Characterization and Epidemiological Study of Newly Emerging Lactococcus garvieae Serotype III in Farmed Fish in Japan

Abstract

Lactococcus garvieae serotype III, a newly emerging pathogen that causes lactococcal infection, has been detected in fish farms since 2021. This pathogen has caused severe damage to the striped jack Pseudocaranx dentex and greater amberjack Seriola dumerili. This study collected strains from various fish species on farms between 2021 and 2023 and conducted an epidemiological and characterization study of L. garvieae serotype III using biochemical characterization tests, agglutinating titers against diagnostic antisera, biased sinusoidal field gel electrophoresis (BSFGE), and drug susceptibility tests. Capsule-like structures were found on the cell surfaces of L. garvieae serotype III strains, and strains showed similar characteristics except for the reaction of acidification ribose in biochemical characterization test using the API 20 STREP system. All strains were agglutinated with antiserum raised against L. garvieae serotype III and lower or non-agglutinated with antiserum raised against L. garvieae serotype I. BSFGE analysis identified three types of electrophoretic pattern: A, B, and C. BSFGE types A and B spread to fish farms in 2021, while type C found in 2022. All strains analyzed in this study were thought to be resistant to lincomycin but not to erythromycin.

Lactococcal infections caused by Lactococcus garvieae serotype I and L. formosensis (formerly L. garvieae serotype II) are prevalent on marine fish farms. Vaccines against these pathogens have been licensed and used to prevent infections in fish farms. In August 2021, unidentified bacteria were isolated from greater amberjack, Seriola dumerili that had been immunized with a commercial vaccine for L. garvieae serotype I and L. formosensis in Miyazaki, Japan. The clinical symptoms caused by this newly emerging pathogen in diseased fish were similar to those caused by L. garvieae serotype I or L. formosensis. Slide agglutination tests were performed using rabbit antisera (anti-L. garvieae serotype I and anti-L. formosensis sera) to diagnose this pathogen; however, these isolates did not agglutinate with either of these antisera (Minami et al., 2023). These non-agglutinating isolates were later identified as the newly emerging L. garvieae serotype III by multilocus sequence analysis (MLSA) using five genes and an agglutination test using an antiserum raised against the newly emerging pathogen (Minami et al., 2023). Although the newly emerging pathogen was identified as L. garvieae by MLSA, it had not belonged to serotype I (Minami et al., 2023; Araki et al., 2024). The newly emerging pathogen has prevailed in different fish species, including the greater amberjack and striped jack Pseudocaranx dentex in 2023, resulting in severe damage to these species. Minami et al. (2023) and Iwao et al. (2024) revealed that the newly emerging pathogen infects greater amberjack and striped jack and that its lethal dose 50 (LD₅₀) was lower than that for yellowtail S. quinqueradiata. Although the LD₅₀ was higher in yellowtail than in greater amberjack and striped jack, this newly emerging pathogen has been isolated from yellowtail since 2022.

Araki et al. (2024) developed a multiplex PCR (mPCR) method to identify this emerging pathogen and rapidly discriminate it from similar pathogens such as L. garvieae serotype I and L. formosensis. Emerging serotype III pathogens can be rapidly diagnosed using this mPCR method and subjected to epidemiological studies. This study aimed to elucidate the characteristic and current epidemiological situation of L. garvieae serotype III strains isolated from diseased fish in Japan between 2021 and 2023 by biochemical characterization tests (n = 16), determining agglutinating titers (n = 154), performing molecular epidemiological analyses using biased sinusoidal field gel electrophoresis (BSFGE) (n = 154), and drug susceptibility tests (n = 153).

Materials and Methods

Bacterial strains and identification

Lactococcus garvieae serotype III strains were isolated from nine fish species between 2021 and 2023 (Table 1). All L. garvieae strains used in this study are listed in Table 2. All strains were identified using a slide agglutination test with diagnostic antiserum and mPCR. The slide agglutination test method was performed as described by Oinaka et al. (2015). Antisera against L. garvieae serotype Ia (KG^– type, EH5803 strain) was prepared according to Yoshida et al. (1996) and against L. formosensis (= L. garvieae serotype II: 121836 strain) and L. garvieae serotype III (MS210922A strain) were obtained as described by Oinaka et al. (2015) and Minami et al. (2023), respectively. The mPCR was performed according to the protocol described by Araki et al. (2024). Strains showing clear agglutination of anti-L. garvieae serotype III serum and confirmed to have an amplification product (approximately 500 bp) by mPCR were identified as L. garvieae serotype III. All the strains were cultured in Todd-Hewitt (Difco, Becton, Dickinson and Company) agar (THA) at 25°C for more than 24 h. Single colony was inoculated into Todd-Hewitt broth (THB), cultured and kept at –80°C until use.

