Cell-Surface Properties of Lactococcus Garvieae Strains and Their Immunogenicity in the Yellowtail Seriola Quinqueradiata
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DISEASES OF AQUATIC ORGANISMS Vol. 51: 169–177, 2002 Published October 4 Dis Aquat Org Cell-surface properties of Lactococcus garvieae strains and their immunogenicity in the yellowtail Seriola quinqueradiata Tsuyoshi Ooyama1, Yusuke Hirokawa2, Takayuki Minami1, Hiroshi Yasuda1, Toshihiro Nakai3, Makoto Endo2, Lila Ruangpan4, Terutoyo Yoshida2,* 1Miyazaki Prefectural Fisheries Experimental Station, Miyazaki Prefecture, Aoshima, Miyazaki 6-16-3, Japan 2Department of Fisheries, Faculty of Agriculture, Miyazaki University, Miyazaki 889-2192, Japan 3Faculty of Applied Biological Science, Hiroshima University, Higashihiroshima 739-8528, Japan 4Samutsakhon Coastal Aquaculture Development Centre Kkkham, Muang, Samutsakhon 74 000, Thailand ABSTRACT: 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 publisher INTRODUCTION agglutinating (KG– phenotype) and agglutinating (KG+ phenotype) phenotypes using anti-KG+ phenotype Lactococcus garvieae is a serious bacterial pathogen serum (Kitao 1982, Yoshida et al. 1996, 1997, Ooyama of the yellowtail Seriola quinqueradiata and amberjack et al. 1999). The KG– phenotype was agglutinated by S. dumullei in Japan (Kusuda et al. 1991, Kitao 1993). anti KG– phenotype cell serum (but not by antisera It has also been isolated from the rainbow trout to the KG+ phenotype, KG7409). However, KG+ pheno- Oncorhynchus mykiss in Italy (Eldar et al. 1996, 1999) type strains were agglutinated with antisera to both and Australia (Carson et al. 1993, Schmidtke & Carson KG+ and KG– phenotypes (Kitao 1982, 1993). Further- 1999), and from the prawn Macrobranchium rosen- more, in KG– phenotype cells incubated with anti KG– bergii in Taiwan (Chen et al. 2001). L. garvieae isolated phenotype serum and stained with ruthenium red, cell from S. quinqueradiata has been divided into non- capsules adjacent to the cell wall were visible by elec- tron microscopy. This capsulated phenotype strain is more virulent to the yellowtail than non-capsulated *Corresponding author. E-mail: [email protected] strains (Alim et al. 1996). Therefore, these capsules are © Inter-Research 2002 · www.int-res.com 170 Dis Aquat Org 51: 169–177, 2002 thought to play roles in the pathogenicity of L. garvieae MATERIALS AND METHODS infection, possibly by increasing resistance to fish phagocytosis (Yoshida et al. 1996, 1997). The bacterial strains are listed in Table 1. All were Control of Lactococcus garvieae infection in yellowtail cultured in Todd-Hewitt broth (THB; Difco) or on culture has depended on chemotherapy with macrolides. agar (THA). Immune sera against the NG8206 KG+ The identification of multiple drug-resistant strains has phenotype (non-capsulated cells) and KG9408 KG– indicated future problems in controlling the pathogen phenotype (capsulated cells) were raised in rabbits, as (Aoki et al. 1990). Recently, oral and injectable vaccines described by Yoshida et al. (1996). The KG+ pheno- against L. garvieae infection in Seriola quinqueradiata types (NG8206, NSS9310 and MS93003) stemmed have been developed and commercialized in Japan. from a subculture of the KG– parent on THA supple- Experimental vaccination against L. garvieae has been mented with 2,3,5-triphenyltetrazolium chloride (TTC) described (Iida et al. 1982, Sato et al. 1996) and has as previously described by Ooyama et al. (1999). been reported to provide immunity and increased The yellowtail fish Seriola quinqueradiata used in opsonic activity in the fish. However, no detailed in- the experiment were bred at Miyazaki Experimental formation on the antigenicity of L. garvieae phenotypes Fisheries Station, Aoshima, Miyazaki, Japan, and kept nor the duration of the immunity was given. The pro- in concrete tanks with sand-filtered sea water. The fish tective mechanisms of the L. garvieae vaccine remains were fed commercial dried pellets once a day. Before unknown. the experiment, the fish (n = 10) were subjected to bac- In a previous