Insights on the virulence and genomic features of Lactococcus garvieae isolated from giant freshwater prawn Macrobrachium rosenbergii (de Man 1879).

Giant freshwater prawn (Macrobrachium rosenbergii (MR)) is a significant aquafarm species commercially cultured in Taiwan. Intensive farming practices have led to the outbreak of Lactococcus garvieae (LG), which causes Lactococcosis in MR. Recently, LG has re-emerged and the number of mortalities in prawn farms has increased in Taiwan. However, there is no preventative strategy described and a lack of knowledge on virulence factors and pathogenesis from LG in MR. The most virulent strain of L. garvieae from M. rosenbergii was screened in vivo among seven isolates selected for infectivity testing injecting 0.1 mL of 108 CFU/mL bacterial concentration. Among the seven isolates screened, L. garvieae 109-6 resulted in 100% mortality within 3 days post-infection. Furthermore, 109-6 L. garvieae LD50 dosage from in MR was found to be 106 CFU/mL. Subsequently, the most virulent strain 109-6 was sequenced using MinIon Nanopore sequencing. Results indicated that the LG genome yielded a protein-coding of 3857 with 59 tRNA and 16 rRNA and no plasmid. Interestingly, the distribution of subsystems in the annotated genome revealed genes related to virulence, defence, and disease among LG 50 genes. Altogether, the virulent strain and its genome data revealed distinctive features of LG, which hinted toward its pathogenicity and could facilitate for better preventive strategies.

Recipient sepsis caused by Lactococcus garvieae contamination of platelets from a donor with colon cancer.

Lactococcus garvieae is a well-known fish pathogen that has low virulence in humans and is rarely isolated from the blood cultures of endocarditis patients. We describe herein the first reported case of transfusion-transmitted L. garvieae sepsis caused by a contaminated platelet concentrate from a donor who consumed raw octopus before blood donation. Retrospective examination of the laboratory results of the index donor revealed that his haemoglobin levels had been steadily decreasing, which led to the detection of a latent colon cancer. The donors with colon lesions involving a latent cancer may relate an asymptomatic bacteremia.

Effects of Enterococcus faecalis UGRA10 and the enterocin AS-48 against the fish pathogen Lactococcus garvieae. Studies in vitro and in vivo.

The aim of this study was to evaluate the effects of Enterococcus faecalis UGRA10 and its enterocin AS-48 against the fish pathogen Lactococcus garvieae. The minimum bactericidal concentrations of AS-48 against L. garvieae CECT 5807, 5806, and 5274 were 15.62, 15.62, and 7.81 µg/ml respectively. In broth cultures, enterocin at 100, 50, and 25 µg/ml reduced 108 CFU/ml lactococci after 2, 5, and 10 h, respectively. In co-cultures of UGRA10/L. garvieae at a 1/10 CFU/ml ratio, lactococci were eliminated after 24 h. Studies on UGRA10 biosafety and AS-48 toxicity in R1 cells and in rainbow trout have shown a lack of adverse effects from both the strain and bacteriocin. Trout challenged with L. garvieae and UGRA10 administered in diet 30 days before infection had a cumulative survival rate of 50% compared with 0% for control fish. Trout inoculated with the pathogen and treated by regular dipping in AS-48 baths had a survival rate of 60% after 20 days compared with that of untreated fish (0%). These results indicate the protective effect of the UGRA10 strain and the bacteriocin AS-48 against L. garvieae and the potential of these natural products as alternatives to antibiotics for controlling diseases in aquaculture.

The effect of banana (Musa acuminata) peels hot-water extract on the immunity and resistance of giant freshwater prawn, Macrobrachium rosenbergii via dietary administration for a long term: Activity and gene transcription.

