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NO49 - 4

Failure to Protect Yellowtail Seriola quinqueradiata against Nocardiosis by Vaccination with a Recombinant Protein and pcDNA4 Expression Vector of Edwardsiella tarda Glyceraldehyde-3-phosphate Dehydrogenase

Masayuki Imajoh1*, Kazuki Tamura1, Kazuki Oguro1, Sumire Kurihara1, Jin Yamane1, Masato Shimizu1, Syun-ichirou Oshima2 and Kenji Kawai3

1Laboratory of Fish Disease, Faculty of Agriculture, Kochi University, Kochi 783-8502, Japan
2Graduate School of Kuroshio Science, Kochi University, Kochi 783-8502, Japan
3The United Graduate School of Agricultural Sciences, Ehime University, Ehime 790-8566, Japan

(Received December 16, 2013)

ABSTRACT―Vaccination with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Edwardsiella tarda has been demonstrated to have cross-protection against some other bacterial pathogens. We prepared a recombinant protein and pcDNA4 expression vector of GAPDH of E. tarda and injected those to 0-age yellowtail Seriola quinqueradiata to evaluate their protective efficacies against Nocardia seriolae. The 60-d survival rates ranged from 0% to 6.7% among vaccinated and control groups, suggesting that the protein and expression vector of E. tarda GAPDH had no protective effect against nocardiosis. However, the copy numbers of the 16S ribosomal RNA gene of N. seriolae were significantly lower in the gill, kidney, and spleen of vaccinated fish 9 and 18 days post-infection compared with those in the control group that was also challenged with N. seriolae. Additionally, the CC chemokine, major histocompatibility complex class II α antigen, interleukin 1β, and immunoglobulin M genes were significantly upregulated in the vaccinated groups. Four predicted B-cell epitopes of GAPDH in E. tarda, which may play an important role in cross-protection, differed in amino acid sequences from those of GAPDH in N. seriolae. This may explain the lack of a protective effect of E. tarda GAPDH against N. seriolae.

Key words: Edwardsiella tarda, Nocardia seriolae, GAPDH, vaccine, adjuvant, yellowtail, B-cell epitope


Development of PCR Diagnosis for Shrimp Acute Hepatopancreatic Necrosis Disease (AHPND) Strain of Vibrio parahaemolyticus

Sasiwipa Tinwongger1,2, Porranee Proespraiwong3, Jumroensri Thawonsuwan4, Preeyanan Sriwanayos4, Janejit Kongkumnerd4, Tidaporn Chaweepack5, Rapeepat Mavichak3, Sasimanas Unajak6, Reiko Nozaki1, Hidehiro Kondo1 and Ikuo Hirono1*

1Laboratory of Genome Science, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan
2Coastal Fisheries Research and Development Bureau, Department of Fisheries Kasetklang Chatuchak, Bangkok 10900, Thailand
3Charoen Pokphand Foods Public CO., LTD. Aquatic Animal Health Research Center, Samutsakorn 74000, Thailand
4Coastal Aquatic Animal Health Research Institute, Coastal Fisheries Research and Development Bureau, Department of Fisheries, Songkhla 90100, Thailand
5Chanthaburi Coastal Fisheries Research and Development Center, Coastal Fisheries, Research and Development Bureau, Department of Fisheries, Chanthaburi, 22000, Thailand
6Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok, 10900, Thailand

(Received July 9, 2014)

ABSTRACT―Acute hepatopancreatic necrosis disease (AHPND) is caused by a unique strain of Vibrio parahaemolyticus that has a plasmid harboring virulent genes. A rapid and accurate diagnosis method is necessary for surveillance of this infectious disease in aquaculture. In this study, three primer sets (TUMSAT-Vp1, Vp2 and Vp3) were designed based on the plasmid DNA sequence. We examined 98 strains of bacteria isolated from shrimp farms in different areas in Thailand. These included 48 strains of V. parahaemolyticus (AHPND strain), 38 strains of non-AHPND V. parahaemolyticus and 12 strains of non-V. parahaemolyticus. All the AHPND strains were detected by TUMSAT-Vp1, Vp2 and Vp3. However, one non-AHPND strain tested positive for TUMSAT-Vp1 and Vp2. The accuracy of AHPND detection was validated with two other primer publicly available sets (AP1 and AP2). AP1 and AP2 primers gave a few false positives in non-AHPND strains. Only TUMSAT-Vp3 primer detected the AHPND strains examined in this study with 100% accuracy.

