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NO51 - 4

Bacterial Cold-water Disease in Salmonid Fish and Ayu

Akira Kumagai

Miyagi Prefecture Fisheries Technology Institute, Miyagi 986-2135, Japan

ABSTRACTFlavobacterium psychrophilum is the causative agent of bacterial cold-water disease (BCWD), inducing severe economic loss in aquaculture worldwide. In Japan, outbreaks of BCWD have often occurred in coho salmon Oncorhynchus kisutch hatcheries and ayu Plecoglossus altivelis farms since the mid-1980s. The disease has been causing serious damages to wild populations of ayu in rivers, and F. psychrophilum has been detected from not only ayu but also other wild fish species. F. psychrophilum in the ovarian fluid can contaminate the surfaces of salmonid eggs and passively enter eggs during water-hardening. The pre-water-hardening disinfection with povidone iodine (50 ppm in isotonic solution, 15 min) of fertilized eggs is a promising method for preventing the transmission of BCWD by eggs. Recently, the disinfection procedure begins to prevail in salmonid hatcheries in Japan.

Viral Nervous Necrosis (VNN): a Short Review

Toshihiro Nakai1* and Koh-ichiro Mori2

1Graduate School of Biosphere Science, Hiroshima University, Hiroshima 739-8528, Japan
2National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency, Mie 516-0193, Japan

ABSTRACT― Viral nervous necrosis (VNN) caused by betanodaviruses (Nodaviridae) was first described in 1990 in Japan and Australia, and has since caused high mortality in a variety of hatchery-reared or cultured marine fishes, worldwide. Tons and tons of paper on VNN and related subjects have been published in almost a quarter century. Current knowledge on the disease and causative virus accumulated through these studies has led to the development of many useful diagnostic and control methods in addition to virological new findings.

Kudoosis of Marine Fish in Japan

Hiroshi Yokoyama

The University of Tokyo, Bunkyo, Yayoi 1-1-1, Tokyo 113-8657, Japan

ABSTRACT― Kudoosis of marine fish is caused by the multivalvulid myxosporean parasites belonging to the genus Kudoa (Myxozoa). Muscular kudoosis generally lowers the commercial value of host fish, while brain and pericardial kudoosis may directly affect the health conditions of infected fish. Recent topics on Kudoa septempunctata causing the food poisoning of humans which consume raw olive flounder have presented a new threat to public health. The bi-phasic life cycles of bivalvulid myxosporeans alternating fish and annelid hosts have been revealed, but the lack of knowledge on the life cycles of multivalvulid myxosporeans is hindering the progress of Kudoa research. In this paper, several major kudoosis including K. amamiensis, K. iwatai, K. thyrsites, K. septempunctata, K. yasunagai and K. shiomitsui in Japanese fishes are reviewed from the viewpoint of the disease history, pathobiology, diagnosis, and the control measures.

First Report of Occurrences of Two Cyprinid Herpesvirus 3 Variants, I++ II and I++ II, in China

Zhenxing Liu1,3, Hao Ke1,3*, Yanping Ma1,3, Le Hao1,3, Jiangyao Ma1,3, Zhiling Liang1,3 and Zhongjian Chen2

1Institute of Animal Heath, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
2Agro-biological Gene Research Center, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
3Guangdong Common Lab of Veterinary Public Health, Guangzhou 510640, China

ABSTRACT― Cyprinid herpesvirus 3 (CyHV-3) is the cause of a lethal disease named as koi herpesvirus disease (KHVD) in carp worldwide. Even though CyHV-3 has been found in China, little is known about the variety of CyHV-3 genotypes distributing in this area. In the present study, to improve the sensitivity of current genotyping approaches, a nested-PCR method was constructed based on the duplex PCR reported previously. The nested-PCR targeting the markers I/II loci was used in genotype identification of CyHV-3 infected fish in Guangdong Province in 2010-2015 and Henan Province in 2013. We detected 3 genotypes including an Asian dominant type (I++ II+ ) and two emerging genotypes in East Asian and South-East Asian regions (I++ II and I++ II). I++ II was the predominant genotype in this study. Two CyHV-3 variants, ZS410 and HZ419 isolates, were successfully isolated in cell culture and exhibited the genotype of I++ II− and I++ II, respectively. This is the first report of both I++ II and I++ II genotypes isolated in China. These data can contribute consideration and exploration of mode of CyHV-3 spread in China.