The non-specific immune parameters, disease resistance and immune genes expressions in Macrobrachium rosenbergii were evaluated at 120 days of post feeding the diets containing the extracts of banana, Musa acuminate, fruit's peel (banana peels extract, BPE) at 0, 1.0, 3.0 and 6.0 g kg(-1). Results showed that prawns fed with a diet containing BPE at the level of 1.0, 3.0 and 6.0 g kg(-1) for 120 days had a significantly higher survival rate (30.0%, 40.0% and 56.7%, respectively) than those fed with the control diet after challenge with Lactococcus garvieae for 144 h, and the respective relative survival percentages were 22.2%, 33.3%, and 51.9%, respectively. Dietary BPE supplementation at 3.0 and/or 6.0 g kg(-1) for 120 days showed a significant increase total haemocyte count (THC), granular cell (GC), superoxide dismutase (SOD) activity, phenoloxidase (PO) activity, transglutaminase (TG) activity, and phagocytic activity and clearance efficiency to L. garvieae infection, and meanwhile, the significant decrease in haemolymph clotting times and respiratory bursts (RBs) per haemocyte of prawns were revealed. Furthermore, the mRNA expressions of prophenoloxidase (proPO), lipopolysaccharide and ß-1,3-glucan binding protein (LGBP), peroxinectin (PE), transglutaminase (TG), and crustin (CT) were significantly increased. We therefore recommend that BPE can be used as an immunomodulator for prawns through dietary administration at 6.0 g kg(-1) for a long term (over 120 days) to modify immune responses and genes expression following the enhanced resistance against pathogens.

Co-infection of Lactococcus garvieae and Aeromonas hydrophila in cultured Nile Tilapia in Kerala, India.

Co-infection of Lactococcus garvieae and Aeromonas hydrophila, has been confirmed from diseased Nile Tilapia (Oreochromis niloticus), Chithralada strain cultured in a freshwater rearing pond of Alappuzha district of Kerala, India. The aetiological agents behind the disease outbreak were bacteriologically proven and confirmed by 16SrRNA sequencing and phylogenetic analysis. PCR detection of the virulent genes, showed existence of adhesin and hemolysin in L. garvieae and aerolysin in A. hydrophila strain obtained. To fulfil Koch's postulates, challenge experiments were conducted and median lethal dose (LD50) of L. garvieae and A. hydrophila was calculated as 1 × 105.91 CFU per mL and 1 × 105.2 CFU per mL respectively. Histopathologically, eyes, spleen, and kidney were the predominantly infected organs by L. garvieae and A. hydrophila. Out of the 13 antibiotics tested to check antibiotic susceptibility, L. garvieae showed resistance to almost 7 antibiotics tested, with a resistance to Ciprofloxacin while A. hydrophila was found resistant to Streptomycin and Erythromycin. Understanding the complex interaction between Gram-positive and Gram-negative bacteria in the disease process and pathogenesis in fish host will contribute to efficient treatment strategies. As a preliminary investigation into this complex interaction, the present study is aimed at phenotypic and genotypic characterization, pathogenicity evaluation, and antibiotic susceptibility of the co-infecting pathogens in a diseased sample of freshwater-farmed Nile tilapia.

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

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.

Comparison of genetic characteristics and pathogenicity of Lactococcus garvieae isolated from aquatic animals in Taiwan.

Seventy-six Taiwanese bacterial isolates including 74 from diseased, cultured, aquatic animals (54 grey mullet Mugil cephalus, 3 basket mullet Chelon alatus, 2 tilapia Oreochromis niloticus, 1 grouper Epinephelus coioides, 2 yellowfin seabream Acanthopagrus latus, 1 Borneo mullet Chelon macrolepis, 1 bullfrog Rana catesbeiana, 1 Japanese eel Anguilla japonica, and 9 giant freshwater prawns Macrobrachium rosenbergii), 1 wild-caught seafood species (squid muscle collected from a restaurant) and 1 human isolate (from a patient with a history of consuming raw squid in the previously mentioned restaurant), all collected between 1999 and 2006, were confirmed by PCR assay to be Lactococcus garvieae. The phenotypic characterization was determined by rabbit anti-KG+ and KG- serums, and 74 of the 76 Taiwanese strains displayed a KG- phenotype. The genetic characterization was investigated by pulsed-field gel electrophoresis (PFGE). Genomic DNA was digested with restriction endonucleases ApaI and SmaI and separated by PFGE. Ten different L. garvieae pulsotypes were identified. Predominant pulsotypes A1a/S1a were obtained from >96% of strains (52 of 54) from grey mullet, demonstrating a clonal dissemination of L. garvieae in grey mullet in Taiwan. In experimental challenges with grey mullet and tilapia, L. garvieae pulsotypes A1/S1 and A11/S11 showed higher virulence compared with other pulsotypes.

