP-16.2∷GFP expression. Quantitative real-time PCR results showed that the up-regulation of aging-associated genes such as daf-16, sod-3, and skn-1 could also contribute to the stress resistance attributed to EGCG. As the death rate of a population is closely related to the mortality caused by external stress, it could be concluded that the survival-enhancing effects of EGCG on C. elegans under stress are very important for antiaging research.     

Microbial diversity and potential pathogens in ornamental fish aquarium water

Ornamental fishes are among the most popular and fastest growing categories of pets in the United States (U.S.). The global scope and scale of the ornamental fish trade and growing popularity of pet fish in the U.S. are strong indicators of the myriad economic and social benefits the pet industry provides. Relatively little is known about the microbial communities associated with these ornamental fishes or the aquarium water in which they are transported and housed. Using conventional molecular approaches and next generation high-throughput amplicon sequencing of 16S ribosomal RNA gene hypervariable regions, we characterized the bacterial community of aquarium water containing common goldfish (Carassius auratus) and Chinese algae eaters (Gyrinocheilus aymonieri) purchased from seven pet/aquarium shops in Rhode Island and identified the presence of potential pathogens. Our survey identified a total of 30 phyla, the most common being Proteobacteria (52%), Bacteroidetes (18%) and Planctomycetes (6%), with the top four phyla representing >80% of all sequences. Sequences from our water samples were most closely related to eleven bacterial species that have the potential to cause disease in fishes, humans and other species: Coxiella burnetii, Flavobacterium columnare, Legionella birminghamensis, L. pneumophila, Vibrio cholerae, V. mimicus. V. vulnificus, Aeromonas schubertii, A. veronii, A. hydrophila and Plesiomonas shigelloides. Our results, combined with evidence from the literature, suggest aquarium tank water harboring ornamental fish are an understudied source for novel microbial communities and pathogens that pose potential risks to the pet industry, fishes in trade, humans and other species.     

Age- and weight-dependent susceptibility of rainbow trout Oncorhynchus mykiss to isolates of infectious haematopoietic necrosis virus (IHNV) of varying virulence

The virulence of 5 European and 1 North American isolate of infectious haematopoietic necrosis virus (IHNV) was compared by infecting female sibling rainbow trout ('Isle of Man' strain) of different weights and ages (2, 20 and 50 g). The fish were exposed to 10(4) TCID50 IHNV per ml of water by immersion, and the mortality was recorded for 28 d. Two new IHNV isolates from Germany were included in the investigation. One was isolated from European eels kept at 23 degrees C (+/- 2 degrees C) and the other was not detectable by immunofluorescence with commercially available monoclonal antibodies recognising the viral G protein. The results showed that IHNV isolates of high or low virulence persisted in rainbow trout of all ages/weights for 28 d, with the exception of fish over 15 g in the eel IHNV (DF [diagnostic fish] 13/98)-infected groups from which the virus could not be reisolated on Day 28. The smallest fish were most susceptible to an infection with any of the IHNV isolates. The lowest cumulative mortality (18%) was observed in fingerlings infected with the North American isolate HAG (obtained from Hagerman Valley), and the highest mortality (100%) in DF 04/99 infected fish. The DF 04/99 and O-13/95 viruses caused mortality in fish independent of their weight or age. The isolates FR-32/87 and I-4008 were virulent in fish up to a weight of 20 g and caused no mortality in larger fish. In the IHNV HAG- and DF 13/98 (eel)-infected rainbow trout, no signs of disease were observed in fish weighing between 15 and 50 g. An age/weight related susceptibility of rainbow trout was demonstrated under the defined conditions for all IHNV isolates tested.     

