Kudoa ogawai (Myxosporea: Kudoidae) Infection in Cultured Olive Flounder Paralichthys olivaceus

Since Kudoa septempuntata was identified as a causative agent of food poisoning associated with raw olive flounder Paralichthys olivaceus, interest and concern regarding the parasite have increased. However, there have been no investigations or reports of other Kudoa species infecting the fish (except for K. paralichthys, which infects the brain) in Korea. We found cysts filled with myxospores of Kudoa species in muscles of cultured olive flounder specimens and identified these to the species level. Mature spores were quadrate, measuring 8.7±0.5 μm in length, 9.2±0.4 μm in thickness, and 12.9±0.6 μm in width. The spores containing 4 polar capsules had a length of 2.1±0.2 μm and a width of 1.8±0.3 μm. The partial 18S and 28S rDNA of isolates showed 99–100% similarities with K. ogawai. Using these morphological and molecular analyses, the species was identified as K. ogawai. This study is the first report of K. ogawai infection in cultured olive flounder in Korea.

Sinuolinea capsularis (Myxosporea: Sinuolineidae) Isolated from Urinary Bladder of Cultured Olive Flounder Paralichthys olivaceus

Sinuolinea capsularis Davis, 1917 is myxosporean that infect the urinary system of the host fish. Insufficient morphological and molecular data of S. capsularis exits, and it is therefore difficult to make an accurate identification of the parasite. We tried a series of morphological and molecular analysis to identify an myxosporean isolated from urinary bladder of cultured olive flounder, Paralichthys olivaceus, from Jeju island in the Republic of Korea. Some of them were observed under a light microscope and SEM, and remain samples were used molecular and phylogenetic analysis. Mature spores were subspherical, measuring 13.9±0.6 μm in length and 13.8±0.8 μm in width. Two spherical polar capsules on opposite sides in the middle of the spore had a diameter range of 4.3±0.4 μm. Scanning electron microscopy revealed that spores a severely twisted the suture line. By the morphological comparison and analysis, it was identified as S. capsularis. In addition, we obtained the partial 18S rDNA of S. capsularis and first registered it in NCBI. Phylogenetic analysis showed that S. capsularis clustered with Zschokkella subclade infecting the urinary system of marine fish, and it supported the infection site tropism effect on phylogeny of marine myxosporeans as well as the origin of Sinuolinea is not monophyly.

Ocurrencia de escuticociliatosis en el lenguado Paralichthys adspersus causado por Miamiensis avidus, en Perú / Occurrence of scuticociliatosis in the flounder Paralichthys adspersus caused by Miamiensis avidus, in Peru

La escuticociliatosis es una enfermedad causada por ciliados del orden Scuticociliatida los cuales se caracterizan por su alto potencial para invadir al huésped, significando un serio problema en la acuicultura marina. El presente trabajo describe la infección con escuticociliados en lenguado Paralichthys adspersus en cautiverio. Los signos externos e internos de la infección incluyen zonas necróticas en el tegumento, abundante mucosidad, abultamiento de la cavidad visceral con acumulación de líquido ascítico, necrosis de las fibras musculares, licuefacción del cerebro, entre otros. Se identificó y caracterizó molecularmente al parásito ciliado como M. avidus, utilizando secuencias de gen mitocondrial citocromo oxidasa I (COI), y de los genes nucleares β-tubulina y la región de la subunidad menor rRNA 18S, mostrando una sinonimia con P. dicentrarchi. Todas las lesiones estuvieron invadidas de ciliados. A nivel histológico, se detectaron ciliados en casi todos los tejidos siendo el cerebro el órgano más parasitado. Los ejemplares infectados, acompañados de infecciones bacterianas secundarias predominantemente por Vibrio alginolyticus, después de un periodo de letargia mueren.

