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NO45 - 4

Influence of Perkinsus Infection on the Physiology and Behavior of Adult Manila Clam Ruditapes philippinarum

<>Tomoyoshi Yoshinaga*, Shinji Watanabe, Tsukasa Waki, Shigeru Aoki and Kazuo Ogawa<>Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan
(Received September 14, 2009)

ABSTRACT―Annual catch landings of the Manila clam Ruditapes philippinarum have decreased in Japan since the early 1980s because of the decreased size of many Manila clam populations. It is hypothesized that Perkinsus olseni infection contributed to these decreases. However, the effect of Perkinsus infection on the survival and physiology of Manila clam is poorly understood. We examined the influence of Perkinsus infection on clearance rate, tolerance against high water temperature, and burrowing activity in a captive adult clam population. Infection intensity did not affect clearance rates, burrowing activity, or tolerance against high water temperature. In addition, we monitored infection intensity in wild, adult clams from a tidal flat lacated in western Seto Inland Sea over a one-year period. Infection levels were relatively stable in the wild population between June 2004 and May 2005, but we noted a significant decrease in the infection intensity at the end of the spawning period in November 2004. However, in the present study, clear evidence showing the negative impact of Perkinsus on clam physiology and survival was not obtained.

Key words: Perkinsus olseni, Perkinsus honshuensis, Ruditapes philippinarum, Manila clam, survival, tolerance, burrowing activity, clearance rate

Viable but Non-culturable State of Bacterial Cold-water Disease Pathogen Flavobacterium psychrophilum at Various Temperatures

<>Kazuhiro Sugahara1,2*, Erina Fujiwara-Nagata3, Ayami Fukuda2 and Mitsuru Eguchi3<>1Shiga Prefectural Fisheries Experimental Station, Shiga 522-0057, Japan
2Graduate School of Agriculture, Kinki University, Nara 631-8505, Japan
3Department of Fisheries, Kinki University, Nara 631-8505, Japan
(Received June 12, 2010)

ABSTRACT―Bacterial cold-water disease (BCWD) caused by Flavobacterium psychrophilum leads to heavy mortality of ayu Plecoglossus altivelis in Japan. Previously, a 28ーC warmed water treatment was shown to be an effective treatment for BCWD, since F. psychrophilum can not grow and maintain colonies at this temperature. However, it was unclear whether the bacteria might resuscitate after the treatment was over. Therefore, we investigated colony formation, membrane potential and pathogenicity of F. psychrophilum at various temperatures (15ーC, 23ーC, 28ーC and 33ーC) in sterilized underground water. Within 2 days at 28ーC, F. psychrophilum completely lost their colony-forming abilities but still maintained their membrane potentials. It seemed that these cells entered into viable but non-culturable (VBNC) state. However, experimental ayu infection revealed that VBNC F. psychrophilum cells were unable to cause BCWD, suggesting that the cells were progressing towards death at 28ーC.

Key words: viable but non-culturable, Flavobacterium psychrophilum, bacterial cold-water disease, Plecoglossus altivelis, VBNC, BCWD

Prevention of Flavobacterium psychrophilum Vertical Transmission by Iodophor Treatment of Unfertilized Eggs in Salmonids

<>Akira Kumagai1* and Akatsuki Nawata2<>1Miyagi Prefecture Fisheries Technology Institute, Miyagi 986-2135, Japan
2Miyagi Prefecture Fisheries Technology Institute Freshwater Fisheries
Experimental Station, Miyagi 981-3625, Japan
(Received July 5, 2010)

ABSTRACT―We evaluated the efficacy of povidone-iodine treatments in preventing vertical transmission of Flavobacterium psychrophilum in the eggs of masu salmon Oncorhynchus masou, Japanese char Salvelinus pluvius, coho salmon O. kisutch and rainbow trout O. mykiss. When newly spawned eggs were immersion-challenged with F. psychrophilum (> 109 CFU/mL) and subsequently disinfected with povidone-iodine (50 ppm in PBS, 15 min) prior to fertilization, the pathogen was not detected in the contents of the eggs. Without this pre-fertilization disinfection with povidone-iodine, however, procedures such as water-hardening of immersion-challenged eggs in povidone-iodine (50-100 ppm in water) and rinsing of the F. psychrophilum-contaminated eggs with PBS were insufficient to control the intra-ovum infection. In a separate experiment using coho salmon eggs challenged with F. psychrophilum, pre-fertilization disinfection was effective to prevent bacterial cold-water disease in the produced fry. Surface disinfection of unfertilized eggs immediately after spawning is thus essential to prevent vertical transmission of F. psychrophilum.