Application of suppressive subtractive hybridization to the identification of genetic differences between two Lactococcus garvieae strains showing distinct differences in virulence for rainbow trout and mouse.

Lactococcus garvieae is the causative microbial agent of lactococcosis, an important and damaging fish disease in aquaculture. This bacterium has also been isolated from vegetables, milk, cheese, meat and sausages, from cow and buffalo as a mastitis agent, and even from humans, as an opportunistic infectious agent. In this work pathogenicity experiments were performed in rainbow trout and mouse models with strains isolated from human (L. garvieae HF) and rainbow trout (L. garvieae UNIUDO74; henceforth referred to as 074). The mean LD(50) value in rainbow trout obtained for strain 074 was 2.1 × 10(2) ± 84 per fish. High doses of the bacteria caused specific signs of disease as well as histological alterations in mice. In contrast, strain HF did not prove to be pathogenic either for rainbow trout or for mice. Based on these virulence differences, two suppressive subtractive hybridizations were carried out to identify unique genetic sequences present in L. garvieae HF (SSHI) and L. garvieae 074 (SSHII). Differential dot-blot screening of the subtracted libraries allowed the identification of 26 and 13 putative ORFs specific for L. garvieae HF and L. garvieae 074, respectively. Additionally, a PCR-based screening of 12 of the 26 HF-specific putative ORFs and the 13 074-specific ones was conducted to identify their presence/absence in 25 L. garvieae strains isolated from different origins and geographical areas. This study demonstrates the existence of genetic heterogeneity within L. garvieae isolates and provides a more complete picture of the genetic background of this bacterium.

Primary infective spondylodiscitis caused by Lactococcus garvieae and a review of human L. garvieae infections.

We report the first case of primary infective spondylodiscitis due to Lactococcus garvieae, confirmed by 16S rRNA gene sequencing, in the absence of concomitant endocarditis in a patient with long-standing gastritis on famotidine. He responded to a 6-week course of ampicillin. The gastrointestinal tract is probably the source of infection.

First isolation and characterization of Lactococcus garvieae from Brazilian Nile tilapia, Oreochromis niloticus (L.), and pintado, Pseudoplathystoma corruscans (Spix & Agassiz).

Lactococcus garvieae infection in cultured Nile tilapia, Oreochromis niloticus (L.), and pintado, Pseudoplathystoma corruscans (Spix & Agassiz), from Brazil is reported. The commercial bacterial identification system, Biolog Microlog, confirmed the identity of L. garvieae. Infectivity trials conducted in Nile tilapia using Brazilian Nile tilapia L. garvieae isolates resulted in a median lethal dose-50 of 1.4 x 10(5) colony-forming units (CFU)/fish. This is the first evidence of the presence of this pathogen from Brazilian fish. In addition, this is the first report of L. garvieae infection in either Nile tilapia or pintado. Collectively, this evidence expands the geographical range of fish hosts, number of fish hosts harbouring L. garvieae and carbon source utilization by L. garvieae fish isolates. Furthermore, the Biolog system may be an alternative technique to polymerase chain reaction for the identification of L. garvieae and discrimination between closely related bacterial species.

Susceptibility of selected freshwater fish species to a UK Lactococcus garvieae isolate.