Key words:yprinid herpesvirus 3, KHV, genotype, variant, epidemiology, China, nested-PCR

Pathogenicity of Striped Jack Nervous Necrosis Virus (SJNNV)
Isolated from Asymptomatic Wild Japanese Jack Mackerel Trachurus japonicus

Toyohiro Nishioka1, Takuma Sugaya2, Yasuhiko Kawato3, Koh-ichiro Mori3 and Toshihiro Nakai4*

1Kamiura Laboratory, National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency, Oita 879-2602, Japan
2Momoshima Laboratory, National Research Institute of Fisheries and Environment of Inland Sea, Japan Fisheries Research and Education Agency, Hiroshima 722-0061, Japan
3National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency, Mie 516-0193, Japan
4Graduate School of Biosphere Science, Hiroshima University, Hiroshima 739-8528, Japan

ABSTRACT― We isolated striped jack nervous necrosis virus (SJNNV), a causative agent (betanodavirus) of viral nervous necrosis (VNN), from asymptomatic wild Japanese jack mackerel Trachurus japonicus, which is one of the most popular feed fish for marine aquaculture in Japan. The five selected SJNNV isolates and a reference SJNNV strain (SJNag93) isolated from larval striped jack Pseudocaranx dentex were examined for their pathogenicity in hatchery-produced larvae of Japanese jack mackerel and striped jack by an immersion challenge method. All of the SJNNV isolates and SJNag93 were able to infect larvae of both species, although the infective titers in the dead larvae of striped jack were significantly higher than those of Japanese jack mackerel. One isolate (05SaiJJM-3) was particularly virulent to striped jack larvae and was comparable to SJNag93. However, phylogenetic analysis on a genotype-specific sequence region (T4) of the coat protein gene (RNA2) showed that the 05SaiJJM-3 isolate was clustered differently from not only the other four isolates of Japanese jack mackerel but also from SJNag93. The present study revealed that SJNNV in subclinically infected wild fish was pathogenic, suggesting that wild fish can be a potential infection source for cultured fish via water- or food-borne transmission.

Key words:betanodavirus, striped jack nervous necrosis virus, SJNNV, pathogenicity, wild fish, Trachurus japonicus, Pseudocaranx dentex

Infection Status of Ayu and Other Wild Fish with Flavobacterium psychrophilum and Edwardsiella ictaluri in the Tama River, Japan

Hisato Takeuchi1, Motoyuki Hiratsuka1,a, Hiroki Oinuma1, Yoshiyuki Umino1,2, Daiki Nakano1, Mayu Iwadare1, Ryuji Tomono1, Kazutomo Hori1, Toshihiro Imai1, Takanori Ishikawa1,3, Aki Namba1,4, Noriyuki Takai1, Takehiro Ryuu5, Hiroshi Maeda5, Toshihiro Nakai2 and Nobuhiro Mano1*

1Department of Marine Science and Resources, College of Bioresource Sciences, Nihon University, Kanagawa 252-0880, Japan
2Graduate School of Biosphere Science, Hiroshima University, Hiroshima 739-8528, Japan
3Tochigi Prefectural Fisheries Experimental Station, Tochigi 324-0404, Japan
4Research Fellow of the Japan Society for the Promotion of Science, Tokyo 102-0083, Japan
5Tokyo Metropolitan Islands Area Research and Development Center of Agriculture, Forestry and Fisheries, Minato, Tokyo 105-0022, Japan

ABSTRACT― The Tama River is an urban river flowing throughout the Tokyo Metropolis. The water quality has been improved and a large number of wild ayu Plecoglossus altivelis migrate upstream in recent years. We conducted an epidemiological survey on two fish pathogens, Flavobacterium psychrophilum and Edwardsiella ictaluri, among ayu and other wild fish species based on PCR and culture methods at four sites (lower-, middle-, upper-reaches and tributary) in the Tama River basin from May 2011 to November 2012. F. psychrophilum was detected in 15 fish species including ayu throughout the survey period, and the occurrence of F. psychrophilum infection (bacterial cold-water disease) among ayu was observed in upper-reaches and tributary areas between mid- and late June of both years. On the other hand, E. ictaluri was detected in 7 fish species mainly in summer and late fall of both years, and E. ictaluri infection of ayu with mass mortality occurred in the tributary area in late August 2012 only. It is speculated that the unusually high water temperature during August 2012 (approximately 6°C higher than that of 2011) served as a predisposing factor in this mortality. Disease occurrence in riverine ayu might be attributable to the subclinical infection status of fish and stress conditions imposed by the environment.

