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NO50 - 2

Isolation of Lactococcus garvieae Showing No Agglutination with Anti-KG- Phenotype Rabbit Serum

Daisaku Oinaka1, Naoto Yoshimura1, Yutaka Fukuda2, Azumi Yamashita3, Shintaro Urasaki4, Yoshinobu Wada5 and Terutoyo Yoshida1*

1Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan
2Fisheries Research Division, Oita Prefectural Agriculture, Forestry and Fisheries Research Center, Oita, 879-2602, Japan
3Fisheries Research Center, Ehime research Institute of Agriculture, Forestry and Fisheries, Ehime 798-0104, Japan
4Fisheries Division, Ainan Town Office, Ehime 798-4196, Japan
5Intervet KK Central Research Laboratories, Ibaraki 300-0013, Japan

(Received November 18, 2014)

ABSTRACTLactococcus garvieae-like bacteria, which did not agglutinate with a rabbit antiserum against L. garvieae capsulated cells (KG- phenotype cells), were isolated from diseased yellowtail Seriola quinqueradiata in 2012. The non-agglutinating L. garvieae-like bacteria reacted positively in a species-specific PCR targeting the 16S rRNA gene of L. garvieae. DNA-DNA relatedness values between reference L. garvieae strains and the non-agglutinating strains were higher than 70%. Almost complete sequencing of the 16S rRNA gene of the non-agglutinating strains revealed high similarity with that of L. garvieae ATCC49156. The non-agglutinating strains showed the same morphological and biochemical characteristics as reference L. garvieae strains. These results confirm that the non-agglutinating strains are L. garvieae. However, the present strains differed from the reference strains in terms of the lytic bacteriophage susceptibility. Serological analysis revealed that autoclave-extracted cellular antigens of the non-agglutinating strains did not react to antiserum raised against L. garvieae KG phenotype cells. Although the non-agglutinating strains were isolated from different prefectures, they showed similar patterns in biased sinusoidal field gel electrophoresis (BSFGE).

Key words: non-agglutinating, Lactococcus garvieae, Seriola quinqueradiata, yellowtail

Infection Kinetics of Tenacibaculum maritimum on the Abraded Skin of Japanese Flounder Paralichthys olivaceus

Tanvir Rahman1, Koushirou Suga1, Kinya Kanai1* and Yukitaka Sugihara2

1Graduate School of Fisheries Science and Environmental Studies, Nagasaki University, Nagasaki 852-8521, Japan
2Nagasaki Prefectural Institute of Fisheries, Nagasaki 851-2213, Japan

(Received December 6, 2014)

ABSTRACTTenacibaculum maritimum is the gliding bacterium that causes tenacibaculosis, an ulcerative disease in marine fish. In this study, we conducted a pathogenicity test to assess the effect of skin abrasion on the infectivity of gliding and non-gliding strains of T. maritimum, NUF1128 and NUF1129, respectively, and investigated the infection kinetics by enumeration and immunohistochemical observation of the adhered and proliferated T. maritimum on the abraded skin of Japanese flounder Paralichthys olivaceus. In the pathogenicity test, Japanese flounder whose dorsal skin was abraded with a cotton swab or blade or tip of dorsal fin was clipped with scissors were immersed for 30 min in seawater containing 106 CFU/mL of cultured NUF1128 or NUF1129 cells. As a result 100% mortality was achieved in the fish groups pretreated with blades or scissors followed by challenged with NUF1128. NUF1129 was unable to induce infection regardless of the treatments applied. The infection kinetic studies revealed that NUF1128 adhered more readily than NUF1129 to dermal connective tissues which were exposed by abrasion with blades and subsequently proliferated mainly in the dermal connective tissues and perimysium.