Gram-positive cocci recovered from diseased rainbow trout from a farm in England were characterized by different methods, including pulsed field gel electrophoresis, as virulent Lactococcus garvieae serogroup 2 (pulsotype A1). Groups of rainbow trout were kept at a range of temperatures and injected intraperitoneally (i.p.) with one of the UK isolates, L. garvieae 00021. The 18 degrees C and 16 degrees C groups showed 67% and 28% mortality, respectively, by day 27 post-injection. Fish kept at 14 degrees C or lower were less susceptible (< or =3% mortality). Raising the temperature of all groups to 18 degrees C at day 27 post-injection did not result in recurrence of the disease, even though viable bacteria were recovered from all groups 42 days later. Grayling were highly susceptible, with 65% mortalities when challenged with 200 colony forming unit fish(-1) by i.p. injection and 37% mortalities when exposed to effluent water from tanks containing affected rainbow trout. Other fish species tested, Atlantic salmon, brown trout and seven cyprinid species, were less susceptible. Viable L. garvieae was isolated from the internal organs of all species tested at the end of the trials, suggesting that they may pose a threat as possible carriers to susceptible farmed and wild fish.

Detection of virulence-related genes in Lactococcus garvieae and their expression in response to different conditions.

Lactococcus garvieae has emerged as an important zoonotic pathogen. However, information regarding mechanisms and factors related to its pathogenicity is lacking. In the present study, we investigated the distribution and functionality of genes related to virulence factors in L. garvieae strains isolated from different niches (diseased fish, humans, meat and dairy products, vegetables), using both post-genomic and genotypic analysis. Putative genes encoding hemolysin, fibronectin-binding protein, and penicillin acylase were detected in all analyzed genomes/strains. Their expression was significantly induced by bile salt stress. Putative genes encoding bile salt hydrolase were found in a few strains from dairy and human sources, as well as the mobilizable tet genes. Finally, all genomes possessed a folate gene cluster, in which mutations in the dihydropteroate synthase gene (folP) could be related to sulfonamide resistance. To the best of our knowledge, this is the first study aimed to explore the pathogenic potential of L. garvieae through the analysis of numerous L. garvieae genomes/strains, coming from different sources. This approach allowed the detection of virulence-related genes not yet investigated in the species and the study of their expression after exposure to different environmental stresses. The results obtained suggest a virulence potential in some L. garvieae strains that can be exploited for survival in the human gastrointestinal tract.

Detection of virulence factors of South African Lactococcus garvieae isolated from rainbow trout, Oncorhynchus mykiss (Walbaum).

Lactococcus garvieae is a Gram-positive bacterium that causes mortalities in freshwater and marine fish worldwide and therefore results in severe economic losses in the aquaculture industry. Apart from the apparent integral role of the exopolysaccharide (EPS) capsule in pathogenesis, factors associated with virulence of this bacterium are poorly understood. However, recent studies have indicated that the ability of L. garvieae to cause disease does not depend on the presence of the EPS capsule. Lack of knowledge of virulence factors, pathogenesis and serology of L. garvieae is an impediment to the development of effective typing methods and control measures. This study, therefore, aimed to detect the presence of EPS capsules and other putative virulence factors in South African L. garvieae fish pathogenic isolates and a non-virulent isolate, and to identify possible candidates for subunit vaccine development. No indication of the presence of the EPS capsule was detected by negative staining or amplification of the EPS biosynthesis gene cluster in the virulent isolates or the avirulent strain, discrediting the notion that the EPS capsule is the sole determinant of virulence. However, a set of putative virulence factor genes was detected in all isolates, and candidates for subunit vaccine development (enolase, lactate dehydrogenase phosphoenolpyruvate-protein phosphotransferase) were identified by identification of extracellular proteins of virulent strains.

First report on characterization and pathogenicity study of emerging Lactococcus garvieae infection in farmed rainbow trout, Oncorhynchus mykiss (Walbaum), from India.