Introduction history of non-native freshwater fish in Okinawa-jima Island: ornamental aquarium fish pose the greatest risk for future invasions

We examined the introduction history of non-native freshwater fish in Okinawa-jima Island, Japan, to identify the pathway posing the greater risk for future invasions and to measure the strength of ornamental fish introductions as a vector of new invasions using all available records of freshwater fish introductions to date. At least 45 non-native freshwater fish have been introduced to Okinawa-jima Island. Of those, 22 species have successfully established in natural waters and, consequently, non-native fish species represent 76 % of the total freshwater fish fauna. The most important pathway was via the dumping of unwanted ornamental aquarium fish, which accounted for 71 % of the total introductions. The introduction of ornamental fish has occurred extensively in recent decades, and all fish introduced since the 1980s have been ornamental aquarium fish. The overall rate of establishment success was estimated at 51 %, although this is undoubtedly an overestimate because of unreported failed introductions. Establishment rates varied between stocked fish (100 %) and dumped ornamental fish (39 %), which may reflect the low propagule pressure of ornamental fish. From the observed trends in freshwater fish introductions, the dumping of ornamental fish into natural waters is expected to continue if no action is taken to reduce this most important pathway. We recommend regulations against the release of unwanted pet fish into natural waters, better programs for returning unwanted pet fish to stores or local zoos, educational programs for aquarists and retailers, and regulations against selling and trading ornamental fish that are judged to pose a high risk of causing economic or ecological harm.     

Inhibitory Effects of Persimmon (Diospyros kaki) Extract and Related Polyphenol Compounds on Growth of Human Lymphoid Leukemia Cells

We have investigated the effects of persimmon (Diospyros kaki) extract (PS) and related polyphenol compounds such as catechin (C), epicatechin (EC), epicatechingallate (ECG), epigallocatechin (EGC), and epigallocatechingallate (EGCG) on the growth of human lymphoid leukemia Molt 4B cells. We found that PS, ECG, EGC, and EGCG strongly inhibited the growth of the cells in a dose-dependent manner, while C and EC inhibited the growth of the cells only moderately. Ornithine decarboxylase (ODC), a rate-limiting enzyme of polyamine biosynthesis, was inhibited by 10–20% by these polyphenol compounds. The morphology of the Molt 4B cells indicated severe damage 3 days after treatment with PS, ECG, EGC, and EGCG. Irregular shape of the cells and DNA fragmentation were observed in PS, ECG, EGC, or EGCG-treated cells. These results suggest that PS, ECG, EGC, and EGCG induce apoptosis (programmed cell death) of Molt 4B cells.     

The importance of the marine ornamental reef fish trade in the wider Caribbean

The marine ornamental fish trade began in the 1930s in Sri Lanka, spread to Hawaii and the Philippines in the 1950s, and expanded to a multi-million dollar industry in the 1970s with fisheries established throughout the tropical Pacific, Indian and Atlantic Oceans. Currently, 45 countries supply global markets an estimated 14-30 million fish annually, with an import value of US$28-44 million. The largest suppliers are Indonesia and the Philippines, followed by Brazil, Maldives, Vietnam, Sri Lanka and Hawaii. In the tropical Western Atlantic, 16 countries have export fisheries, including the U.S. (Florida and Puerto Rico). The U.S. is the world's largest buyer, followed by the European Union and Japan. The global trade consists of over 1400 species of reef fishes, of which only about 25 are captive bred on a commercial scale. Damselfish, anemonefish, and angelfish constitute over 50% of the global volume; butterflyfish, wrasses, blennies, gobies, triggerfish, filcfish, hawkfishes, groupers and basselets account for 31% of the trade, and the remaining 16% is represented by 33 families. The most important fishes from the Caribbean are angelfish (six species), seahorses (two species), royal gramma, jawfish, queen triggerfish, redlip blenny, puddingwife, bluehead wrasse, and blue chromis. The Caribbean currently supplies a small percentage of the global trade in marine ornamental species, but ornamental fisheries in this region represent important emerging industries. It is critical that effective ornamental fishery management plans and regulations are developed and enforced, and fishery-dependent and fishery-independent data are collected and utilized in decision making processes to ensure sustainable ornamental fisheries throughout the region.     

Epigallocatechin gallate (EGCG) combined with zinc sulfate inhibits Peste des petits ruminants virus entry and replication

