A virulent bacteriophage of Lactococcus garvieae (formerly Enterococcus seriolicida) isolated from yellowtail Seriola quinqueradiata

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The cell-surface properties of strains of Lactococcus garvieae were examined. Two capsular types were found, one with a highly developed capsule (KG9408) and one with a micro-capsule (MS93003) carrying fimbriae-like components projecting from the cell surface. One strain (NSS9310) had neither cell capsular nor fimbriae-like structures on its cell surface. The strains with the highly developed capsule were more virulent to fish than either the micro-capsular or non-capsular strains. The KG9408, MS93003 and NSS9310 strains could be clearly differentiated by their susceptibility to bacteriophages. Protection against L. garvieae infection was induced in the yellowtail Seriola quinqueradiata by immunization with formalin-killed L. garvieae KG9408 and MS93003 cells. Although protection was also induced by immunization with NSS9310, the level of protection was significantly lower than that with KG9408 and MS93003 vaccines. Passive immunization with yellowtail immune sera raised against KG9408 and MS93003 conferred strong protection on yellowtail with rapid bacterial clearance after challenge with L. garvieae. Immunoblotting analysis of protein antigens extracted from L. garvieae strains using rabbit anti-KG9408 and anti-MS93003 sera and yellowtail anti-KG9408 and anti-MS93003 sera indicated that some bands in KG9408 and MS93003 strains were not detectable in NSS9310.KEY WORDS: Immunogenicity ⋅ Lactococcus garvieae ⋅ Seriola quinqueradiata ⋅ Cell capsule ⋅ Fimbriae Resale or republication not permitted without written consent of the publisherDis Aquat Org 51: [169][170][171][172][173][174][175][176][177] 2002 thought to play roles in the pathogenicity of L. garvieae infection, possibly by increasing resistance to fish phagocytosis (Yoshida et al. 1996(Yoshida et al. , 1997.Control of Lactococcus garvieae infection in yellowtail culture has depended on chemotherapy with macrolides. The identification of multiple drug-resistant strains has indicated future problems in controlling the pathogen (Aoki et al. 1990). Recently, oral and injectable vaccines against L. garvieae infection in Seriola quinqueradiata have been developed and commercialized in Japan. Experimental vaccination against L. garvieae has been described (Iida et al. 1982, Sato et al. 1996 and has been reported to provide immunity and increased opsonic activity in the fish. However, no detailed information on the antigenicity of L. garvieae phenotypes nor the duration of the immunity was given. The protective mechanisms of the L. garvieae vaccine remains unknown.In a previous study, Ooyama et al. (1999) reported that formalin-killed Lactococcus garvieae KG-phenotype (capsulated phenotypes) cells and KG+ phenotype (noncapsulated) cells induced strong immunity in Seriola quinqueradiata against artificial infection and longlasting agglutinating titres against non-capsulated cells (avirulent KG+ phenotype). Furthermore, appendages (fimbriae-like structures) were seen extending from the cell surface of L. garvieae KG-phenotype, with some destruction of...

Section: Discussionmentioning

confidence: 64%

mentioning

confidence: 64%

Cell-surface properties of Lactococcus garvieae strains and their immunogenicity in the yellowtail Seriola quinqueradiata

Ooyama¹,

Hirokawa²,

Minami³

et al. 2002

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“…L. garvieae is also a zoonotic pathogen, occurring rarely and with a low virulence in human infections (Wang et al 2007). This disease infects farmed fish during the summer when the water temperature exceeds 20°C (Park et al 1997). L. garvieae induces a fatal hemorrhagic septicemia of fish.…”

Section: Introductionmentioning

confidence: 99%

Development of a sensitive and specific LAMP PCR assay for detection of fish pathogen Lactococcus garvieae