Table 1. Number of Lactococcus garvieae serotype III strains that were isolated from different sources in Japan between 2021 and 2023

Sources		Year of isolation			Total
		2021	2022	2023
Striped jack
	Pseudocaranx dentex	11	4	57	72
Greater amberjack
	Seriola dumerili	11	24	15	50
Yellowtail
	Seriola quinqueradiata		7	8	15
Japanese horse mackerel
	Trachurus japonicus		9	2	11
Cobia
	Rachycentron canadum			2	2
Chicken grunt
	Parapristipoma trilineatum	1			1
Black scraper
	Thamnaconus modestus		1		1
Chub mackerel
	Scomber japonicus			1	1
Japanese flounder
	Paralichthys olivaceus			1	1
Total		23	45	86	154

Table 2. All Lactococcus garvieae strains used in this study and results of agglutinating titers and BSFGE analysis

Strain	Source	Region	Agglutinating titers (1:)		BSFGE Type
			serotype I antiserum	serotype III antiserum
Lactococcus garvieae serotype I
Isolation of 1974
ATCC49156	Yellowtail	Kochi	NT	NT	NT
Isolation of 1994
KG9408	Yellowtail	Kagoshima	640	< 20	NT
Lactococcus garvieae serotype III
Isolation of 2021 (n = 23)
MS210819*	Greater amberjack	Miyazaki	20	320	B
MS210827SJ1	Striped jack	Miyazaki	20	1280	B
MS210913A	Greater amberjack	Miyazaki	20	640	A
MS210922A*a, b	Greater amberjack	Miyazaki	< 20	160	A
MS210922SJ	Striped jack	Miyazaki	40	640	A
MS211005A	Greater amberjack	Miyazaki	40	1280	A
MS211001SJb	Striped jack	Miyazaki	160	640	B
MS211013A1	Greater amberjack	Miyazaki	40	1280	A
MS211013A2	Greater amberjack	Miyazaki	20	640	A
MS211013A3	Greater amberjack	Miyazaki	20	1280	A
MS211013*	Chicken grunt	Miyazaki	20	320	A
MS211020A	Greater amberjack	Miyazaki	< 20	320	A
MS211202A	Greater amberjack	Miyazaki	20	640	A
MS211206A1	Greater amberjack	Miyazaki	20	640	A
MS211206A2	Greater amberjack	Miyazaki	< 20	320	A
SS210928SJ1*	Striped jack	Sizuoka	20	2560	B
SS210928SJ2	Striped jack	Sizuoka	20	640	A
L21-68	Striped jack	Ehime	20	1280	A
214611b	Striped jack	Oita	40	1280	A
214612	Striped jack	Oita	20	640	A
214631	Striped jack	Oita	20	640	A
214632	Striped jack	Oita	20	320	A
214633	Striped jack	Oita	< 20	320	A
Lactococcus garvieae serotype III
Isolation of 2022 (n = 45)
KS220516A*b	Greater amberjack	Kochi	< 20	320	B
MS220607Aa	Greater amberjack	Miyazaki	80	2560	A
MS220609A	Greater amberjack	Miyazaki	< 20	320	A
MS220609SJ	Striped jack	Miyazaki	< 20	320	A
220403*	Black scraper	Oita	20	640	A
MS220615A	Greater amberjack	Miyazaki	160	2560	A
MS220628A	Greater amberjack	Miyazaki	< 20	640	B
OT220587a	Greater amberjack	Oita	160	2560	A
MS220719A	Greater amberjack	Miyazaki	20	640	A
MS220726A	Greater amberjack	Miyazaki	20	320	A