Key words: acute hepatopancreatic necrosis disease, AHPND, early mortality syndrome, EMS, Vibrio parahaemolyticus, shrimp, PCR diagnosis, plasmid


Development of DNA Vaccines against Nocardia seriolae Infection in Fish

Goshi Kato1, Keitaro Kato2, Walissara Jirapongpairoj1, Hidehiro Kondo1 and Ikuo Hirono1*

1Laboratory of Genome Science, Graduate School of Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan
2Fisheries laboratory, Kinki University, Wakayama 649-2211, Japan

(Received July 11, 2014)

ABSTRACT―This study presents the development of DNA vaccines against nocardiosis, using the Antigen 85-like (Ag85L) gene in Nocardia seriolae. An expression plasmid encoding Ag85L (pAg85Lwt) and codon-optimized Ag85L (pAg85Lopt) was intramuscularly injected into amberjack Seriola dumerili. Survival rates of the pAg85Lwt, pAg85Lopt vaccinated group and PBS-injected group as a negative control were 88%, 98% and 51%, respectively, at 40 days after the N. seriolae challenge. In addition, the N. seriolae bacterial count in the spleen was significantly lower in the pAg85Lwt and pAg85Lopt vaccinated fish than in the PBS-injected fish (P < 0.05). These results suggest that the DNA vaccines pAg85Lwt and pAg85Lopt conferred protective efficacy against N. seriolae infection in amberjack.

Key words: DNA vaccine, Antigen 85 complex, mycolyl-transferase, Nocardia seriolae, nocardiosis, amberjack, Seriola dumerili, codon optimization


Use of Acridine Orange to Visually Improve the Loop-mediated Isothermal Amplification for Detection of Infectious Spleen and Kidney Necrosis Virus

Kuttichantran Subramaniam1, Mohamed Shariff1*,2, Abdul Rahman Omar1,2, Mohd Hair-Bejo1 and Bee Lee Ong1

1Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400, Selangor, Malaysia
2Institute of Bioscience, Universiti Putra Malaysia, 43400, Selangor, Malaysia

(Received July 20, 2014)

ABSTRACT―The advancement in nucleic acid amplification has improved the diagnostic methods for many diseases. In the current study, an improved technique for the detection of infectious spleen and kidney necrosis virus (ISKNV) based on loop-mediated isothermal amplification (LAMP) is described. The addition of acridine orange at the end of the reaction causes distinct color change which can be recognized by the naked eye. In addition, a new set of primers was designed based on the ISKNV major capsid protein (MCP) gene sequences. The primers are highly specific for ISKNV and there was no cross amplification with red sea bream iridovirus (RSIV), white spot syndrome virus (WSSV), Aeromonas hydrophila or Vibrio parahaemolyticus. The detection limit of LAMP assay was 1.4 × 104 copies of the virus DNA and the optimum temperature and time for assay was 65°C and 60 min respectively, and this protocol could successfully detect the virus from asymptomatically infected fish. This improved LAMP assay is a simple and inexpensive diagnostic tool for the detection of ISKNV without the need of specialized equipment.

Key words: infectious spleen and kidney necrosis virus, Megalocytivirus, major capsid protein gene, loop-mediated isothermal amplification, acridine orange, LAMP


A Novel Paramyxean Parasite, Marteilia granula sp. nov. (Cercozoa), from the Digestive Gland of Manila Clam Ruditapes philippinarum in Japan

Naoki Itoh1,2*, Toshihiro Yamamoto3, Hyun-Sil Kang4, Kwang-Sik Choi4, Timothy J. Green5, Noelia Carrasco6, Masahiko Awaji7 and Seinen Chow8

1Laboratory of Aquacultural Biology, Graduate School of Agricultural Science, Tohoku University, Miyagi 981-8555, Japan
2Laboratory of Fish Diseases, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan
3National Research Institute of Aquaculture, Fisheries Research Agency, Kanagawa 238-0316, Japan
4School of Marine Biomedical Science, Jeju National University, Jeju 690-756, Republic of Korea
5School of Biological Sciences, Flinders University, SA 5001, Australia 6IRTA Sant Carles de la Rapita, 43540, Tarrogona, Spain
7National Research Institute of Aquaculture, Fisheries Research Agency, Mie 516-0193, Japan
8National Research Institute of Fisheries Science, Fisheries Research Agency, Kanagawa 236-8648, Japan

(Received September 4, 2014)

ABSTRACT―A paramyxean parasite infection was observed in the epithelial cells of the stomach, intestine, and digestive diverticula of Manila clam Ruditapes philippinarum collected from Odawa Bay, Japan. The mean detection frequency between April 2010 and September 2011 as revealed by histology was 8.9% with a maximum monthly frequency of 28.6%, but remarkable mass mortality was not recognized during the study period. Sporulation of the parasites involves repeated production of intracellular cells, and a primary cell contains 8 secondary cells, in which 4 multicellular spore cells are produced. Several large eosinophilic granules formed in the cytoplasm of secondary cells during the final maturation process. Transmission electron microscopy (TEM) revealed that spore cells consisted of the innermost, intermediate and outermost cells, indicating that this parasite is a member of the genus Marteilia. TEM observations also revealed that the cell membrane of mature spores was lined with an electron-dense monolayer, which has not been reported from other species in the genus Marteilia. Determined SSU rRNA gene sequence of this parasite was apparently different from previously described Marteilia species, and this species was identified as Marteilia granula n. sp., a new species in the genus Marteilia, Order Paramyxida, Phylum Cercozoa. Our results identify for the first time the parasite species of the genus Marteilia infecting the Manila clam in Japan.