The Scuticociliatosis, a disease caused by ciliates from the order Scuticociliatida characterized by their high potential to in vade the host, is a serious problem in marine aquaculture. This paper describes scuticociliatosis infection in farmed flounder Paralichthys adspersus. External and internal signs of infection include necrotic areas in the tegument, abundant mucus, swelling of the visceral cavity with ascitic fluid accumulation, necrotic muscle fibers and brain liquefaction, among others. The ciliate parasite was molecularly identified and characterized as M. avidus, using sequences of mitochondrial gene cytochrome oxidase I (COI), and nuclear genes β-tubulin and the region of the small subunit 18S rRNA, showing synonymy with P. dicentrarchi . All lesions were infested by ciliates. Histologically, ciliates are detected in almost all tissues being the brain the organ more parasitized. The infected specimens associated with secondary bacterial infections, predominantly Vibrio alginolyticus, died after a lethargy period.

Recombination-activating gene 1 and 2 (RAG1 and RAG2) in flounder (Paralichthys olivaceus).

During the development of B and T lymphocytes, Ig and TCR variable region genes are assembled from germline V, D, and J gene segments by a site-specific recombination reaction known as V(D)J recombination. The process of somatic V(D)J recombination, mediated by the recombination-activating gene (RAG) products, is the most significant characteristic of adaptive immunity in jawed vertebrates. Flounder (Paralichthys olivaceus) RAG1 and RAG2 were isolated by Genome Walker and RT-PCR, and their expression patterns were analysed by RT-PCR and in situ hybridization on sections. RAG1 spans over 7.0 kb, containing 4 exons and 3 introns, and the full-length ORF is 3207 bp, encoding a peptide of 1068 amino acids. The first exon lies in the 5′-UTR, which is an alternative exon. RAG2 full-length ORF is 1062 bp, encodes a peptide of 533 amino acids, and lacks introns in the coding region. In 6-monthold flounders, the expression of RAG1 and RAG2 was essentially restricted to the pronephros (head kidney) and mesonephros (truck kidney). Additionally, both of them were mainly expressed in the thymus. These results revealed that the thymus and kidney most likely serve as the primary lymphoid tissues in the flounder.

Development of a multiplex PCR assay to detect Edwardsiella tarda, Streptococcus parauberis, and Streptococcus iniae in olive flounder (Paralichthys olivaceus)

A multiplex PCR protocol was established to simultaneously detect major bacterial pathogens in olive flounder (Paralichthys olivaceus) including Edwardsiella (E.) tarda, Streptococcus (S.) parauberis, and S. iniae. The PCR assay was able to detect 0.01 ng of E. tarda, 0.1 ng of S. parauberis, and 1 ng of S. iniae genomic DNA. Furthermore, this technique was found to have high specificity when tested with related bacterial species. This method represents a cheaper, faster, and reliable alternative for identifying major bacterial pathogens in olive flounder, the most important farmed fish in Korea.

External Application of Fermented Olive Flounder (Paralichthys olivaceus) Oil Alleviates Inflammatory Responses in 2,4-Dinitrochlorobenzene-induced Atopic Dermatitis Mouse Model

Allergic skin inflammation such as atopic dermatitis (AD) is characterized by edema and infiltration with various inflammatory cells such as mast cells, basophils, eosinophils and T cells. Thymic stromal lymphopoietin (TSLP) is produced mainly by epidermal keratinocytes, as well as dermal fibroblasts and mast cells in the skin lesions of AD. Omega-3 polyunsaturated fatty acids in fish oil can reduce inflammation in allergic patients. Fermentation has a tremendous capacity to transform chemical structures. The anti-inflammatory effects of fish oil have been described in many diseases, but the beneficial effects by which fermented olive flounder oil (FOF) modulates the allergic response is poorly understood. In this study, we produced FOF and tested its ability to suppress the various allergic inflammatory responses. The ability of FOF to modulate the immune system was investigated using a mouse model of AD. The FOF-treated group showed significantly decreased immunoglobulin E (IgE) and histamine in serum. Also, the increased TSLP expression was significantly inhibited in the FOF group; the FOF-treated group was not appreciably different from the hydrocort cream treatment group. In addition, FOF treatment resulted in a smaller spleen size with reduced the thickness and length compared to the induction group. Splenocytes from mice treated with FOF produced significantly less IFN-gamma, IL-4, T-box transcription factor (T-bet) and GATA binding protein 3 (GATA3) expression compared with the induction group. These results suggest that FOF may be effective in treating the allergic symptoms of AD. 5.