MS220802A	Greater amberjack	Miyazaki	< 20	320	A
MS220803Y	Yellowtail	Miyazaki	< 20	320	B
MS220804Y*b	Yellowtail	Miyazaki	< 20	320	B
MS220805A	Greater amberjack	Miyazaki	20	640	A
MS220809A	Greater amberjack	Miyazaki	20	320	A
MS220809A1	Greater amberjack	Miyazaki	20	640	A
MS220809A2	Greater amberjack	Miyazaki	< 20	640	B
MS220808Y	Yellowtail	Miyazaki	< 20	640	B
MS220812Y1	Yellowtail	Miyazaki	< 20	640	B
MS220812Y2	Yellowtail	Miyazaki	< 20	640	B
MS220816A1	Greater amberjack	Miyazaki	80	2560	A
MS220816A2	Greater amberjack	Miyazaki	< 20	320	A
MS220816A3	Greater amberjack	Miyazaki	< 20	640	A
MS220816A4	Greater amberjack	Miyazaki	20	640	A
221411	Striped jack	Oita	40	1280	A
220911	Yellowtail	Oita	40	2560	A
MS220831A1	Greater amberjack	Miyazaki	< 20	320	A
MS220831A2	Greater amberjack	Miyazaki	20	640	A
MS220831Y	Yellowtail	Miyazaki	< 20	320	B
KG220831A*	Greater amberjack	Kagoshima	< 20	640	B
KG220901A1	Greater amberjack	Kagoshima	< 20	320	B
KG220901A2	Greater amberjack	Kagoshima	< 20	320	B
MS220930SJ	Striped jack	Miyazaki	< 20	320	A
SS221003HM1*b	Japanese horse mackerel	Shizuoka	< 20	640	A
SS221003HM2	Japanese horse mackerel	Shizuoka	< 20	640	B
SS221003HM4	Japanese horse mackerel	Shizuoka	20	640	A
MS221017SJ	Striped jack	Miyazaki	20	640	A
SS221004HM2b	Japanese horse mackerel	Shizuoka	40	2560	A
SS221004HM4	Japanese horse mackerel	Shizuoka	< 20	1280	A
SS221004HM8	Japanese horse mackerel	Shizuoka	20	1280	B
SS221004HM9	Japanese horse mackerel	Shizuoka	< 20	1280	A
SS221004HM11	Japanese horse mackerel	Shizuoka	< 20	1280	A
SS221018HM5*b	Japanese horse mackerel	Shizuoka	40	1280	C
MS221124A	Greater amberjack	Miyazaki	20	640	B
MS221129A	Greater amberjack	Miyazaki	20	2560	A
Lactococcus garvieae serotype III
Isolation of 2023 (n = 86)
MS230111A	Greater amberjack	Miyazaki	< 20	640	A
MS230424A	Greater amberjack	Miyazaki	< 20	640	A
MS230531A	Greater amberjack	Miyazaki	20	640	A
KN230612SJ1*	Striped jack	Kanagawa	20	1280	A
KN230612SJ2	Striped jack	Kanagawa	20	640	A
MS230628SJ	Striped jack	Miyazaki	< 20	640	A
MS230703SJ	Striped jack	Miyazaki	< 20	320	A
MS230711A	Greater amberjack	Miyazaki	< 20	320	A
MS230720A	Greater amberjack	Miyazaki	40	640	A
MS230720A2	Greater amberjack	Miyazaki	< 20	320	A
MS230713A1	Greater amberjack	Miyazaki	< 20	320	A
MS230713A2	Greater amberjack	Miyazaki	< 20	320	A
MS230725A1	Greater amberjack	Miyazaki	< 20	320	A
MS230725A2	Greater amberjack	Miyazaki	< 20	320	A
ME230802SJ	Striped jack	Mie	< 20	320	A
MS230817A	Greater amberjack	Miyazaki	< 20	320	B
MS230906Y	Yellowtail	Miyazaki	< 20	320	A
MYS230731A	Greater amberjack	Miyazaki	< 20	320	A
