2025 年 60 巻 2 号 p. 88-91
This study evaluated the protective efficacy of two vaccines against lactococcosis in chub mackerel Scomber japonicus: a commercial vaccine for Lactococcus garvieae serotype I approved for use in yellowtail Seriola quinqueradiata, and an inactivated vaccine prepared from L. garvieae serotype I strain L17-2, isolated from S. japonicus. Following experimental infection via intraperitoneal inoculation with strain L17-2, the relative percentage of survival for both vaccines was 55.6%. The serum agglutination antibody titers against L. garvieae were significantly higher in vaccinated fish than in the negative control, with no significant differences between the two vaccines. These results indicate that the commercial vaccine against L. garvieae serotype I is effective in chub mackerel.
Lactococcosis, caused by Lactococcus spp., has resulted in serious economic losses worldwide. Lactococcus spp. that cause damage to Japanese aquaculture have been classified as serotypes I, II, and III for convenience (Mahmoud et al., 2023; Minami et al., 2023). In addition, serotypes I and III have been identified as L. garvieae and type II as L. formosensis (Fukuda et al., 2015; Minami et al., 2023). Among these serotypes, L. garvieae serotype I infects a wide host range and causes serious damage (Yoshida, 2016; Meyburgh et al., 2017). Inoculation with the inactivated vaccine, formalin-killed cells (FKC) of L. garvieae, is an effective preventive method against L. garvieae infection in Seriola species, i.e., Japanese yellowtail Seriola quinqueradiata and greater amberjack S. dumerili, threadsail filefish Stephanolepis cirrhifer, and rainbow trout Oncorhynchus mykiss (Ceschia et al., 1998; Ooyama et al., 1999; Minami et al., 2013). In fact, inactivated vaccines against L. garvieae serotype I for Seriola species and threadsail filefish have been approved in Japan. As a result, the damage caused by the bacteria has been greatly reduced among these fish species in Japanese aquaculture. However, the damage caused by L. garvieae continues to have a significant impact on Japanese aquaculture in fish species for which no vaccine has been approved.
In June 2017, an outbreak of L. garvieae serotype I occurred at a chub mackerel Scomber japonicus farm in Uwajima City, Ehime Prefecture, with approximately 10% of the entire farm dying within 3 days of disease onset (unpublished). Currently, the outbreak in chub mackerel has also occurred in other regions of Japan outside Uwajima City (Kanzaki, 2023; Yamada et al., 2023). However, there are still no useful control measures have been developed for L. garvieae infection in chub mackerel, and the development of a vaccine against this disease and evaluation of its efficacy in chub mackerel are urgently needed.
In this study, we evaluated the pathogenicity of L. garvieae isolated from chub mackerel. In addition, the protective efficacy of a commercially available L. garvieae vaccine approved for Japanese yellowtail was examined using a challenge test and antibody titers in chub mackerel.
Chub mackerel were purchased from MarineTech Co., and kept for use in the experiments. The fish were maintained in 1 kL tanks with sand-filtered flow-through seawater. The fish were fed twice daily with commercial pellets.
All fish used for sampling were handled according to the policy designated by the Institutional Animal Care and Use Committee of the Fisheries Technology Institute. All fish experiments were approved by the committee (no. 23002 and 24002).
BacteriaLactococcus garvieae L17-2, isolated in June 2017 from diseased chub mackerel (body weight [BW]: 484 g) cultured in Uwajima city, Ehime, Japan, was used in this study. The strain was identified as L. garvieae serotype I by the Ehime Fisheries Research Center, Ehime Research Institute of Agriculture, Forestry and Fisheries, using serum agglutination, distributed by the Japan Fisheries Resource Conservation Association. The strain was stored in Todd-Hewitt (TH) broth with 15% glycerol at −80°C until use in the experiments.
Pathogenicity testLactococcus garvieae L17-2 was inoculated on TH agar (Becton, Dickinson and Company) from the stock and incubated for 24 h at 25°C. The grown colonies were collected sterilely and suspended in sterile phosphate-buffered saline (-) (PBS) (Takara) at concentrations of 100 ng/mL (106 CFU/mL), 1 ng/mL (104 CFU/mL), and 100 pg/mL (102 CFU/mL). Fifteen chub mackerel (mean BW: 39.01 ± 9.97 g) per group were infected with 100 μL of each bacterial suspension by injection intraperitoneally at concentrations of 105, 103, and 101 CFU/fish. The control group was inoculated intraperitoneally with 100 μL of PBS. After infection, the fish were kept in 0.5 kL tanks and fed twice daily with running seawater at 24–26°C. Dead fish were observed for external symptoms and necropsy, and bacteria were re-isolated from the brains and kidneys. Bacteria were also re-isolated from the brains and kidneys of all surviving fish 21 days after infection, when the pathogenicity test was completed. Isolated bacteria were confirmed to be L. garvieae serotype I using multiplex PCR, which identified and distinguished three serotypes of L. garvieae and L. formosensis (Araki et al., 2024).