Despite the fact that the Peste des petits ruminants virus (PPRV) leads to high morbidity and mortality (up to 100%), antiviral drugs against PPRV are not available. The aim of this study was to estimate the dose of epigallocatechin gallate (EGCG) co-administered with zinc (II) ions as an antiviral agent against PPRV. Treatment of PPRV-infectedVero cells with EGCG and zinc sulfate (zinc II) was administered, and antiviral activities against PPRV in infected Vero cells was evaluated by determination of virus yields, expressed as logTCID₅₀/mL. Cytotoxicity was determined using the tetrazolium-based MTS test. Zinc sulfate at 1.1 mg/mL and EGCG at 25 μM showed low potentiated and potentiated antiviral activities against PPRV, respectively. These agents caused significant inhibition of PPRV in Vero cells (p < 0.05) with a reduction in logTCID₅₀/mL by up to 3-fold. The combination of EGCG (25 μM) and zinc sulfate (1.1 mg/mL) was observed to have strong antiviral activity (p < 0.01) against PPRV with a reduction in logTCID₅₀/mL of the virus up to 4-times without causing any host cell cytotoxicity. This study is the first one to prove that the zinc II has the capability of stimulating EGCG to inhibit in vitro PPRV entry. Moreover, this combination appears capable of reducing infection resistance by hindering viral adaptation.     

Tissue-Specific Analysis of Alternative Splicing Events and Differential Isoform Expression in Large Yellow Croaker (Larimichthys crocea) After Cryptocaryon irritans Infection

Marine Biotechnology - The large yellow croaker (Larimichthys crocea) is one of the most important mariculture fish in China. Recently, cryptocaryonosis caused by Cryptocryon irritans infection has...     

Inhibitory Effects of Green Tea and (–)-Epigallocatechin Gallate on Transport by OATP1B1, OATP1B3, OCT1, OCT2, MATE1, MATE2-K and P-Glycoprotein

Green tea catechins inhibit the function of organic anion transporting polypeptides (OATPs) that mediate the uptake of a diverse group of drugs and endogenous compounds into cells. The present study was aimed at investigating the effect of green tea and its most abundant catechin epigallocatechin gallate (EGCG) on the transport activity of several drug transporters expressed in enterocytes, hepatocytes and renal proximal tubular cells such as OATPs, organic cation transporters (OCTs), multidrug and toxin extrusion proteins (MATEs), and P-glycoprotein (P-gp). Uptake of the typical substrates metformin for OCTs and MATEs and bromosulphophthalein (BSP) and atorvastatin for OATPs was measured in the absence and presence of a commercially available green tea and EGCG. Transcellular transport of digoxin, a typical substrate of P-gp, was measured over 4 hours in the absence and presence of green tea or EGCG in Caco-2 cell monolayers. OCT1-, OCT2-, MATE1- and MATE2-K-mediated metformin uptake was significantly reduced in the presence of green tea and EGCG (P < 0.05). BSP net uptake by OATP1B1 and OATP1B3 was inhibited by green tea [IC₅₀ 2.6% (v/v) and 0.39% (v/v), respectively]. Green tea also inhibited OATP1B1- and OATP1B3-mediated atorvastatin net uptake with IC₅₀ values of 1.9% (v/v) and 1.0% (v/v), respectively. Basolateral to apical transport of digoxin was significantly decreased in the presence of green tea and EGCG. These findings indicate that green tea and EGCG inhibit multiple drug transporters in vitro. Further studies are necessary to investigate the effects of green tea on prototoypical substrates of these transporters in humans, in particular on substrates of hepatic uptake transporters (e.g. statins) as well as on P-glycoprotein substrates.     

The use of catechins in Chinese hamster ovary cell media for the improvement of monoclonal antibody yields and a reduction of acidic species

Catechin compounds have potential benefits for recombinant monoclonal antibody (Mab) production as chemical additives in cell culture media. In this study, four catechin compounds catechin (Cat), epicatechin (EC), gallocatechin-gallate (GCG), and epigallocatechin-gallate (EGCG) were added to cell culture media (at 50 μM) and their effects on the recombinant Chinese hamster ovary (CHO) cell culture, specific productivity, and Mab quality were assessed. The results indicate that the improvement of specific productivity was linked to cell growth inhibition. All catechins caused cell phase growth arrest by lowering the number of cells in the G1/G0 phase and increasing the cells in the S and G2/M phases. Late addition of the catechin resulted in a significantly higher final IgG concentration. Cat and EC caused an improvement in the final antibody titer of 1.5 ± 0.1 and 1.3 ± 0.1 fold, respectively. Catechins with a galloyl group (GCG and EGCG) arrested cell growth and reduced cell specific productivity at the concentrations tested. The Cat-treated IgG was found to have reduced acidic species with a corresponding increase in the main peak.