MYS230813A	Greater amberjack	Miyazaki	< 20	320	A
MYS230823A	Greater amberjack	Miyazaki	< 20	320	A
MYS230921Ab	Greater amberjack	Miyazaki	20	320	A
MS230907SJ	Striped jack	Miyazaki	< 20	320	A
L23-07	Striped jack	Ehime	20	320	A
L23-10	Striped jack	Ehime	< 20	320	A
L23-13	Striped jack	Ehime	< 20	320	A
L23-15	Striped jack	Ehime	< 20	640	A
L23-16	Striped jack	Ehime	20	320	A
L23-19	Striped jack	Ehime	< 20	640	B
L23-22	Striped jack	Ehime	< 20	320	A
L23-23	Striped jack	Ehime	< 20	320	A
L23-25	Striped jack	Ehime	< 20	320	A
L23-26	Striped jack	Ehime	40	640	A
L23-28	Striped jack	Ehime	< 20	640	A
L23-29	Striped jack	Ehime	20	640	A
L23-30	Striped jack	Ehime	< 20	640	A
L23-32	Yellowtail	Ehime	< 20	320	A
L23-35	Striped jack	Ehime	< 20	640	A
L23-45	Striped jack	Ehime	< 20	640	B
L23-46	Striped jack	Ehime	20	640	A
L23-49	Striped jack	Ehime	< 20	640	A
L23-50	Striped jack	Ehime	< 20	320	B
L23-52	Striped jack	Ehime	20	320	A
L23-55	Striped jack	Ehime	20	640	A
L23-58	Striped jack	Ehime	20	640	A
L23-59	Striped jack	Ehime	< 20	320	A
L23-68	Striped jack	Ehime	20	320	A
L23-69	Striped jack	Ehime	20	640	A
L23-70	Striped jack	Ehime	< 20	320	A
L23-72*	Chub mackerel	Ehime	< 20	320	A
L23-73	Striped jack	Ehime	20	640	A
L23-74	Striped jack	Ehime	< 20	640	A
L23-79	Striped jack	Ehime	< 20	320	B
L23-80	Striped jack	Ehime	< 20	640	A
L23-81	Striped jack	Ehime	< 20	640	A
L23-84	Striped jack	Ehime	20	1280	A
L23-85	Striped jack	Ehime	< 20	320	A
MS231031JF*	Japanese flounder	Miyazaki	20	640	A
MS231108SJ	Striped jack	Miyazaki	20	320	A
OS231025C*b	Cobia	Okinawa	< 20	640	B
OS231026C	Cobia	Okinawa	< 20	640	B
MEL2305	Striped jack	Mie	< 20	640	A
MEL2306	Striped jack	Mie	< 20	320	B
MEL2309	Yellowtail	Mie	< 20	640	A
MEL2310	Striped jack	Mie	< 20	640	B
MEL2311	Striped jack	Mie	< 20	640	A
MEL2312	Striped jack	Mie	< 20	640	B
MEL2313	Striped jack	Mie	< 20	320	B
MEhL2302*b	Japanese horse mackerel	Mie	< 20	320	B
MEhL2303	Striped jack	Mie	20	320	B
MEhL2304	Striped jack	Mie	20	640	B
MEhL2306	Striped jack	Mie	< 20	320	B
MEhL2307	Striped jack	Mie	< 20	320	B
MEhL2308	Striped jack	Mie	< 20	320	B
MEhL2310	Striped jack	Mie	< 20	320	B
KS230710SJ	Striped jack	Kochi	< 20	640	A
KS230905Y	Yellowtail	Kochi	< 20	320	A
KS230928SJ	Striped jack	Kochi	< 20	320	A
KS231012SJ	Striped jack	Kochi	< 20	640	A
SS230525HM	Japanese horse mackerel	Shizuoka	< 20	160	A
SS230804SJ	Striped jack	Shizuoka	< 20	320	B
SS230902SJ	Striped jack	Shizuoka	< 20	320	B
231201	Yellowtail	Oita	< 20	320	A
232041*b	Yellowtail	Oita	< 20	320	A
232043	Yellowtail	Oita	< 20	320	A
232044	Yellowtail	Oita	< 20	320	A
233401	Striped jack	Oita	< 20	640	A