Key words: vertical transmission, Flavobacterium psychrophilum, bacterial cold-water disease,
povidone-iodine, salmonid egg, BCWD

Optimization of WSSV rVP Expression in E. coli Cells and Minimum Dose of rVPs for Oral Vaccination in Kuruma Shrimp

<>Jun Satoh1, Hyoung Jun Kim2, Takanori Matsui3 and Toyohiko Nishizawa4*<>1Aquatic Animal Health Division, National Research Institute of Aquaculture, Fisheries Research Agency,
Oita 879-2602, Japan
2Incheon Branch Office, National Fisheries Production Inspection Service, Incheon, 400-800, Korea
3Fisheries Research Division, Oita Prefectural Agriculture, Forestry and Fisheries Research Center,
Oita 879-2602, Japan
4Department of Aqualife Medicine, Chonnam National University, Yeosu, 550-749, Korea
(Received July 30, 2010)
ABSTRACT―The optimum conditions for the expression of WSSV (= PRDV) rVP26 and rVP28 in E. coli cells were investigated, especially the IPTG concentration (0.001-1.0 mM) and inducement temperatures (25ーC and 37ーC). For both rVP26 and rVP28, the strongest intensity of expression was observed in medium containing 0.01 mM IPTG at 37ーC. The growth of the transformed bacteria expressing rVP26 or rVP28 in media containing 0.01 mM IPTG was the almost same as that in medium without IPTG, but clearly delayed than that in the medium containing 1.0 mM IPTG. The productivity was increased 9-fold in rVP26 and 6-fold in rVP28 when the IPTG concentration was reduced from 1.0 mM to 0.01 mM. Next, we investigated the minimum doses of rVPs required to induce phylaxis of kuruma shrimp Penaeus japonicus against WSD (= PAV) by an experimental challenge. The results show that at least 10 μg/g shrimp/day of rVPs should be orally administered to kuruma shrimp for 15 days in order to induce effective phylaxis.

Key words: WSSV, recombinant protein, VP26, VP28, vaccine, expression, PRDV, Penaeus japonicus

Development of Heterobothrium ecuadori (Monogenea: Diclidophoridae) in Bullseye Puffer Fish Sphoeroides annulatus under Experimental Conditions

<>Mayra Grano-Maldonado1,2*, Ana Roque3 and Emma J. Fajer-Avila2<>11National Autonomous University of Mexico, Institute of Marine and Limnology Research, Mazatlan Unit,
Av.Joel Montes Camarena S/N A.P. 811, CP 82040 Mazatlan, Sinaloa, Mexico
2Research Center for Food and Development, Mazatlan Unit for Aquaculture, A.P. 711,
CP 82010 Mazatlan, Sinaloa, Mexico
3Institute of Research and Technology in Food and Agriculture, C/ al Poblenou SN Km 5, 5 43540
Sant Carles de la Rapita, Spain
(Recieved December 6, 2009)
ABSTRACT―Heterobothrium ecuadori is a gill monogenean infecting the gills of the bullseye puffer fish Sphoeroides annulatus and feeding fish blood. This experimental study was performed to describe the juvenile stages of H. ecuadori and to determine the time required to reach the adult stage at 20-23ーC. Uninfected bullseye puffer fish were exposed to oncomiracidia and sampled at intervals to obtain worms of different developmental stages. Between 5 and 10 days after the exposure, the first and second pairs of clamps were observed in worms along with the emergence of the third clamp. Four pairs of clamps were observed on day 15, and primordial male copulatory organ, testes and ovary were found on day 20. Adult parasites started to appear between 25 and 30 days post-infection. The time required for egg laying was estimated to be 33 days, based on the first collection of eggs in the rearing water.

Key words: Monogenea, Heterobothrium ecuadori, Diclidophoridae, Sphoeroides annulatus, bullseye puffer fish

Study of Adherence, Invasion and Survival of Hafnia alvei in RTG-2

<>Daniel Padilla1*, Felix Acosta1, Judit Vega1, Lita Sorroza1, Lorena Roman1, Jimena Bravo1, Fernando Real1 and Jose Vivas2<>1University Institute of Animal Health (IUSA), University of Las Palmas de Gran Canaria, 35416, Arucas, Las Palmas, Spain
2Santa Cruz de Liencres Hospital, Marques de Valdecilla-IFIMAV Foundation, Department of Microbiology, 39120, Liencres, Cantabria, Spain
(Received July 1, 2010)

ABSTRACT―Hafnia alvei is a Gram-negative bacillus that belongs to the family Enterobacteriaceae and has been isolated from different fish species. H. alvei causes a wide variety of diseases, including hemorrhagic septicemia, kidney lesions and mortality in different fish species, as rainbow trout Oncorhynchus mykiss, cherry salmon O. masou, and brown trout Salmo trutta. The aim of this study is to investigate the interaction of different H. alvei strains with RTG-2 by gentamicin protection and immunofluorescence assays. Our results demonstrated a considerable potential of H. alvei to attach to RTG-2 cells, and that the internalized strains remained viable in the cells for 48 h.

Key words: Hafnia alvei, adherence, invasion, RTG-2