"Warm water lactococcosis" in farm-reared rainbow trout, Oncorhynchus mykiss (Walbaum) in the northern Himalayan region of India, caused by bacterium Lactococcus garvieae is described in this study. Nine bacterial isolates were recovered from the organs of haemorrhagic septicaemia rainbow trout and were subjected to biochemical and molecular identification. Cell surface characteristics and virulence of the bacterial isolates are also described. All the nine bacterial isolates had homogenous biochemical characteristics and were Gram-positive, short chains forming (two to eight cells long), α-haemolytic, non-motile ovoid cocci. Partial 16S rDNA nucleotide sequence (~1,400 bp) of current isolates shared 99% identities with the 16S rDNA nucleotide sequence of L. garvieae R421, L. garvieae FMA395 and L. garvieae CAU:1730. The identity of the bacterial isolates was further confirmed by PCR amplification of L. garvieae-specific ~1,100 bp fragment. Transmission electron microscopy (TEM) of one representative isolate, L. garvieae RTCLI04, indicates that the isolated strain lacks thick outer capsule and is of KG+ (non-capsulates) phenotype. An intraperitoneal and intramuscular injection (2.6 × 105 CFU ml-1 ) and also immersion in bacterial suspension @ of 2.6 × 105 CFU ml-1 to healthy rainbow trout juveniles (body weight: 27.5 ± 3.7 g) with L. garvieae RTCLI04 caused 80%, 60% and 10% cumulative mortality in challenged fish, respectively, within 15 days post-infection. The haemorrhagic septicaemic disease was reproduced experimentally. Histopathological examination of organs of experimentally infected fish revealed extensive degenerative and inflammatory changes in eye, kidney, gill and liver. PCR amplification of several putative virulence genes such as haemolysins, adhesins, LPxTG-containing surface proteins and adhesins cluster confirms the virulence of our Indian L. garvieae isolates. To the best of our knowledge, we are reporting for the first time that L. garvieae is associated with fatal haemorrhagic septicaemia in farmed rainbow trout in India.

Native-valve bacterial endocarditis caused by Lactococcus garvieae.

We report a case of definite Lactococcus garvieae native-valve endocarditis. The diagnosis was suspected in a patient presenting with congestive heart failure and found to have Enterococcus hirae bacteremia, with a history of L. garvieae bacteremia 1 month prior. Diagnosis was confirmed by 16S rRNA gene sequencing of the 2 isolates and the demonstration of aortic valve vegetations.

The zoonotic potential of Lactococcus garvieae: An overview on microbiology, epidemiology, virulence factors and relationship with its presence in foods.

Lactococcus garvieae is a relevant worldwide fish pathogen affecting various farmed and wild marine and freshwater species. It has also been isolated from other animals, such as ruminants with subclinical mastitis and pigs with pneumonia. From the early 90s, L. garvieae has been associated with different human infections, mainly endocarditis. During the last five years, human infections by this bacterium appear to be increasing, likely due to the improvement in microbiological methods for bacterial identification and the alertness of this bacterium by physicians. Human L. garvieae infections have been associated with the consumption or the handling of contaminated raw fish or seafood, and recently, a genetic study showed that meat, raw milk and dairy products may also be food sources of human L. garvieae infections. However, the status of L. garvieae as a potential zoonotic bacterium is still controversial to date. In this work, we describe four new human infections by L. garvieae in elderly and inmunocompromised patients, and we show an overview on L. garvieae microbiology, epidemiology, virulence factors and relationship with its presence in foods.

Safety of industrial lactic acid bacteria.

Lactic acid bacteria (LAB) are ubiquitous in fermented and non-fermented foods and are common components of the human commensal microflora. This long history of human exposure and consumption has led to the reasonable conclusion that they are generally safe. Recent attention has also focused on their possible role as probiotic bacteria, promoting beneficial health effects. There have, however, been a number of reports of human infections caused by LAB and these are reviewed. In most cases, the source of the infection was the commensal LAB flora rather than ingested bacteria and the patient had some underlying disease or predisposing condition. Even as opportunistic pathogens, the LAB, with the notable exception of the enterococci, are much less successful than a number of other members of the commensal microflora. The use of new strains for probiotic use is likely to require more detailed evidence for their safety, particularly if the strains have been genetically modified or have been derived from animals. Procedures that have been proposed for assessing the safety of new strains are described.