Fisher’s exact test was used for the statistical analysis of pathogenicity, and the median lethal dose (LD50) was determined using the logit model.
Vaccine testA commercial vaccine and FKC of L. garvieae L17-2 were used as test vaccines. The commercial vaccine, Piscivac® 5 oil (Kyoritsu Seiyaku Co.), an oil adjuvanted 5-mixture vaccine against lactococcosis (serotype I and II), iridovirus disease, pseudotuberculosis and J-O-3 vibriosis that is approved for Japanese yellowtail, was used. FKC were prepared as follows. Lactococcus garvieae L17-2 was spread from the stock onto TH agar and incubated for 24 h at 25°C. The growing colony was inoculated into TH broth and cultured for 24 h at 25°C in a shaker, BIO-SHAKER BR-40LF (TAITEC). Before inactivation of the bacteria, the titer of the L. garvieae was measured using the colony count method and was 1.8 × 109 CFU/mL. Then, Formalin (Wako) was added to a final concentration of 0.5% and incubated for 24 h at 25°C to inactivate the bacterial cells. FKCs were collected by centrifugation at 5,000 × g for 5 min at 4°C and washed three times with sterilized PBS. The washed cells were resuspended in PBS and adjusted to 109 CFU/mL, to match the antigen dose of the commercial vaccine, and used as the test vaccine.
Thirty-five chub mackerel (mean BW: 71.47 ± 19.14 g) per tank were injected intraperitoneally with 100 μL of commercial vaccine, FKC, or PBS in each tank. These fish were kept in 0.5 kL tanks with sand-filtered running seawater at 24–26°C. After 21 days of vaccination, 9–10 fish from each tank were collected, and their serum was sampled. Vaccinated chub mackerels were anesthetized with 2-Phenoxyethanol (Wako), and blood was collected from the caudal peduncle using a syringe fitted with 25G needle. The blood was kept for 2 h on ice for coagulation and centrifuged at 820 × g for 10 min at 4°C. The serum was collected from the supernatant and inactivated for 30 min at 45°C for measurement of agglutinating antibody titers (Matsuyama et al., 2016).
Then, the remaining 25 vaccinated fish from each group were intraperitoneally injected with 100 μL of the L. garvieae L17-2 suspended in sterile PBS at 10 ng/mL (105 CFU/mL). Symptoms were observed up to 14 days after infection, and isolation of the bacteria was attempted from the kidneys of all test fish.
The log-rank test was used for the statistical analysis of the vaccine test, and the relative percentage of survival (RPS) was calculated using the following formula: RPS = (1 – mortality in vaccinated group/mortality in control group) × 100.
Agglutinating antibody titersAgglutinating antibody titers were determined using the microtiter method in 96-well V-bottom plates (STEM). The antigen for agglutinating antibody titer was the FKC of L. garvieae L17-2, which was washed three times and adjusted to OD595 = 0.8 with sterile PBS. Each serum sample was diluted 2-fold stepwise from 1 : 8 to 1 : 1024 in sterile PBS, and 100 μL of each diluted serum was loaded into a well. Then, 10 μL of the antigen was added to each well containing diluted serum. The plates were incubated for 1 h at 25°C and overnight at 4°C, and the aggregation antibody titers were measured.
Pathogenicity test results are shown in Fig. 1A. Cumulative mortality rates were 46.6% in 7.65 × 101 CFU/fish group, 66.7% in 7.65 × 103 CFU/fish group, 80.0% in 7.65 × 105 CFU/fish group, and 0.0% in the control groups, respectively. The LD50 of L. garvieae L17-2 in chub mackerel was estimated to be 1.41 × 102 CFU/fish in this study. Similarly, the LD50 of L. garvieae serotype I isolated from each fish species in yellowtail (Ooyama et al., 2002) and threadsail filefish (Minami et al., 2013) was reported to be 5.2 × 103 CFU/fish and 1.0 × 101.6 CFU/fish, respectively. These observations suggest that L17-2 is highly pathogenic to chub mackerel, comparable to L. garvieae infections in other fish species. The external symptoms and autopsy of the fish that died during the infection included pop-eyes, ocular haemorrages, reddening of the head, pale gills, haemorrages of the each fin and intestinal tract, and epicarditis (Fig. 1B–G). These symptoms were similar to those observed in Japanese yellowtail lactococcosis (Yoshida, 2016). Lactococcus garvieae was re-isolated from the brains and kidneys of all fish that died or survived during the test period in the groups infected with the bacteria. Twenty-one days after the infection, when the mortality had stopped, L. garvieae was still being re-isolated from the surviving chub mackerel. These results indicate that L. garvieae causes persistent infection. In threadsail filefish, bacteria may persist in surviving fish after L. garvieae infection for a long period, leading to chronic disease progression (Minami et al., 2013). Therefore, it is necessary in the future to analyze the long-term effects of the L. garvieae that continue to infect chub mackerel.
