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NO50 - 1

Detection of Pseudomonas anguilliseptica from Olive Flounder Paralichthys olivaceus Using Real-time PCR with a TaqMan Fluorescent Probe

Bong-Jo Kang1, Subramanian Dharaneedharan2, Yeoung-Hwan Jang1, Seung-Hwan Won1 and Moon-Soo Heo2*

1Jeju Special Self-Governing Province Ocean and Fisheries Research Institute, Jeju 697-914, South Korea
2Department of Aquatic Biomedical Sciences, Jeju National University, Jeju 690-756, South Korea

(Received May 28, 2014)

ABSTRACTPseudomonas anguilliseptica is a dreadful fish pathogen causing severe problem in olive flounder Paralichthys olivaceus farms in Jeju Island, Korea. Diagnosis of this disease required a time-consuming and labor intensive culturing step. Hence more rapid and sensitive detection methods are needed. In this study, a TaqMan probe real-time PCR-based detection assay targeting the 16S rRNA gene of P. anguilliseptica was developed. This assay was specific for P. anguilliseptica and sensitive in detecting as little as 300 pg P. anguilliseptica genomic DNA and 2.4 × 100 plasmid copie. Challenge experiments of healthy flounder with a virulent strain of P. anguilliseptica confirmed the specificity of the designed molecular probe for detection of P. anguilliseptica. This assay represents a significant approach for diagnosing P. anguilliseptica infection in olive flounder.

Key words: TaqMan probe, diagnosis, Pseudomonas anguilliseptica, Paralichthys olivaceus, real-time PCR

luxS/AI-2 Quorum Sensing Is Involved in Antimicrobial Susceptibility in Streptococcus agalactiae

Yan Ping Ma1, Hao Ke1*, Le Hao1, Zhen Xing Liu1, Zhi Ling Liang1, Jiang Yao Ma1, Hu Cheng Yang2 and Yu Gu Li3

1Guangdong Public Laboratory of Veterinary Public Health and Guangdong Laboratory for Animal Diseases, Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, PR China
2Hunan Agricultural University, Changsha 410128, PR China
3South China Agricultural University, Guangzhou 510642, PR China

(Received July 11, 2014)

ABSTRACTluxS-mediated autoinducer-2 (AI-2) directly or indirectly regulates important physiologic function in a variety of bacteria, including bioluminescent effect, antimicrobial resistance, virulence factor control, biofilm structure stability and so on. In this study, the regulation of susceptibility to antimicrobials by luxS/AI-2 was investigated in Streptococcus agalactiae. A luxS- knockout mutant strain named SX1 was constructed by homologous recombination from S. agalactiae CNEP110823 (wild type strain), and a luxS-complementation strain named SX2 was constructed from SX1 through transducing a plasmid with the luxS gene and its promoter. Then the susceptibility to cefradine and norfloxacin of these two strains, SX1 exposed to AI-2 precursor molecule 4,5-dihydroxy-2,3-pentanedione (DPD) and the wild type strain was determined by viable bacterial counting. SX1 decreased susceptibility to cefradine and norfloxacin, SX2 returned the susceptibility to the same level as the wild type strain and DPD restored the susceptibility of SX1. The results indicate that luxS/AI-2 quorum sensing is involved in antimicrobial susceptibility in S. agalactiae, which may provide novel clues for antimicrobial therapy in the infection.

Key words: Streptococcus agalactiae, luxS mutant, AI-2, antimicrobial susceptibility, molecular mechanism

Suppressive Effects of Low Salinity and Low Temperature on In-vivo Propagation of the Protozoan Perkinsus olseni in Manila Clam

Tsukasa Waki and Tomoyoshi Yoshinaga*

Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan

(Received October 2, 2014)

ABSTRACT―Infection levels of Pekinsus olseni in Manila clam Ruditapes phillipinarum much vary among different locations. In order to know the effects of environmental factors on the infection of the parasite, we maintained Manila clams at different salinities (15, 18, 21 and 30 psu) and temperatures (13°C, 18°C and 23°C) after challenge with parasites and examined the infection intensities in the clams at intervals. In clams maintained at different salinities, no association was detected between salinity and infection intensity except that the intensity was significantly lower at 15 psu than at any other salinities on 54 day post challenge only, when the experiment was terminated. A significant difference was observed in the survival rate of clams between challenged and control groups at 30 psu only, suggesting that the negative effect of P. olseni on the survival of clams was attenuated with decreasing salinity. In clams maintained at different temperatures, the infection intensity little changed at 13°C, although it increased remarkably at 18°C and 23°C. The result indicates that in-vivo propagation is suppressed at 13°C and lower. A significant decrease was observed in the survival of clams at 23°C only, indicating that the virulence of P. olseni depends on temperature.

Key words: Perkinsus olseni, Ruditapes phillipinarum, Manila clam, in-vivo propagation, salinity, temperature


Egg Laying and Hatching Rhythms of the Skin Fluke Neobenedenia girellae

Chihaya Hirano, Katsuya Ishimaru and Sho Shirakashi*

Fisheries Laboratory, Kinki University, Wakayama 649-2211, Japan

(Received December 9, 2014)

ABSTRACT―Diurnal rhythms in egg laying and hatching of the skin fluke Neobenedenia girellae were investigated under different light conditions. Egg production was monitored every 2 h for 3 days under two light conditions (darkness and natural light) using Seriola dumerili infected with multiple N. girellae as well as black molly Poecilia sphenops that had been transplanted by a single mature fluke. In any cases, oviposition was continuously observed with no obvious diurnal rhythm. The maximum egg production was approximately 60 eggs/h/worm for both multiple- and single-infection. Such high fecundity emphasizes the importance of the prompt eradication of mature flukes from culture sites to prevent parasite multiplication. The hatching rhythm was monitored under natural light condition and controlled light condition with LED lamp (24L, 12L:12D, 24D). An obvious hatching rhythm with a monomodal peak in the morning was detected under natural light condition. This rhythm was different from that previously reported for Benedenia seriolae whose hatching has bimodal peaks in the early morning and evening. Under controlled light conditions, no such pattern was observed and the overall hatching was considerably less than that under natural light condition.
Key words: skin fluke, Neobenedenia girellae, oviposition, hatching, diurnal rhythm, monogenean