Induction, characterisation and pathogenicity in rainbow trout Oncorhynchus mykiss (Walbaum) of Lactococcus garvieae L-forms.

Difficulties with induction and cultivation of L-forms, particularly those derived from Gram positive parent cells, have constrained to some degree the ability to evaluate the pathogenicity of these morphotypes. Induction of L-forms of Lactococcus garvieae was undertaken using either charcoal or inactivated horse serum media supplemented with ampicillin, benzylpenicillin or erythromycin, the drug of choice for treatment of infections in rainbow trout, Oncorhynchus mykiss, (Walbaum), and NaCl as an osmotic stabiliser. Lysozyme treated cells could be cultured in a cell wall deficient state using media consisting of charcoal, NaCl and either ampicillin or benzylpenicillin. The influence of some amino acids for induction of L-forms was assessed by disc diffusion and combined interaction. Analysis of variance of colony counts indicated that the amino acids glycine, DL-methionine, L-threonine and L-serine (P<0.03), and the presence of charcoal were beneficial and that inactivated horse serum was detrimental to L-form development. Electron microscopy revealed that the cell wall of L-forms was missing and this cell had a greatly expanded volume compared to parent cells. Electrophoresis of whole cell proteins showed some variation of electropherotype between parent and L-form cells. L-forms expressed greater quantities of proteins with molecular mass of 36 and 66 kDa and parent cells contained greater quantities of proteins of molecular mass 29, 43 and 60 kDa. Additional proteins of molecular mass 32, 44 and 53 kDa were present in L-form extracts, and in parent cells of 34, 38, 40, 42, 85 and 123 kDa which may represent cell wall associated proteins or alterations in expression due to different growth rates. Intraperitoneal challenge of rainbow trout with L-forms failed to produce overt infection even in immune-suppressed fish, but L-forms were shown by indirect fluorescent antibody test to remain inkidney tissue. Fish were susceptible to infection when challenged with parent cells of L. garvieae.

Technological performance of several Lactococcus and Enterococcus strains of dairy origin in milk.

Thirty-nine strains (29 Lactococcus strains and 10 Enterococcus strains) isolated from five different artisanal cheeses were subjected to technological characterization. Several strains of lactococci and enterococci produced lactic acid at a rate and final concentration suitable for large-scale cheesemaking. However, extensive phenotypic differences between strains were encountered. Proteolytic activity correlated quite well with acidification for all strains, with the more proteolytic strains being the best acidifiers. The strains were also assayed for the production of organic acids and volatile components in milk. With few exceptions, enterococcus isolates produced more formic acid and acetic acid than did lactococcus isolates. The volatile-compound profiles obtained were rather simple. The main volatile component produced by most strains was ethanol. Since the inclusion of enterococcus strains in food systems is controversial, tests were also performed to detect recognized determinants of virulence (namely, aggregation, gelatinase and hemolysin production, and antibiotic resistance). Aggregation in both liquid and solid media was observed only for two Enterococcus durans isolates. None of the strains studied produced gelatinase under the conditions of the assay. Beta-hemolysin activity was clearly detected in two Enterococcus faecalis strains, which also produced the biogenic amine tyramine from tyrosine in a laboratory medium. In general, the enterococcus strains were more resistant to the antibiotics assayed than were the lactococcus strains. Both the minimum inhibitory concentration (MIC) modes and the highest MIC values were consistently higher for the enterococci. Nevertheless, particular strains of lactococci were resistant to antibiotics such as bacitracin, cephalothin, clindamycin, streptomycin, and tetracycline.

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

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.