No swimming abnormalities were observed in the chub mackerel inoculated with the test vaccines or in the control fish inoculated with PBS, suggesting that the adverse effects of the inoculated vaccines were negligible. Additionally, the survival rates of fish immunized with the commercial vaccine and FKC after challenge with isolate L17-2 were 68.0% and 68.0%, respectively, which were significantly higher than the 28.0% survival rate in the negative control group (Table 1). The RPS in both the commercial and FKC vaccination groups was 55.6%. In cases where even higher vaccine efficacy is required, multiple administrations of the vaccine may improve the protective efficacy. In rainbow trout, for instance, the administration of an oral booster vaccine in addition to the initial vaccination increases the efficacy of the vaccine against lactococcosis (Romalde et al., 2004). Bacteria were re-isolated from the kidneys of all dead fish during the experimental period. The ratio of surviving fish from which bacteria were re-isolated differed between pathogenicity and vaccine tests. This may have been due to differences in the sizes of the chub mackerel used in each test which was approximately 40 and 70 g, respectively. There are no previous reports of challenge tests performed on chub mackerel with L. garvieae, and it is necessary to investigate the differences in susceptibility and carrier rates by size in the future.
| Treatment | Challenge dose (CFU/fish) | Number of dead fish* | Survival rate (%) | RPS (%) | Number of reisolated fish** |
|---|---|---|---|---|---|
| commercial vaccine | 6.45 × 104 | 8/25 | 68.0 | 55.6 | 1/17 |
| FKC | 6.45 × 104 | 8/25 | 68.0 | 55.6 | 2/17 |
| control | 6.45 × 104 | 18/25 | 28.0 | – | 1/7 |
Similar to the present study on chub mackerel, mortality due to L. garvieae infections was reduced in rainbow trout and threadsail filefish by vaccination with a commercially available vaccine for yellowtail (Takahashi and Yamamoto, 2007; Minami et al., 2013). Therefore, the immunogenicity of L. garvieae in a commercial vaccine approved for Japanese yellowtail could be verified against L. garvieae isolates from other fish species, including L17-2. Accordingly, we measured the titers of agglutinating antibodies against L17-2 in chub mackerel sera immunized with a commercial vaccine for Japanese yellowtail and found that the agglutination antibody titers were equivalent to those of sera immunized with FKC of L17-2 (Fig. 2). As reported for L. garvieae in the Japanese yellowtail (Ooyama et al., 2002; Nakajima et al., 2014), serum antibodies induced by a commercial vaccine or FKC inoculation may be important for protection against L. garvieae infection in chub mackerel. However, the agglutinating antibody titers tended to be lower in the commercial vaccine group than in the FKC group (Fig. 2). A possible reason for this is the difference in immunogenicity of the strains contained in the commercial and FKC vaccines. It has been reported that the recognition of antigens by serum antibodies differs among the different L. garvieae strains that are immunized in Japanese yellowtail (Ooyama et al., 2002). There may be such differences in antibody recognition between the strains of the commercial vaccine and the FKC used in this study.
In addition to L. garvieae serotype I infection, which is the target of this study, outbreaks of L. formosensis infection, iridovirus disease, and vibriosis caused by Vibrio anguillarum infection have been observed occasionally in farmed Japanese chub mackerel. Although there are no reports of pseudotuberculosis in farmed chub mackerel, the risk of disease outbreaks exists since the causative organism, Photobacterium damselae subsp. piscicida, infects a wide range of marine fish (Romalde, 2002). The commercial vaccine used in this study contains antigens for these infectious diseases, making it even more practical if its efficacy against these diseases is demonstrated in the future.
This study was supported by the Fisheries Technology Institute, Japan Fisheries Research and Education Agency, and funded by the Food Safety and Consumer Affairs Bureau, Ministry of Agriculture, Forestry, and Fisheries of Japan. We would like to thank Editage (www.editage.jp) for English language editing.
