Identification, Growth Profile and Probiotic Properties of Autochthonous Intestinal Bacteria of Sagor catfish (Hexanematichthys sagor).

　The prevalence of antibiotic resistant bacteria in aquaculture has reached alarming proportions and intensified the search for microbe derived antimicrobial compounds. This study isolated bacteria from the intestine of Sagor catfish (Hexanematichthys sagor) and screened it for antagonistic properties. Five out of 334 bacterial isolates inhibited growth of fish pathogens. The 5 bacterial strains included relatives of Shewanella haliotis, Myroides odoratimimus, Vibrio harveyi, Vibrio alginolyticus and Alcaligenes faecalis. The growth profiles and probiotic properties of these bacteria were examined. The results showed that the isolate 9 (3) 7.5.2.1, whose closest relative was S. haliotis exhibited growth and probiotic advantage compared to the other bacterial strains, such as highest doubling time and the ability to survive at all experimental temperatures (18 to 60℃) , and bile concentrations (0.01 to 1.00%) and pH (pH2 to 9) . While the bacteria with probiotic properties were successfully isolated. Further study is necessary to examine the efficiency of the probiotic candidate bacteria in boosting fish immunity against pathogens.

Leucoxenols A and B, two new phenolics from Bornean medicinal plant Syzygium leucoxylon.

The medicinal plant, Syzygium leucoxylon or commonly known as Obah found in North Borneo was considered as traditional medicine by local committee. Two new phenolics, leucoxenols A (1) and B (2) were isolated and identified as major secondary metabolites from the leaves of S. leucoxylon. Their chemical structures were elucidated based on spectroscopic data such as NMR and HRESIMS. Furthermore, these compounds were active against selected strains of fungi.

First Report of Achlya oblongata Infection in Freshwater-Reared Asian Seabass Lates calcarifer.

In September 2014, a freshwater oomycete was first isolated from Asian Seabass Lates calcarifer fry that were reared in freshwater at a fish hatchery in Sabah, Malaysia. A fungal strain was isolated from infected fry by using glucose yeast extract (GY) agar. From morphological identification, the strain belonged to the genus Achlya based on the mode of zoospore release. Molecular phylogenetic analysis of the internal transcribed spacer region sequences from the strain showed high similarity (99-100%) to Achlya oblongata. The isolate was able to grow on GY agar incubated at 15-35°C, in GY broth adjusted to pH 3.0-11.0, and in up to 1.0% NaCl. This is the first report of Achlya infection in freshwater-reared Asian Seabass in Malaysia.

In vitro Inhibitory Effects of Two Bornean Medicinal Wild Gingers against Pathogenic Lagenidium thermophilum Infected Mud Crab Scylla tranquebarica.

　The antifungal activity of two Bornean medicinal wild gingers Plagiostachys megacarpa and Zingiber phillippsiae were examined against Lagenidium thermophilum. The most active extract was P. megacarpa at concentration of 320 µg/mL inhibiting both hyphal growth and zoospore production of L. thermophilum in 24 h. Toxicity tests were conducted using mud crab (Scylla tranquebarica) larva. Bath treatment of P. megacarpa at concentrations of 320 and 640 µg/mL for 24 h were highly effective against hyphae and zoospores of the strain and it is non-toxic to mud crab larva. Therefore, crude extracts P. megacarpa may be used as alternative treatment for marine Oomycete infection of mud crab.

Complete genome sequence of a giant Vibrio phage ValKK3 infecting Vibrio alginolyticus.

This paper describes the complete sequence of a giant lytic marine myophage, Vibrio phage ValKK3 that is specific to Vibrio alginolyticus ATCC(®) 17749™. Vibrio phage ValKK3 was subjected to whole genome sequencing on MiSeq sequencing platform and annotated using Blast2Go. The complete sequence of ValKK3 genome was deposited in DBBJ/EMBL/GenBank under accession number KP671755.

Genotypic characteristics of a Mycobacterium sp. isolated from yellowtail Seriola quinqueradiata and striped jack Pseudocaranx dentex in Japan.