NT: not tested.

*   These strains were used for biochemical characterization.

^a   These strains were used for cell morphology by transmission electron microscopy (TEM).

^b   These strains were used in Fig. 2.

Biochemical characterization

Lactococcus garvieae serotype III strains (n = 16) were examined by performing bacteriological tests in the API 20 STREP system (bioMérieux) according to the manufacturer’s protocol after its modification. All L. garvieae strains were cultured aerobically on THA at 25°C for 24 h for the API 20 STREP system. Bacterial growth was examined using THB medium at different conditions, 10°C and 45°C, 6.5% NaCl, and pH 9.6 as described in a previous paper (Kitao, 1982).

Cell morphology

Bacterial cell morphology was examined by an optical microscope with Gram staining and by an electron microscope. Gram staining was carried out on a representative strain (MS210922A) and performed using the neo-B&M Wako (FUJIFILM Wako Pure Chemical Corporation).

Three representative strains (MS210922A, MS220607A, and OT220587) were selected for cell morphology analysis using transmission electron microscopy (TEM). One strain (MS210922A) agglutinated against L. garvieae serotype III antiserum (1:160) but not against L. garvieae serotype I antiserum (1:< 20). Two strains (MS220607A and OT220587) agglutinated against both serotypes of the antisera (Table 2). The bacterial cells of each strain were grown overnight in 10 mL of THB at 25°C for 24 h and collected bacteria via centrifugation. The collected cells were suspended in 1.5 mL of physiological saline solution, embedded in 3% agarose, excised into around 1 mm³, immersed in the antiserum, and kept at 4°C for 2 h. The cells were then washed twice with 0.1 M cacodylate buffer (pH 7.4), fixed with 1% glutaraldehyde solution for 2 h, and washed three times with the same buffer. The capsules on the cell surface were stained with ruthenium red in a glutaraldehyde solution for 2 h and washed with the same buffer until the color of the agarose faded. The cells were fixed with an osmium oxide solution, washed three times with the same buffer, dehydrated in ethanol, increased in concentration from 50% to 100%, and embedded in Epon 812 (TAAB Laboratories Equipment). Subsequent processes of ultrathin sectioning, electronic staining, observation by transmission electron microscopy, and photography were performed by Tokai Electron Microscopy, an electron microscopy analysis company.

Agglutinating titer

The newly emerging L. garvieae isolates were close to the L. garvieae serotype I strain in MLSA (Minami et al., 2023; Araki et al., 2024). Thus, serotype I and III antisera were used to determine agglutinating titers. Antisera against L. garvieae serotype Ia (KG^– type, EH5803 strain) and III (MS210922A strain) were the same as those used for the slide agglutination test. The agglutinating titers against these antisera were determined using microtiter assay according to the method described by Fukuda et al. (2015). Lactococcus garvieae serotype Ia (KG^– type, KG9408 strain) was used as a control (Ooyama et al., 2002).

Biased sinusoidal field gel electrophoresis (BSFGE) analysis

BSFGE analysis was performed using the method of Nishiki et al. (2011) with slight modifications. Colonies on THA were suspended in 1 mL of TE buffer (10 mM Tris-HCl and 1 mM EDTA; pH 8.0) to approximately equal the MacFarland No.1 standard density, and the suspended cells were collected by centrifugation. The collected cells were resuspended in 150 μL of TE buffer and mixed with 150 μL of 2% low melting point agarose (Agarose L; NIPPON GENE). Immediately after, the mixture was poured into plug molds and the molds were kept at 4°C for 30 min. The obtained plugs were placed in 1.0 mL of lysis buffer (0.1 M EDTA, 7.5 mg/mL lysozyme, and 30 units/mL mutanolysin) and incubated at 37°C for 18 h with gentle rotation. The plugs were replaced in 1.0 mL of protein-denatured buffer (0.1 M EDTA, 1% sodium dodecyl sulfate, and 50 units/mL proteinase K) and incubated at 55°C for 18 h with gentle rotation. After denaturation, the plugs were washed with 1.0 mL of TE buffer and TE buffer containing 1 mM phenylmethylsulfonyl fluoride at 55°C for 1 h with gentle rotation. The plugs were washed six times with 2.0 mL of TE buffer at room temperature. Each of the first five washes was performed for 30 min and the last wash was performed overnight with gentle shaking.

The restriction enzyme SmaI (Takara Bio) was used before BSFGE typing. The plugs were cut into two pieces and placed in 200 μL of reaction buffer with 50 units of the SmaI. DNA digestion was carried out for 24 h at a reaction temperature was set at 37°C. The plugs were loaded into wells containing 1% agarose gel (SeaKem Gold Agarose; Lonza) and 0.5 × TBE buffer (44.5 mM Tris, 45.2 mM boric acid, and 2.2 mM EDTA). Lambda Ladders (ProMega-Markers; Promega) were used as molecular markers. Genomic DNA fragments were separated using a BSFGE system (Genofield; ATTO).