study, Ooyama et al. (1999) reported that terial examination to determine the presence of Lacto- formalin-killed Lactococcus garvieae KG– phenotype coccus garvieae. (capsulated phenotypes) cells and KG+ phenotype (non- The bacteria were cultured in THB at 25°C without capsulated) cells induced strong immunity in Seriola shaking for 24 h. Cells were harvested by centrifugation quinqueradiata against artificial infection and long- and suspended in sterile saline solution (0.85% NaCl). lasting agglutinating titres against non-capsulated cells Serial dilutions were prepared in the saline solution, and (avirulent KG+ phenotype). Furthermore, appendages viable counts were determined by plating on THA. Viru- (fimbriae-like structures) were seen extending from the lence was tested with 10 fish (approximately 20 g) per cell surface of L. garvieae KG– phenotype, with some dilution inoculated intraperitoneally with 0.1 ml of each destruction of the cell capsule, after opsonization with bacterial suspension: 3.2 × 103, 3.2 × 104 and 3.2 × 105 yellowtail immune serum. Both formalin-killed cells of CFU ml–1 for KG9408 (KG– phenotype cells), 5.2 × 105, the KG– and KG+ phenotypes were effective against in- 5.2 × 105 and 6.2 × 105 CFU ml–1 for NSS9310, MS93003 fection with KG– virulent phenotype cells, suggesting and NG8206 (KG+ phenotype cells), respectively. The that the antigen providing immunity against fish were maintained at 24 to 26°C for 14 d in separate L. garvieae infection is located on the cell surface or tanks. Non-treated fish were injected with saline as con- projects into the cell capsules from the surface, and trols. Virulence was expressed as the 50% lethal dose, is not present in the cell capsules themselves. LD50 (Reed & Muench 1938). Recently, some strains of Lactococcus garvieae KG+ Transmission electron microscopy (TEM) was per- phenotype were found that induced a weak immune formed as described by Yoshida et al. (1997). Briefly, response in fish against capsulated virulent KG– phe- Lactococcus garvieae KG– and KG+ (KG9408, MS93003 notype infection. The aim of the present study was to and NSS9310) were grown overnight in 10 ml of THB, re-examine the cell-surface properties of various diluted 1:100 in fresh THB, and incubated for an L. garvieae strains and compare the immune response additional 5 h at 25°C. Bacteria were washed with of Seriola quinqueradiata to these strains. phosphate-buffered saline (PBS) and fixed with 2.5% glutaraldehyde. The cells were washed 3 times with PBS and resus- Table 1. Lactococcus garvieae strains used in this study pended in 10 ml of anti-KG– rabbit serum (agglutinating titre against Strain Year Sources Pheno- Source KG9408, KG– phenotype cells; 1:1280) (prefectures) types diluted 200 times with PBS and incu- bated for an additional hour before NG8206 1982 Nagasaki KG+ Yoshida et al. (1996, 1997), staining with 0.15% ruthenium red in Ooyama et al. (1999), Okada et al. (2000) 0.1 M cacodylate buffer, pH 7.4 for 2 h. MS93003 1993 Miyazaki KG+ Ooyama et al. (1999) Bacteria were washed 3 times with NSS9310 1993 Nagasaki KG+ This study PBS, embedded in 3% agarose, fixed KG9408 1994 Kagoshima KG– This study with 2% osmium tetroxide, washed 5 times with cacodylate buffer, and Ooyama et al.: Immunogenicity of Lactococcus garvieae in fish 171 dehydrated with ethanol. Cells were embedded in type cells at a density of 2.5 × 104 cells fish–1, and were Quetol 652 (Nishin EM, Tokyo, Japan). Thin sections monitored for 14 d. At the end of this period, all fish were cut (60 nm), post-stained with uranyl acetate were subjected to bacteriological examination for the and lead acetate, and observed by transmission presence of Lactococcus garvieae in the brain and electron microscopy (Hitachi-H4800Mu, Japan) at an kidney; 30 h after infection, 3 fish were also sampled accelerating voltage of 100 kV. For investigation of for bacteriological counts in the blood, spleen and fimbriae-like structures on the cell surface, bacterial brain using plate counts. These organs and blood were strains were cultured in 10 ml of normal filter-sterilised aseptically