Key words:Flavobacterium psychrophilum, bacterial cold-water disease, Edwardsiella ictaluri, epidemiology, wild fish, Plecoglossus altivelis, ayu

Multiplication Rate of Red Seabream Iridovirus (RSIV) in Rock Bream Oplegnathus fasciatus at Different Fish Rearing Temperatures

So-Young Oh, Wi-Sik Kim, Myung-Joo Oh and Toyohiko Nishizawa

Department of Aqualife Medicine, Chonnam National University, Yeosu 59626, Republic of Korea

ABSTRACT― Red seabream iridovirus (RSIV) was intramuscularly inoculated to rock bream Oplegnathus fasciatus at 11°C, 16°C, 26°C or 28°C to compare the multiplication rates of RSIV in vivo. The rearing temperature at 11°C and 16°C increased at 0.15°C/day, while that at 26°C decreased at 0.11°C/day, along changes of natural water temperatures. After RSIV inoculation at 28°C and 26°C, fish began to die at 11 and 15 days post inoculation, respectively. Their cumulative mortalities were more than 92%. However, no mortality or less than 5% mortality was observed in fish inoculated with RSIV at 11°C or 16°C. The fastest multiplication rate of RSIV was in fish at 28°C, and it was reduced by decreasing fish rearing temperature. Therefore, the virulence of RSIV was affected by fish rearing temperature and the multiplication rate of RSIV. When fish inoculated with different doses of RSIV at 11°C, all of those fish were persistently infected by RSIV for a long period. After water temperature increased to ≥ 20°C, the fish persistently infected by inoculation of RSIV at high intensities developed disease (≥ 78% of mortality), but no mortality occurred in the fish at low intensities of RSIV inoculation. The initial intensity of RSIV infection influenced the mortality of fish with persistently infection of RSIV.

Key words:Megalocytivirus, red seabream iridovirus, multiplication rate, virulence, persistent infection, water temperature, Oplegnathus fasciat

The Epidemiology of the Trichodinid Ciliate Trichodina truttae on Hatchery-reared and Wild Salmonid Fish in Hokkaido

Shinya Mizuno1*, Shigehiko Urawa2, Mahito Miyamoto1, Makoto Hatakeyama1, Hayato Saneyoshi1, Yoshitaka Sasaki1, Nobuhisa Koide1 and Hiroshi Ueda3

1Salmon and Freshwater Fisheries Research Institute, Hokkaido Research Organization, Hokkaido 061-1433, Japan
2Hokkaido National Fisheries Research Institute, Japan Fisheries Research and Education Agency, Hokkaido 062-0922, Japan
3Field Science Center for Northern Biosphere, Graduate School of Environmental Science, Hokkaido University, Hokkaido 060-0809, Japan

ABSTRACT― Trichodinids are ectoparasitic ciliates that infect the body surface of salmonid fish and occasionally cause high mortality in juvenile chum salmon Oncorhynchus keta reared at hatcheries in northern Japan. The present study examined the occurrence of trichodinid ciliates on wild fish and hatchery-reared juvenile chum salmon in Hokkaido to determine the epidemiology of these parasites. Trichodinids were observed on juvenile chum salmon reared at 28 out of the 87 examined hatcheries and on wild fish, including masu salmon O. masou, rainbow trout O. mykiss, adult chum salmon, white-spotted char Salvelinus leucomaenis, and Dolly Varden S. malma in rivers. All isolates of trichodinid ciliates had the same nucleotide sequences of small subunit ribosomal RNA gene and were identified morphologically as T. truttae. A transmission experiment showed that T. truttae was transmitted horizontally from wild masu salmon to hatchery-reared juvenile chum salmon. Our results demonstrate that a dominant species of trichodinid ciliate infecting salmonid fish in Hokkaido is T. truttae and that wild salmonids can be an infection source of hatchery-reared juvenile chum salmon.

Key words:Trichodina truttae, Oncorhynchus keta, Oncorhynchus mykiss, Salvelinus leucomaenis, Salvelinus malma, hatchery, Hokkaido, Epidemiology

Modification of PCR Program for Detection of Infectious Myonecrosis Virus

Mari Inada1, Christina Retna Handayani2, Yasuhiko Kawato1, Tohru Mekata1, Kei Yuasa1 and Satoshi Miwa1*

1National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency, Mie 516-0193, Japan
2Main Center for Brackishwater Aquacµlture, Jepara 59401, Central Java, Indonesia

ABSTRACT― Infectious myonecrosis (IMN) causes high mortality in the white leg shrimp Litopenaeus vannamei and is listed by the OIE. In the current OIE manual (2016), a commercial kit is recommended to conduct one-step RT-PCR for the detection of IMNV. However, the reagent kit is no longer sold in Japan. We therefore tested different programs for RT-PCR using SuperScript® III One-Step RT-PCR System, which is different from the one that is recommended in the OIE manual for the diagnosis of the disease. The sensitivity of virus detection was clearly higher when the program of 3-step PCR after reverse-transcription at 55°C was adopted than the detection using the program described in the OIE manual, which is 2-step (shuttle) PCR after reverse-transcription at 60°C. The result suggests that the 3-step PCR program with SuperScript® III is useful for the diagnosis of IMN.

Key words: Infectious myonecrosis virus, IMNV, one-step RT-PCR, Litopenaeus vannamei