Key words: Tenacibaculum maritimum, tenacibaculosis, infection kinetics, abraded skin, immersion challenge, Paralichthys olivaceus, Japanese flounder

Histopathological and Immunohistochemical Studies of Gonadal Undifferentiated Carcinoma in Common Carp Cyprinus carpio

Shinya Yasumoto*, Daiji Koga, Kazuyuki Tanaka, Masakazu Kondo and Yukinori Takahashi

Department of Applied Aquabiology, National Fisheries University, Yamaguchi 759-6595, Japan

(Received December 14, 2014)

ABSTRACT―We examined in detail the pathological features of common carp Cyprinus carpio (mature male and immature fish) with neoplastic lesions in the abdominal cavity. Diseased fish had abdominal enlargement, exophthalmos and scale elevation, along with a large tumor and accumulated ascitic fluid. The tumor was located where the gonad would have been in a healthy fish. Histopathologically, the tumor was composed of parenchyma, stroma and necrotic tissue, and the tumor cells had infiltrated the abdominal organs. Gonadal debris, including germ cells, was observed in the tumor parenchyma. The tumor and primary tissues were dissimilar, suggesting that the tumors were undifferentiated. Immunohistochemically, the tumor cells were positive for cytokeratin AE1/AE3 but negative for vimentin. They were positive for proliferating cell nuclear antigen (PCNA) and p53. These features indicated that tumor proliferation was uncontrolled, with a fast cell cycle, resulting in the development of large tumors. The tumor can therefore be diagnosed as an undifferentiated carcinoma originated from gonadal tissue.

Key words: Cyprinus carpio, gonad, immunohistochemistry, tumor, undifferentiated carcinoma, carp

Infection Dynamics of Kudoa septempunctata (Myxozoa: Multivalvulida) in Hatchery-produced Olive Flounder Paralichthys olivaceus

Hiroshi Yokoyama1*, Meibi Lu1, Koh-ichiro Mori2, Jun Satoh2, Tohru Mekata2 and Tomoyoshi Yoshinaga1

1Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan
2National Research Institute of Aquaculture, Kamiura Branch, Oita 879-2602, Japan

(Received January 6, 2015)

ABSTRACT―Food poisoning of humans, caused by the ingestion of raw flesh of the olive flounder Paralichthys olivaceus infected with Kudoa septempunctata, has recently become a public health concern in Japan. The present study investigated the infection dynamics of K. septempunctata in two cohorts of olive flounder produced in a hatchery, where K. septempunctata infection was enzootic, by PCR assay and light microscopy. In less than 1-year-old juveniles of the 2011 cohort (hatched in February 2011), K. septempunctata was not detected in either June or July 2011 even by conventional PCR, but light microscopy detected a heavy infection (> 1 × 106 spores/g) in October 2011. In 2-year-old fish of the 2009 cohort (hatched in February 2009), the prevalence of infection varied from 30% to 90% from April to December 2011, although no clear pattern was observed in the fluctuation of prevalence and intensity. Fish-to-fish transmission of K. septempunctata was not possible orally or by cohabitation. To investigate the infection period and early development of K. septempunctata, uninfected fish were exposed monthly for 2 weeks to the seawater at the infected hatchery. The results indicated that the peak period of infection was July, and that K. septempunctata was detectable in the heart by quantitative PCR assay as early as 1 week post-exposure, then in the blood and somatic muscle at 2 weeks post-exposure.

Key words: Kudoa septempunctata, Myxozoa, Paralichthys olivaceus, infection dynamics, food poisoning, olive flounder

Nontuberculous Mycobacteriosis in Farmed Thread-sail Filefish Stephanolepis cirrhifer

Hanako Fukano1, Shinpei Wada1*, Osamu Kurata1, Kaori Mizuno2, Kazue Nakanaga3 and Yoshihiko Hoshino3

1Laboratory of Aquatic Medicine, Nippon Veterinary and Life Science University, Tokyo 180-8602, Japan
2Fisheries Research Center, Ehime Research Institute of Agriculture, Forestry and Fisheries, Ehime 798-0104, Japan
3Leprosy Research Center, National Institute of Infectious Diseases, Tokyo 189-0002, Japan

(Received February 28, 2015)