Antimicrobial susceptibility testing

The minimum inhibitory concentration (MIC) was determined using the agar dilution method described by Shi et al. (2019). Total 153 strains were used in this study. Five antimicrobials, erythromycin (EM), lincomycin (LCM), oxytetracycline (OTC), florfenicol (FF), and ampicillin (ABPC), were selected. These antimicrobials have been licensed for use on farmed fish. Tiamulin (TML) has also been used to confirm cross- resistance to LCM (Shi et al., 2019, 2023). MIC values were determined after 48 h culture at 25°C.

Results

Host fish species

The fish species infected with L. garvieae serotype III are listed in Table 1. Lactococcus garvieae serotype III strains were mainly isolated from striped jack (n = 72) and greater amberjack (n = 50) since 2021. Since 2022, it has also been isolated from yellowtail (n = 15).

Biochemical characteristics

The results of bacteriological test using the API 20 STREP system are shown in Table 3. All L. garvieae serotype III strains grew in broth at 10°C and 45°C, 6.5% NaCl, and pH 9.6. In the API 20 STREP system, the reactions of L. garvieae serotype III strains (n = 16) were almost identical to those of the L. garvieae serotype I strain. Hippurate hydrolysis and acidification mannitol were not clearly observed in any of the strains. Regarding L. garvieae serotype III strains, the acidification ribose differed depending on the strain.

Table 3. Biochemical characteristics of Lactococcus garvieae serotype III and L. garvieae serotype I

Characteristics		Serotype III strains	Sertoype I
		(n = 16)	ATCC49156
Growth at 10°C		+	+
	45°C	+	+
Growth in 6.5% NaCl		+	+
	pH 9.6	+	+
Voges-Proskauer test (VP)		+	+
Hippurate hydrolysis (HIP)		NC	NC
Esculin hydrolysis (ESC)		+	+
Pyrrolidonyl arylamidase (PYRA)		+	+
α-Galactosidase (α-GAL)		–	–
β-Glucuronidase (β-GUR)		–	–
β-Galactosidase (β-GAL)		–	–
Alkaline phosphatase (PAL)		–	–
Leucine aminopeptidase (LAP)		+	+
Arginine dihydrolase (ADH)		+	+
Acidification Ribose (RIB)		V (+ 5/– 11)	+
	Arabinose (ARA)	–	–
	Mannitol (MAN)	NC	NC
	Sorbitol (AOR)	–	–
	Lactose (LAC)	–	–
	Trehalose (TRE)	+	+
	Inulin (INU)	–	–
	Raffinose (RAF)	–	–
	Amygdaline (AMD)	–	–
	Glycogen (GLYG)	–	–

NC: not clear.

V: variable.

Morphology

Optical microscopy with Gram-stained revealed Gram-positive, which formed chains in the growth medium (Fig. 1a). Electron microscopy revealed capsule-like structures on the cell surface of one strain (MS210922A) that agglutinated only against the anti-L. garvieae serotype III serum (Fig. 1b). Capsule-like structures were also observed on the cell surfaces of two strains (MS220607A and OT220587) that agglutinated against both anti-L.garvieae serotypes I and III serum (Fig. 1c and 1d, respectively).

Fig. 1. Morphological characteristics of Lactococcus garvieae serotype III. Optical microscope image of Gram-stained (a). Transmission electron microscopy (TEM) images. MS2109222A strain that was agglutinated only against anti- L. garvieae serotype III serum (b). MS220607A (c) and OT220587 (d), which were agglutinated against both anti-L. garvieae serotype I and serotype III sera.

Scale bars: approximately 5 μm for (a); 500 nm for (b), (c), and (d).

Agglutinating titer

The agglutinating titers for each strain are shown in Table 2. The agglutinating titer of L. garvieae serotype I strain (KG9408 strain), which was used as a control, was 1:640 against the anti-L. garvieae serotype I serum and 1:< 20 against anti-L. garvieae serotype III. Agglutinating titers of the L. garvieae serotype III strains against anti-L. garvieae serotype I serum were 1:< 20 to 1:160, whereas those against anti-L. garvieae serotype III serum was 1:160 to 1:2560. These results confirmed that the antigenicity of new isolates differed from the L. garvieae serotype I (Table 2).