In Japan, a Mycobacterium marinum-like mycobacterium was isolated from the yellowtail, Seriola quinqueradiata. The species was identified as M. marinum by a commercial mycobacterial DNA-DNA hybridization kit. Nevertheless, PCR restriction analysis of the DNA of its RNA polymerase ß-subunit gene definitively showed that this Mycobacterium sp. was M. ulcerans. PCR analysis revealed the genotypic characteristics of M. ulcerans in the Mycobacterium sp., only the mup053 gene sequence being absent, as has been found previously in other piscine mycobacteria such as M. marinum strains DL240490 and DL045 and M. pseudoshottsii. With one exception, this Mycobacterium sp. and M. pseudoshottsii had identical 16S rRNA gene sequences, which is also probably true of M. marinum strains DL240490 and DL045. Similarly, according to comparisons of the 16S rRNA gene, ITS region, and hsp65 gene sequences, this Mycobacterium sp. is more closely related to M. pseudoshottsii than to M. ulcerans or M. marinum. A PCR product of approximately 2000 bp was amplified from region of difference 9 in the Mycobacterium sp. The nucleotide sequence revealed insertion of IS2404, the sequence of which is 1366 bp long. The novel single nucleotide polymorphisms identified in this region distinguished this Mycobacterium sp. from M. marinum strain DL240490 and M. pseudoshottsii. The present findings raise the possibility that these species have a common ancestor. Further studies are required to improve our understanding of the relationship between their geographical origin and genetic diversity.

Diseases of fish and shellfish caused by marine fungi.

Fungal diseases are problematic in cultured fish and shellfish, their seeds, and sometimes wild marine animals. In this chapter fungal diseases found in marine animals, especially in Japan, are described. Pathogens in the fungal diseases are divided into two groups. One of them is marine Oomycetes, which cause fungal diseases in marine shellfish and abalones. The diseases caused by the fungi of this group and the fungal characteristics are introduced. The pathogens include members of the genera Lagenidium, Haliphthoros, Halocrusticida, Halioticida, Atkinsiella, and Pythium. On the other hand, some fungal diseases caused by mitosporic fungi are also known in marine fish and shellfish. The diseases caused by these fungi and the fungal characteristics are described. The pathogens include members of the genera Fusarium, Ochroconis, Exophiala, Scytalidium, Plectosporium, and Acremonium.

Mycobacterium pseudoshottsii isolated from 24 farmed fishes in western Japan.

Mycobacteria isolated from epizootics of farmed fishes in western Japan were examined for the first time using multigenotypic analysis. By analysis of the sequences of the internal transcribed spacer between the 16S and 23S rRNA genes (ITS) region and the partial 16S rRNA, hsp65 and rpoB genes, M. pseudoshottsii was identified as the causative agent in these infections. Prior to this study, only M. marinum has been known as the causative agent of lethal mycobacterial disease in marine fishes in Japan.

Lymphocytes with T-cell-like properties express the Fas ligand in the Japanese flounder Paralichthys olivaceus.

In this study, we aimed to identify the leukocyte population that expresses Fas ligand (FasL) in the Japanese flounder (Paralichthys olivaceus). The transcriptional activity of FasL was examined for the first time in the fish leukocytes. Transcription of the FasL gene in flounder leukocytes was significantly increased by phytohemagglutinin (PHA) treatment. All the leukocyte populations we tested possessed binding activity for PHA, but this was especially high in the lymphocyte population. However, the lymphocytes consisted of two subsets showing heterogeneity with respect to PHA binding, with the high-binding subset being surface IgM-negative. We also found that only the lymphocyte population showed a significant increase in the expression of the FasL gene after stimulation with PHA. In addition, only the lymphocyte subset showing high binding to PHA showed conspicuous expression of the FasL gene. This subset also had a CD3γ/δ+, CD8α+ and IgM heavy-chain (-) phenotype. These results suggested that lymphocytes with T-cell-like properties are FasL-expressing cells in the Japanese flounder.