Variações ontogênicas e sazonais na dieta de Etropus crossotus Jordan e Gilbert, 1882 (Paralichthydae) na baía de Ubatuba-Enseada, Estado de Santa Catarina, Brasil / Ongenic and seasonal variations in the diet of Etropus crossotus Jordan and Gilbert, 1882 (Paralichthydae) in the bay of Ubatuba-Enseada, State of Santa Catarina, Brazil

Foram investigadas as variações ontogênicas e sazonais na dieta de Etropus crossotus coletados na baía de Ubatuba-Enseada, Estado de Santa Catarina, Brasil. Redes de arrasto com porta foram utilizadas para as coletas mensais, e 60 exemplares foram coletados entre outubro de 2003 e setembro de 2004. Maior similaridade entre os Índices de Importância Alimentar foi observada entre a primavera e o outono. Crustáceos decápodes representaram 50% dos itens alimentares durante a primavera e outono, e crustáceos anfípodes foram os itens mais representativos no inverno. Maior similaridade entre os IAi foi observada entre as classes 1, 2 e 4, pela alta representatividade de Decapoda, e entre as classes 5, 6 e 7, em função da representatividade de Decapoda e Amphipoda. Os resultados obtidos foram similares a outros estudos realizados na região costeira Sul do Brasil.

Ontogenetic and seasonal changes in the diet of Etropus crossotus sampled at Ubatuba -Enseada Bay in Santa Catarina State, Brazil were investigated. A trawl net was used for the surveys, and 60 fish were collected monthly during October (2003) and September (2004). The highest similarity of the Alimentary Importance Index (AII) was observed between spring and autumn. Crustacea Decapoda represented 50% of the feeding items during spring and fall, and Crustacea Amphipoda were the most representative item in winter. The highest AII similarity was observed among length classes 1, 2 and 4, which was related to Decapoda representation, and among length classes 5, 6 and 7, because of Decapoda and Amphipoda importance. Results were similar to other studies in the southern coast of Brazil.

Expression, Purification of Recombinant Flounder MRF4 Protein in Escherichia coli and Analysis of Its Polyclonal Antibodies / 生物化学与生物物理进展

MRF4 is one of muscle regulatory factors and plays critical roles during skeletal muscle development. The muscle development is important for the fish growth which is an important economic factor for the fish culture. To analyze the function of MRF4 in fish, the founder MRF4 antibody was prepared. The flounder MRF4 was cloned, ligated into prokaryotic expression vector pET-30b and expressed in strain E. coli BL21 (DE3). The recombinant flounder MRF4 fusion protein was soluble and purified with cobalt IMAC resins. To prepare MRF4 polyclonal antibodies, rabbits were immunized with the soluble protein and the increasing level of antibodies was determined by Western blot. Also, the endogenous flounder MRF4 was recognized by the anti-serum. The result further proved the existence of the anti-MRF4 antibody in the anti-serum, which will be useful for studies on the function of flounder MRF4.

Identification of Acinetobacter junii Morphovari from Diseased Stone Flounder (Kareius bicoloratus L.) / 微生物学通报

Successive pathogenic bacteria were examined from liver,spleen, kidney and contents of intestine of diseased stone flounder (Kareius bicoloratus L.) occurring bacterial septicaemia infection caused by Aeromonas salmonicida. Identification of extensive phenotypic information to 6 pure cultures and detection of the mol% G+C ratio of the DNA to representative strains,showed that the examined bacteria belonged to a new morphovar of Acinetobacter junii, and was designated as Acinetobacter junii morphovar I. In addition, serotype, antibiotic sensitivity and pathogenicity of isolates were studied, the results showed that the 6 strains have the same K-antigen and O-antigen, there are no obvious differences in sensitivity and resistance to used 37 antimicrobial agents between strains, and have strong pathogenicity to experimental stone flounder and Bastard halibut.