BSFGE analysis

The representative image results of the BSFGE analysis are presented in Fig. 2. The chromosomal DNA of L. garvieae serotype III strains (n = 154) digested with SmaI, were classified into three BSFGE patterns (Table 2). These patterns were represented as BSFGE types A, B, and C, respectively. BSFGE type A was the most common strain (n = 114), followed by type B (n = 39). Only one strain belonged to type C. BSFGE types A and B were observed since 2021 whereas type C was observed only in 2022. This type C strain was isolated from Japanese horse mackerel Trachurus japonicus, which was not main host fish species in this study.

Fig. 2. Biased sinusoidal field gel electrophoresis (BSFGE) separation of SmaI-digested genomic DNA fragments of Lactococcus garvieae serotype III strains. Lane M corresponds to a Lambda DNA size marker. Lanes 1–5 correspond to BSFGE type A strains (MS210922A, 214611, SS221003HM1, MYS230921A, and 230241, respectively). Lanes 6–10 correspond to BSFGE type B strains (MS211001SJ, KS220516A, MS220804Y, MEhL2302, and OS231025C, respectively). Lane 11 corresponds to BSFGE type A strain (SS221004HM2). Lane 12 corresponds to BSFGE type C strain (SS221018HM5).

Antimicrobial susceptibility test

The distribution of the MIC values of the six antimicrobials against all L. garvieae serotype III strains used in this study is shown in Table 4. Although none of two peaks distribution of MIC values were found in six antimicrobials, the relative high MIC values (100 to 400 μg/mL) were observed against LCM and TML in all the serotype III strains. None of the strains were supposed to be resistant to the other four antimicrobials.

Table 4. MIC values of ABPC, EM, FF, OTC, LCM, and TML against Lactococcus garvieae serotype III strains

Antimicrobials	MIC (μg/mL)														Total
	0.05	0.1	0.2	0.39	0.78	1.56	3.13	6.25	12.5	25	50	100	200	400
ABPC						35	116	2							153
EM		16	87	21	29										153
FF							81	68	4						153
OTC					46	62	40	5							153
LCM												78	41	34	153
TML													15	138	153

ABPC: ampicilin, EM: erythromycin, FF: florfenicol, OTC: oxytetracycline, LCM: lincomycin, TML: tiamulin

Discussion

Streptcoccosis, which is caused by the genera Streptococcus and Lactococcus, occurs in aquaculture in Japan. Pathogens such as S. iniae, S. dysgalactiae, S. parauberis, and Lactococcus species have been observed in marine aquaculture (Yoshida, 2016). Recently, lactococcal infections in Japanese fish farms were found to be caused by three different pathogens: Lactococcus garvieae serotype I, L. formosensis (formerly L. garvieae serotype II), and the newly emerging L. garvieae serotype III. This newly emerging lactococcal pathogen was isolated in 2021 for the first time. Since then, the pathogen has spread to several fish farms and species by 2023. This study aimed to elucidate the current epidemiological situation of this newly emerging pathogen in fish farms.

The newly emerging pathogen strains were mostly collected from striped jack (n = 72), followed by greater amberjack (n = 50). This pathogen was first isolated from yellowtail in 2022, and total 15 strains were collected. Iwao et al. (2024) revealed that the serotype III infects greater amberjack and striped jack and that its lethal dose 50 (LD₅₀) was lower for greater amberjack and striped jack than for yellowtail. The different susceptibilities in these fish species against the newly emerging pathogens may reflect sample numbers from fish farms.

In susceptibility tests for the five antimicrobials, the newly emerging strains showed relatively high MIC values for LCM from their first occurrence on fish farms. EM and LCM are popular chemotherapeutics used to control lactococcal infections on fish farms. Recently, many strains of L. garvieae serotype I and L. formosensis isolated from fish farms were found to be resistant to LCM. The LCM resistance of L. formosensis in the absence of EM resistance ranged from 12.5 to 100 μg/mL in terms of the MIC value, while LCM-sensitive strains were less than 1 μg/mL (Frushita et al., 2015; Shi et al., 2019; Akmal et al., 2023). Furushita et al. (2015) reported that the tentative break point of the LCM-resistant strains of L. garvieae serotype I was 4 μg/mL in terms of the MIC value. These results suggested that all L. garvieae serotype III strains with high MIC values for LCM were natural resistant to LCM and TML during the first outbreak on fish farms.