Key words: Paramyxida, Marteilia, Manila clam, protozoan parasite, digestive gland


Fins Are an Applicable Organ for PCR-based Diagnosis of Koi Herpesvirus Disease in Clinical Fish

Takafumi Ito1*, Hiroshi Hirakiuchi2 and Kei Yuasa3

1Tamaki Laboratory, Aquatic Animal Health Division, National Research Institute of Aquaculture, Fisheries Research Agency, Mie 519-0423, Japan
2Coastal Fisheries Group, Shimane Prefectural Fisheries Technology Center, Shimane 690-0322, Japan
3Diagnosis and Training Center for Fish Diseases, National Research Institute of Aquaculture, Fisheries Research Agency, Mie 516-0193, Japan

(Received May 15, 2014)

ABSTRACT―We investigated the applicable organ to detect DNA of cyprinid herpesvirus 3 (CyHV-3), the causative agent of koi herpesvirus disease (KHVD). CyHV-3 DNA was detected at the highest positive rate in the caudal fin among several organs including gills, kidney and spleen of the fish sequentially sampled after experimental exposure to the virus. Moreover, CyHV-3 DNA was detected in the caudal and pectoral fins of all dead fish collected in a natural KHVD case. Since fins are an easily accessible organ, they can be an applicable organ for PCR-based diagnosis of KHVD in clinical fish.

Key words: fin, cyprinid herpesvirus 3, KHV, detection, carp


Investigation of the Infection with Diphyllobothrium nihonkaiense Plerocercoids and Metagonimus Metacercariae in Freshwater Salmonids Cultured in Japan

Takeo Watanabe1*, Morinobu Sawada1, Tetsuya Yanagida2** and Kazuo Ogaw3

1Tochigi Prefectural Fisheries Experiment Station, Tochigi 324-0404, Japan
2Asahikawa Medical University, Asahikawa 078-8510, Japan
3Meguro Parasitological Museum, Tokyo 153-0064, Japan

(Received May 28, 2014)

ABSTRACT―We investigated the existence of Diphyllobothrium nihonkaiense plerocercoids and trematode metacercariae of the genus Metagonimus in salmonid fishes (N = 2,187) cultured in freshwater areas of Japan. The infection of D. nihonkaiense plerocercoid larvae was not detected in cultured freshwater salmonids, whilst five out of 60 wild anadromous masu salmon Oncorhynchus masou masou captured during the spawning migration in the Miomote River was infected with D. nihonkaiense. No Metagonimus metacercariae were detected in any salmonids examined. The present study corroborates the previous report indicating that salmonids cultured in freshwater areas of Japan have no chance of infection with D. nihonkaiense.

Key words: salmonids, Diphyllobothrium nihonkaiense, plerocercoid, Metagonimus, Metacercaria


Mass Mortality of Cage-cultured Orange-spotted Grouper Epinephelus coioides Associated with Renal Sphaerosporosis Caused by Sphaerospora epinepheli in South China Sea

Li-Wen Xu1, Jing-Yong Zhang2*, Juan Feng1 and Jian-Guo Wang2

1Key Laboratory of South China Sea Fishery Resources Exploitation & Utilization (Ministry of Agriculture), South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, P. R. China
2Fish Diseases Laboratory, State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, P. R. China

(Received July 7, 2014)

ABSTRACT―In November 2011, the renal sphaerosporosis was found in cage-cultured orange-spotted grouper Epinephelus coioides in a fish farm in Guangdong, China. The infected fish exhibited emaciation, anaemia, anorexia, renomegaly, slight splenomegaly, and occasionally skin ulcer. The cumulative mortality reached at 50% to 80% within 2 weeks, when water temperature ranged from 21°C to 27°C. The renal tubules were almost completely occluded by sporogonic pseudoplasmodia of a myxosporean, and mature spores were found in the lumen. Combined with morphological, histopathological, and molecular analyses, Sphaerospora epinepheli was suggested as the etiological agent. This is the first report of mortality case of cultured E. coioides associated with S. epinepheli.

Key words: renal sphaerosporosis, Sphaerospora epinepheli, Epinephelus coioides, grouper