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NO47 - 2

Molecular Identification of Marine Crustacean-pathogenic Peronosporomycetes Using DNA Sequences of ITS1 and their Pathogenicity for Nauplii of Brine Shrimps

Yasunori Muraosa1*,2, Ayako Sano3/ and Kishio Hatai2
1Division of Clinical Research, Medical Mycology Research Center, Chiba University, Chiba 260-8673, Japan
2Laboratory of Fish Diseases, Nippon Veterinary and Life Science University, Tokyo 180-8602, Japan
3Department of Animal Sciences, Faculty of Agriculture, University of the Ryukyus, Okinawa 903-0213, Japan

(Received November 9, 2011) (Received November 9, 2011)

ABSTRACT―We isolated 27 strains of marine crustacean-pathogenic Peronosporomycetes from various infected marine crustaceans collected in Japan, Thailand, Vietnam and the Philippines, and identified them morphologically. The internal transcribed spacer (ITS) l region of the nuclear rRNA gene of the 27 strains and 12 reference strains of six peronosporomycete species isolated from marine crustaceans were sequenced. The phylogenic relationship inferred from the sequences corresponded well to the morphological identification, indicating that sequencing of the ITS1 region is a useful tool for identification of marine crustacean-pathogenic Peronosporomycetes. Nauplii of brine shrimp Artemia salina were experimentally challenged by bathing in zoospore suspensions (1 × 104 zoospores/mL, 25°C) with 18 strains belonging to nine species in four genera. The 48-h mortalities varied from 1.1% to 99.4% among the species, though all the strains were isolated from diseased animals.

Key words:Peronosporomycetes, Lagenidium, Haliphthoros, Halocrusticida, Atkinsiella, pathogenicity, Artemia salina, brine shrimp

Intra-ovum Infection in Salmonid Eggs Artificially Contaminated with Fish Pathogenic Bacteria: Flavobacterium psychrophilum, Renibacterium salmoninarum and Aeromonas salmonicida

Masakazu Kohara1*, Hisae Kasai2 and Mamoru Yoshimizu2
1Nagano Prefectural Fisheries Experimental Station Saku-branch, Nagano 385-0042, Japan
2Faculty of Fisheries Sciences, Hokkaido University, Hokkaido 041-8611, Japan
(Received January 12, 2012)

ABSTRACT―Intra-ovum infection of artificially contaminated salmonid (rainbow trout or amago salmon) eggs was studied using three fish pathogens (Flavobacterium psychrophilum, Renibacterium salmoninarum and Aeromonas salmonicida). Artificial contamination was performed using egg-surface contamination or immersion-water contamination. For F. psychrophilum and R. salmoninarum, infection occurred by the entry of pathogens from the egg surface to the perivitelline space through the micropyle during water-hardening. The infection rate was higher for egg-surface contamination than for immersion-water contamination. The successful concentration of F. psychrophilum for the occurrence of intra-ovum infection was 107 CFU/mL or more for egg surface contamination, and was 109 CFU/mL or more for immersion-water contamination. After the entry into eggs, F. psychrophilum increased to 107 CFU/egg but A. salmonicida steadily decreased and became undetectable.

Key words:intra-ovum infection, micropyle, perivitelline space, salmonid, Flavobacterium psychrophilum, Renibacterium salmoninarum, Aeromona salmonicida

Multiple Drug-resistant Strains of Aeromonas hydrophila Isolated from Tilapia Farms in Thailand

Nutcharnart Tipmongkolsilp1, Carmelo S. del Castillo2, Jun-ichi Hikima2, Tae-Sung Jung2, Hidehiro Kondo1, Ikuo Hirono1 and Takashi Aoki1,2*
1Laboratory of Genome Science, Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan
2Aquatic Biotechnology Center, College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701, South Korea
(Received February 3, 2012)

ABSTRACT―Fifty-five Aeromonas hydrophila isolates, which were collected from infected tilapia Oreochromis niloticus at aquaculture farms in Thailand, were tested for their antimicrobial susceptibilities against 11 drugs including ampicillin (ABPC), kanamycin (KM), streptomycin (SM), chloramphenicol (CP), florfenicol (FF), tetracycline (TC), sulfamonomethoxine (SMMX), trimethoprim (TMP) and three quinolones (QNs). All strains showed resistance to one or more drugs except FF, and more than half of the strains showed resistance to five drugs and more. These isolates were conjugated with Escherichia coli for detection of the transferable R-plasmid, and only one transferable R-plasmid, which encoded resistance to the combination of ABPC, CP, SM, SMMX and TC, was found from one isolate. This R-plasmid contained blaOXA-35 (ABPC resistance), cat2 (CP resistance), aadA1 (SM resistance), sul1 (SMMX resistance) and tetA (TC resistance).