Tsai¹,

Wang²,

Yoshida³

et al. 2013

Based on use of a loop-mediated isothermal amplification (LAMP) identification protocol, this study attempted to detect Lactococcus garvieae in fish by using primer sets designed from an L. garvieae alpha/beta fold family hydrolase gene. Reaction time and temperatures were optimized for 60 min at 60°C with the resulting amplicons visualized by adding SYBR Green I to the reaction tube. The assay specificity was assessed using 45 different bacterial strains. Positive results were observed in all 30 L. garvieae isolates from various aquatic animals. No false-positive results were observed in 15 non-L. garvieae strains. The detection limit of the LAMP assay was 10-fold more sensitive than the conventional polymerase chain reaction (PCR) targeting 16S rDNA when using purified L. garvieae DNA. The detection limit of the LAMP assay was approximately 300 colony-forming units (CFU) using crude bacterial lysates, 100-fold more sensitive than PCR. Furthermore, L. garvieae in spleen, kidney and brain of experimentally challenged tilapia and grey mullet were detected using this optimized LAMP assay. Results of this study demonstrate the effectiveness of LAMP in providing a rapid yet simple test for detecting L. garvieae in fish.KEY WORDS: Lactococcosis · Alpha/beta fold family · Hydrolase gene · Loop-mediated isothermal amplification · Polymerase chain reaction · SYBR Green I Resale or republication not permitted without written consent of the publisherDis Aquat Org 102: [225][226][227][228][229][230][231][232][233][234][235] 2013 swimming. However, similar clinical signs are also found in Streptococcus sp. infections (Eldar & Ghittino 1999).Molecular detection methods of Lactococcus garvieae have been developed, including polymerase chain reaction (PCR) (Zlotkin et al. 1998, Aoki et al. 2000, Pu et al. 2002 and 16S rRNA and sodA sequence analysis (Fihman et al. 2006). However, although considered rapid and specific methods for detection, these require expensive equipment and other costly materials (Gunimaladevi et al. 2005).A method of amplifying nucleic acid sequences with high specificity, sensitivity and rapidity under isothermal conditions (60 to 65°C), loop-mediated isothermal amplification (LAMP) has been developed more recently (Notomi et al. 2000) and applied to fish pathogen detection (Caipang et al. 2004, Savan et al. 2004, Yeh et al. 2005, Itano et al. 2006. The assay uses a set of 4 or 6 primers that create a targetspecific stem loop DNA structure during initial amplification steps; subsequent LAMP auto-cycling is performed by the Bst DNA polymerase large fragment (Notomi et al. 2000). Since it does not require complicated equipment, the LAMP method provides a cost-effective, simple, and rapid (producing a result within 60 min) DNA amplification method.Recently, a fosmid library of Lactococcus garvieae from grey mullet has been constructed and many new genes of L. garvieae were cloned (M. A. Tsai et al. unpubl. data). The clone flg08-6 contained an alpha/beta fold family hydrolase gene that is also...

“…Three phages having different infectivities, designated as PLgY-16, PLgY-30 and PLgW-l, were used here. The were obtained from L. garvieae cultures isolated from diseased yellowtail, and the PLgW-1 was isolated from natural seawater in the previous studies (Park et al 1997(Park et al , 1998. These phages were propagated in L. garvieae strain No.…”

Section: Methodsmentioning

confidence: 99%

“…Previously, we found a virulent bacteriophage, designated as PLgY, specific to Lactococcus garvieae isolated from diseased fish, and the phage was identified as a member of the family Siphoviridae (Park et al 1997). L. garvieae strains from diseased fish were divided into 14 or more phage types by using several phage strains isolated from fish and culture environments, with a major phage type A (Park et al 1998).…”

Section: Introductionmentioning

confidence: 99%

Protective effects of bacteriophage on experimental Lactococcus garvieae infection in yellowtail

Nakai¹,

Sugimoto²,

Park³

et al. 1999

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174

ABSTRACT. The present study describes the in vitro and in vivo survival of Lactococcus garvieae bacteriophages and the potential of the phage for controlling experimental L. garvieae infection in yellowtail. Anti-L. garvieae phages persisted well in various physicochemical (water temperature, salinity, pH) and biological (feed, serum and alimentary tract extracts of yellowtail) conditions, except for low acidity. In the in vivo, the phage PLgY-16 was detected in the spleens of yellowtail until 24 h after intraperitoneal (i.p.) injection, or the phage was recovered from the intestine of yellowtail 3 h after the oral administration of phage-impregnated feed but undetectable 10 h later. Simultaneous administration of Live L. garvieae and phage enhanced recovery of the phage from the spleen or intestine. The survival rate was much higher in yellowtail that received 1.p. injection of the phage after i.p. challenge with L. garvieae, compared with that of control fish without phage injection. When fish were i.p.-injected with phage at different hours after L. garvieae challenge, higher protective effects were demonstrated in fish that received phage treatment at the earlier time. Protection was also obtained in yellowtail receiving phage-impregnated feed, in which fish were challenged by an anal intubation with L. garvieae. Anal-intubated L. garvieae were detected constantly In the spleens of the control fish, while they were detected sporadically and disappeared from the phage-treated fish 48 h later. On the other hand, orally administered phage was detected at high plaque-forming units from the intestines and spleens of the phage-treated fish until 48 h later. These results indicate that intraperitoneally or orally administered anti-L. garvieae phage prevented fish from experimental L, garvieae infection, suggesting potential use of the phage for controlling the disease.