Fungal infection of mantis shrimp (Oratosquilla oratoria) caused by two anamorphic fungi found in Japan.

Two fungal pathogens of the mantis shrimp (Oratosquilla oratoria) in Yamaguchi and Aichi Prefectures, Japan are described as the new species Plectosporium oratosquillae and Acremonium sp. (a member of the Emericellopsis marine clade). Both fungi infect the gills of the mantis shrimp, which become brown or black due to melanization. The former species is characterized by its slow growth on artificial seawater yeast extract peptone glucose (PYGS) agar, pale yellow to pale orange or grayish yellow colonies, short cylindrical solitary phialides with a wavy tip, and one-celled ellipsoidal conidia. Although lacking the two-celled conidia demonstrated by the type species Plectosporium tabacinum, the taxonomic placement of the new species was confirmed by DNA sequence analysis of the internal transcribed spacer (ITS) region of ribosomal DNA (ITS1, 5.8S rDNA and ITS2). Acremonium sp., the other causal pathogen, differs from P. oratosquillae by its fast growth on PYGS agar, pale orange to salmon-colored colonies, long, slender conidiophores consisting of solitary phialides with tips lacking an undulate outline, and typically cylindrical conidia. Analysis of ITS and beta-tubulin gene sequences placed this fungus within the phylogenetically distinct Emericellopsis (anam. Acremonium) marine clade. Various physiological characteristics of both pathogens were also investigated. This is the first report of a fungal infection found on the mantis shrimp in Japan.

In vitro leukocyte-encapsulation model in rainbow trout (Oncorhynchus mykiss).

We developed an in vitro model to study the cellular and molecular mechanisms of granulomatous inflammation in response to invading pathogens. Ichthyophonus hoferi was used as a target for encapsulation by cultivated leukocytes from the kidney of the rainbow trout (Oncorhynchus mykiss). The encapsulation process was observed over 1 week. The leukocytes were identified as either macrophages in the inner layer, or neutrophils and lymphocytes in the outer layer. The encapsulation response was inhibited by treatment with heat, but not formalin or methanol. The recognition of heat-unstable molecules on the pathogen surface could induce encapsulation. Increased expression of pro-inflammatory cytokines, such as interleukin (IL)-1beta, IL-8 and tumor necrosis factor-alpha2, was observed during encapsulation. These cytokines might play crucial roles in the encapsulation process. In particular, IL-8, which was expressed at a late phase, might recruit specific cell populations, such as the lymphocytes comprising the outer cellular layer around the target.

Transmission of the parasite Ichthyophonus hoferi in cultured rainbow trout and comparison of epidemic models.

The epidemic process of the parasite Ichthyophonus hoferi in cultured rainbow trout Oncorhynchus mykiss was quantitatively estimated by both the cohabitation experiment and two standard models (the Kermarck-McKendrick model and the Reed-Frost model). For analysis of the parasite transmission by cohabitation, fish in two replicate tanks were exposed to 1, 5, or 10 infected fish, and daily mortality was counted for 102 d. Despite simple experiments for artificial exposure to the pathogen, the daily estimate of dead fish in the Kermarck-McKendrick model did not fit the observed number of dead fish in the experiment. In contrast, when the longest possible incubation period (generation time) was assumed to be 51 d in the Reed-Frost model, the estimated number of dead fish in discrete generations was close to the observed number of dead fish. If the time unit was 51 d, the estimated mortalities in the generation-based Kermarck-McKendrick model were significantly correlated with observed mortalities. These results suggest that the deterministic aspects of the epidemic process of the parasite can be quantitatively demonstrated on a 51-d timescale or longer, whereas transmission on a daily timescale is uncertain.

The use of bronopol to control fungal infection in rainbow trout eggs.