Key words: Aeromonas hydrophila, antimicrobial resistance, transferrable R-plasmid, Oreochromis niloticus, tilapia

Subclinical Edwardsiella ictaluri Infection of Wild Ayu Plecoglossus altivelis

Ebtsam Sayed Hassan1,2§, Mahmoud Mostafa Mahmoud1,2§, Yasuhiko Kawato1, Takahiro Nagai3, Osamu Kawaguchi3, Yoshisuke Iida3, Kei Yuasa4 and Toshihiro Nakai1*
1Graduate School of Biosphere Science, Hiroshima University, Hiroshima 739-8528, Japan
2Faculty of Veterinary Medicine, Assiut University, Assiut 71526, Egypt
3Fisheries and Marine Technology Center, Hiroshima Prefectural Technology Research Institute, Hiroshima 737-1207, Japan
4National Research Institute of Aquaculture, Fisheries Research Agency, Mie 516-0193, Japan
(Received February 6, 2012)

ABSTRACT―Edwardsiella ictaluri infection is a newly emerging disease in ayu Plecoglossus altivelis in Japanese rivers, and has been continuously observed since the first outbreak in 2007. The present field study was performed in a river in Hiroshima Prefecture, Japan, where mortality due to E. ictaluri infection was recorded in 2007. Investigations over the course of 3 years (2008-2010) revealed the existence of constant subclinical E. ictaluri infection in apparently healthy ayu in the river, with higher rates (average 45.4%) in September and October, when water temperature drops and fish mature sexually. Among 11 other wild fish species examined at the same time, only one forktail bullhead Pelteobagrus nudiceps was positive for E. ictaluri. The bacterium was not isolated from groups of juvenile ayu just before the release into the river for stock enhancement of this species. On the other hand, the ubiquitous presence of E. ictaluri-specific phages in the river water and sediments suggested the existence of E. ictaluri in the river; that represents an environmental reservoir of the pathogen and may serve as a potential infection source for ayu. The current subclinical status of wild ayu infected with E. ictaluri might turn to overt infection and thus cause mortality under unidentified stress conditions.

Key words: Edwardsiella ictaluri, Plecoglossus altivelis, subclinical infection, epizootiology, wild fish

Virulence of Yersinia ruckeri for Four Indigenous Fish Species in Japan

Takamitsu Sakai1*, Chihaya Nakayasu1, Takafumi Ito1, Satoshi Miwa1, Norihisa Oseko2 and Takaji Iida2
1Tamaki Laboratory, National Research Institute of Aquaculture, Fisheries Research Agency, Mie 519-0423, Japan
2National Research Institute of Aquaculture, Fisheries Research Agency, Mie 516-0193, Japan
(Received April 26, 2012)

ABSTRACT―Enteric redmouth disease (ERM), which is caused by Yersinia ruckeri, has not been reported in Japan. In the present study, virulence of the bacterium were assessed by experimental challenges with the rainbow trout Oncorhynchus mykiss and 4 indigenous fish species in Japan: yamame (masu salmon parr) O. masou masou, amago O. masou macrostomus, whitespotted char Salvelinus leucomaenis and ayu Plecoglossus altivelis. When intraperitoneally (ip) injected with the bacterium at a dose of 7.1 × 102 CFU/g fish body weight (BW), rainbow trout, char, yamame, and amago showed the cumulative mortalities of 100%, 60%, 30% and 30%, respectively. The cumulative mortalities of ip-injected ayu with doses of 1.5 × 106, 1.5 × 105, 1.5 × 104 and 1.5 × 103 CFU/g BW were 87%, 47%, 13% and 0%, respectively. In addition, the cumulative mortalities of ayu immersed in water containing 1.7 × 108 and 1.7 × 107 CFU/mL of the bacterium were 60% and 6.7%, respectively. Typical clinical signs of ERM, such as reddening of the wall of the oral cavity, tongue, upper jaw, lower jaw, operculum, bases of fins, or skin around the anus, were observed in all dead salmonids but not in ayu. Instead, exophthalmos, hemorrhage in the eyes, ascites or hypertrophy of the kidney were often observed in dead ayu. These results indicate that Yersinia ruckeri is a potentially dangerous bacterium to freshwater aquaculture in Japan.

Key words: Yersinia ruckeri, enteric redmouth disease, Oncorhynchus mykiss, Oncorhynchus masou masou, Oncorhynchus masou macrostomus, Salvelinus leucomaenis, Plecoglossus altivelis

Streptococcus parauberis Infection in Turbot Scophthalmus maximus in Northern Portugal

Miguel Filipe Ramos1,2, Joana Filipa Marques1,2, João Vilares Neves3, Teresa Barandela3, José Américo Sousa1,2, Aurelia Saraiva1,2 and Pedro N. Rodrigues3*
1FCUP (Faculty of Science of the University of Porto), 4169-007 Porto, Portugal
2CIIMAR (Interdisciplinary Centre of Marine and Environmental Research), 4050-123 Porto, Portugal
3ICBAS (Institute of Biomedical Sciences Abel Salazar), 4099-003 Porto, Portugal
(Recieved December 7, 2011)

ABSTRACT―High mortalities of turbot Scophthalmus maximus were observed at a fish farm in northern Portugal from May to August in 2004. Moribund fish exhibited typical symptoms of streptococcosis, such as uni- or bilateral exophthalmia and hemorrhages and edema in the dorsal trunk region and the base of fins. Gram-positive a-haemolytic cocci were isolated from moribund fish and identified as Streptococcus parauberis based on physiological, biochemical and serological characteristics and 16S rDNA-targeted PCR. Histopathological examinations revealed occurrence of meningitis and presence of bacteria in the connective tissue of examined organs. This is the first report of S. parauberis infection in cultured turbot in Portugal.

Key words: Streptococcus parauberis, streptococcosis, Scophthalmus maximus, histopathology, turbot