ABSTRACT―In 2009 and 2010, unusually high mortality events were recorded among cultured populations of thread-sail filefish Stephanolepis cirrhifer in Ehime Prefecture, Japan. Diseased fish exhibited abdominal distention and many white nodules filled with thick, pale-yellow material scattered on the surface of the serosae of internal organs and mesentery. Histopathologically, the disease was characterized by variable sized granulomatous lesions with central necrotic core surrounded by thin irregular arrangement of epithelioid cells and the outermost thin rim of connective tissue. The central part of granuloma showed colliquative necrosis with abundant cellular debris and some clusters of long-rods that were positive with Ziehl-Neelsen stain. From the results of microbiological examinations and simplified identification with DNA-DNA hybridization, two representative isolates collected in 2009 and 2010 were classified into rapidly growing "nontuberculous mycobacteria (NTM)" that closely related to Mycobacterium chelonae. A pathogenicity test using one of the isolates successfully reproduced the granulomatous lesions closely resembled to those in the spontaneous cases, suggesting that the rapidly growing NTM is pathogenic to the thread-sail filefish, and is a causative agent of the spontaneous case. This is the first report of the disease caused by a rapidly growing NTM in maricultured fish in Japan.

Key words: nontuberculous mycobacteriosis, NTM, Stephanolepis cirrhifer, thread-sail filefish, DNA-DNA hybridization

Existence of Subserotypes in Streptococcus parauberis Serotype I

Kinya Kanai*, Chuandeng Tu, Naoki Katayama and Koushirou Suga

Graduate School of Fisheries Science and Environmental Studies, Nagasaki University, Nagasaki 852-8521, Japan

(Received March 26, 2015)

ABSTRACTStreptococcus parauberis is the etiologic agent of streptococcosis in Japanese flounder Paralichthys olivaceus. Two serotypes, termed serotypes I and II, are known among the Japanese S. parauberis isolates. In the course of serodiagnosis, we found several strains that did not agglutinate with rabbit anti-serotype I or II sera. In this study, we investigated the serological and genetic relationships among the stocked S. parauberis strains including the non-agglutinating ones using a newly prepared rabbit antiserum against a non-agglutinating strain (NUF1071) as well as previously prepared anti-serotype I and II sera, and pulsed-field gel electrophoresis (PFGE). An antiserum cross-absorption test and microtiter agglutination test revealed that the serotype I was divided into three subserotypes, tentatively designated Ia, Ib and Ic. The non-agglutinating strains belonged to the subserotype Ic. Of the 104 serotype I strains, 6, 91 and 7 strains belonged to subserotypes Ia, Ib and Ic, respectively. Formalin-killed cells of subserotype Ia and Ic strains were agglutinated with the anti-subserotype Ia serum (so far being used as an anti-serotype I serum) and Ic serum, respectively. Subserotype Ib strains were agglutinated with both sera. In PFGE analysis, the stocked 188 S. parauberis strains were divided into three clusters corresponding to subserotypes Ib/Ic, Ia and serotype II.

Key words: Streptococcus parauberis, serotype, subserotype, PFGE, Paralichthys olivaceus, Japanese flounder

Occurrence of Lernaea cyprinacea (Copepoda) in Wild Ayu Plecoglossus altivelis and Several Other Fishes in the Shonai River, Japan

Yu Yoshimine1, Tadashi Isshiki1*, Shizuo Aino1, Kay Lwin Tun2 and Tomoyoshi Yoshinaga2

1Graduate School of Bioresources, Mie University, Mie 514-8507, Japan
2Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan

(Received December 10, 2014)

ABSTRACTLernaea cyprinacea, known as an anchor worm, was found on wild ayu Plecoglossus altivelis collected in the Shonai River, Japan, from early June to middle September 2013, with the maximum prevalence of 64.3% in late August. The prevalence was positively correlated with water temperature and standard length of the fish. Most (90%) of parasites were found at the base of the fins. Impact of the parasite on ayu populations might be supported by high prevalence in individuals detained downstream. The parasite also infected four other fishes including Rhinogobius giurinus, Pseudogobius esocinus, Mugil cephalus cephalus, and Carassius auratus langsdorfii; the former three fishes were new hosts for L. cyprinacea in Japan.

Key words: Lernaea cyprinacea, anchor worm, Plecoglossus altivelis, ayu, new hosts