We evaluated bronopol as a practical alternative anti-fungal agent to malachite green for use in hatcheries. Repeated exposure everyday, just after fertilization to the stage of eggs showing eye development to 50 mg/L and 100 mg/L bronopol for 30 min showed an efficacy comparable to 0 mg/L (control) for the inhibition of fungal infection in rainbow trout (Oncorhynchus mykiss) eggs. The 100 mg/L bronopol treatment groups showed a control of fungal infection up to the stage of eye development in eggs. Significant differences in terms of the number of eggs showing eye development were seen between 0 mg/L and treatment at 50 and 100 mg/L bronopol.

Lecythophora hoffmannii isolated from a case of canine osteomyelitis in Japan.

A 2-year-old spayed female mongrel dog showed claudication with abnormal ossification containing fungal cells detected by biopsy. The dog was treated with ketoconazole and itraconazole perorally for 5 months; however, the osteomyelitis became aggravated, and an amputation from the scapula was performed. The right superficial cervical lymph node became swollen 5 months after the operation. The lymph node contained PAS positive fungal elements and a portion of tissue produced mycelial fungal growth on potato dextrose agar supplemented with chloramphenicol. The culture was identified as Lecythophora hoffmannii based on morphology, physiology and 100% identity in the sequence of the D1/D2 domain of the large subunit ribosomal RNA gene of the fungal species in the GenBank database (accession number AB100627). In addition, the sequence from the present isolate was submitted as AB189164. The isolate showed resistance to antifungal agents, i.e., amphotericin B, 5-FC, fluconazole, itraconazole, miconazole and micafungin. The dog developed cachexia 2 months after the onset of lymphadenopathy, and was euthanized on the 459th day after onset of clinical symptoms. This was the first disseminated case of L. hoffmannii infection in Japan.

Antifungal activities of bronopol and 2-methyl-4-isothiazolin-3-one (MT) against saprolegnia.

This study compared the antifungal activities of bronopol and 2-methyl-4-isothiazolin-3-one (MT) against members of the genus Saprolegnia in the family saprolegniaceae, which have the ability to infect fish. The minimum inhibitory concentration of bronopol was 100-200 mg/l but for MT it was 31 mg/l. Concerning fungicidal effects against the hyphae, treatment with bronopol at 100-200 mg/l for 30 min or treatment with MT at 25-50 mg/l for 30 min was effective in killing the vegetative stage of all fungal strains tested. Treatment with bronopol at 100 mg/l for 60 min or treatment with MT at 25-50 mg/l for 60 min was effective in killing the zoospores of all fungal strains tested. These results showed that MT was a more effective antifungal agent than bronopol against infectious members of the genus Saprolegnia.

Activation of carp leukocytes by a galactose-binding protein from Aphanomyces piscicida.

This study demonstrated that a galactose-binding protein (GBP) produced by a fish pathogenic water mold, Aphanomyces piscicida, activates carp leukocytes. Leukocytes were separated from the head kidney and peripheral blood using Percoll density centrifugation. A flow cytometric analysis revealed that GBP binds with many cells and a variety of cell types including lymphocytes, granulocytes and thrombocytes. Intracellular calcium flux of the peripheral blood leukocytes induced by stimulation with GBP was confirmed by counting the fluo-3 loaded cells whose fluorescence increased after the stimulation using flow cytometry. The percentage of cells in which a calcium flux was induced peaked 1 min after the stimulation. Approximately 6% of the cells specifically responded 1 min after the stimulation. The proliferation response was determined by the level of BrdU uptake by the leukocytes after the stimulation. Cell proliferation was observed 2, 4 and 6 days after stimulation with GBP. The expression of cytokines IL-1beta and TGF-beta1 in the peripheral blood leukocytes, after the stimulation was evaluated by a semi-quantitative reverse-transcriptase polymerase chain reaction. Increased expression of IL-1beta was observed 4h after stimulation with GBP. Variation of TGF-beta1 expression under the same conditions was not observed. The kinetics of intracellular calcium flux and the level of IL-1beta expression induced by GBP stimulation were different from those induced by phytohemagglutinin stimulation. These results confirmed that GBP is a pathogenic microbial component that can induce cell activation. GBP seems to induce the inflammatory response observed in the Aphanomyces infection.