Recently, the single LCM resistance mechanism in the absence of EM resistance was shown to be caused by lsa(D) or lsa(D) variant in the chromosomes of L. formosensis and L. garvieae serotype I, respectively. The LCM resistance by lsa(D) or lsa(D) variant was also cross-resistant to TML (Shi et al., 2021, 2023). The lsa(D) variant has also been found in the L. garvieae serotype III genome. Therefore, it thought to be expressed to resist LCM and TML in serotype III. EM is a useful drug for controlling lactococcal infections (L. garvieae serotype I and L. formosensis) caused by LCM-resistant strains in fish farms. Lactococcus formosensis (formerly L. garvieae serotype II) was first identified in 2012 and EM resistance was observed in this species in 2019 (Akmal et al., 2023). Although EM-resistant strains of L. garvieae serotype I and L. formosensis have spread to fish farms, no strains of L. garvieae serotype III were found to be resistant to EM until 2023. An epidemiological survey to monitor the possible occurrence of EM-resistant L. garvieae serotype III in the future should be continued.

Electron microscopy revealed capsule-like structures on the cell surfaces of L. garvieae serotype III strains with different agglutinating properties. A previous study revealed that there were two sub-serotypes of Ia (KG^–) and Ib (KG⁺) in L. garvieae serotype I due to the different agglutinating properties of the antiserum raised against KG⁺ type cells. KG^– type cells develop cell capsule on their surface, but KG⁺ type cells do not (Kitao, 1982; Yoshida et al., 1996, 1997; Fukuda et al., 2015). The cell capsule plays a role in resisting phagocytosis by phagocytic cells (Yoshida et al., 1996, 1997). In this study, capsule-like structures were found on the cell surfaces of all the serotype III strains, regardless of their agglutination titers, the cell capsule may contribute to the virulence of this pathogen.

The bacteriological characteristics of L. garvieae serotype III strains coincided with each other except for acidification ribose and their reactions were similar to those of L. garvieae serotype I strains. Bacteriological characteristics alone could not differentiate L. garvieae serotype III strains from L. garvieae serotype I strains. The results of agglutination titer were clearly divided into serotype I and serotype III using antisera (serotype I and serotype III). Thus, the slide agglutination test using L. garvieae serotype III appropriately-diluted antiserum would be routinely available for the diagnosis of serotype III infection except for some isolates which showed cross reaction to serotype I antiserum.

In a previous epidemiological study for the five-year survey of L. formosensis (formerly L. garvieae serotype II), BSFGE analysis revealed that homogeneous strain had spread to fish farms since the first outbreak of L. formosensis infection (Shi et al., 2019). In this study, BSFGE analysis revealed that three BSFGE types (A, B, and C) of L. garvieae serotype III were spread on fish farms. These patterns were not coincided with those of L. garvieae serotype I and L. formosensis (formerly L. garvieae serotype II), which were reported previously (Nishiki et al., 2011; Shi et al., 2019). BSFGE types A and B were found and spread since 2021, whereas type C was found firstly in 2022. Type C was not found in 2023. These results suggest that there are some routes for the spreading of this emerging pathogen. Further monitoring is needed to study the spread of this pathogen in fish farms.

In conclusion, this is the first epidemiological study for newly emerging L. garvieae serotype III in fish farms. This study revealed that three different BSFGE types prevailed in fish farms, all of which were resistant to LCM, but not to EM. Routine slide agglutination tests with L. garvieae serotype III antiserum were also useful to diagnose this infection as well as mPCR assays.

Acknowledgments

This study was supported by Grants-in-Aid for Scientific Research from JSPS KAKENHI (grant number 21H02287 and 23K05396). This study was also supported by the Food Safety and Consumer Affairs Bureau, Ministry of Agriculture, Forestry, and Fisheries of Japan. This work is partially supported by MAFF Commissioned project study on “Development of effective antibiotics administration methods for responding to the risk of new fish diseases which occur with promoting hatchery-reared juvenile in Seriola sp.” Grant Number JPJ012045. The authors are grateful to all prefectural officers in Oita, Miyazaki, Kagoshima, Okinawa, Kochi, Ehime, Mie, Shizuoka, and Kanagawa for kindly providing the bacterial strains.

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