Phenotypic characteristics of Streptococcus iniae and Streptococcus parauberis isolated from olive flounder (Paralichthys olivaceus)

The etiological agents of streptococcosis were isolated from diseased olive flounder collected on the Jeju island of Korea. A total of 151 bacterial isolates were collected between 2003 and 2006. The isolates were examined using various phenotypic and proteomic analyses, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, and glycoprotein assays. In addition, isolates were grown on blood agar to assess hemolytic activity, and biochemical assays were performed using the API20 Strep kit. Our results revealed that all isolates were nonmotile, Gram-positive cocci that displayed negative catalase and oxidase activities. Multiplex PCR assays revealed that 43% and 57% of the isolates were Streptococcus iniae and Streptococcus parauberis , respectively. These results were consistent with those of the SDS-PAGE and immunoblot analyses using whole-cell lysates of bacterial isolates. Significant differences were observed with respect to the Voges–Proskauer, pyrrodonyl arylamidase, alkaline phosphatase, and hemolytic activities of the S. iniae and S. parauberis isolates. Isolates of S. iniae displayed uniform profiles in the immunoblot and glycoprotein assays; however, immunoblot assays of S. parauberis isolates (using a chicken IgY antibody raised against a homologous isolate) revealed three distinct antigenic profiles. Our findings suggest that S. parauberis and S. iniae are endemic pathogens responsible for the development of streptococcosis in olive flounder.     

Application of immunoproteomics in developing a Streptococcus iniae vaccine for olive flounder (Paralichthys olivaceus)

Streptococcus iniae is the major etiological agent of streptococcosis, which is responsible for hemorrhagic septicemia in fish, particularly olive flounder (Paralichthys olivaceus). In the present study, we sought to understand the pathogenicity and immunogenicity of S. iniae in order to develop a vaccine for streptococcosis. Immunoproteomics, a technique involving two-dimensional gel electrophoresis (2-DE) followed by immunoblotting, was employed to investigate the pathogenicity and immunogenicity of two S. iniae isolates, Jeju-13 and Jeju-45, in olive flounder. The virulence of Jeju-13 was moderate whereas that of Jeju-45 was high. A vaccination trial with formalin-killed Jeju-45 demonstrated relatively low protection against the homologous isolate compared with the heterologous isolate. A significant difference in the secretion of extracellular products (ECPs) was noticed between the two S. iniae isolates. ECP antigens were highly immunogenic compared to those from whole cell lysates as determined by 2-DE immunoblot assay of Jeju-13 and Jeju-45 anti-sera collected from post-challenge survival fish. Furthermore, there were differences in the appearance of antigenic spots on 2-DE immunoblot profiles of ECPs of the respective sera. Interestingly, the mixture of killed-cells and concentrated ECPs from Jeju-45 led to significant protection against the homologous isolate of S. iniae in olive flounder. The present study demonstrates the usefulness of immunoproteomics in understanding the pathogenicity of S. iniae to aid the development of a vaccine for fish streptococcosis.     

Streptococcus iniae capsule impairs phagocytic clearance and contributes to virulence in fish

Surface capsular polysaccharides play a critical role in protecting several pathogenic microbes against innate host defenses during infection. Little is known about virulence mechanisms of the fish pathogen Streptococcus iniae, though indirect evidence suggests that capsule could represent an important factor. The putative S. iniae capsule operon contains a homologue of the cpsD gene, which is required for capsule polymerization and export in group B Streptococcus and Streptococcus pneumoniae. To elucidate the role of capsule in the S. iniae infectious process, we deleted cpsD from the genomes of two virulent S. iniae strains by allelic exchange mutagenesis to generate the isogenic capsule-deficient DeltacpsD strains. Compared to wild-type S. iniae, the DeltacpsD mutants had a predicted reduction in buoyancy and cell surface negative charge. Transmission electron microscopy confirmed a decrease in the abundance of extracellular capsular polysaccharide. Gas-liquid chromatography-mass spectrometry analysis of the S. iniae extracellular polysaccharides showed the presence of l-fucose, d-mannose, d-galactose, d-glucose, d-glucuronic acid, N-acetyl-d-galactosamine, and N-acetyl-d-glucosamine, and all except mannose were reduced in concentration in the isogenic mutant. The DeltacpsD mutants were highly attenuated in vivo in a hybrid striped bass infection challenge despite being more adherent and invasive to fish epithelial cells and more resistant to cationic antimicrobial peptides than wild-type S. iniae. Increased susceptibility of the S. iniae DeltacpsD mutants to phagocytic killing in whole fish blood and by a fish macrophage cell line confirmed the role of capsule in virulence and highlighted its antiphagocytic function. In summary, we report a genetically defined study on the role of capsule in S. iniae virulence and provide preliminary analysis of S. iniae capsular polysaccharide sugar components.     

Identification of Streptococcus iniae by commercial bacterial identification systems

The fish pathogen Streptococcus iniae cannot be identified by most commercial bacterial identification systems. The results presented here indicate that over 70% of our S. iniae isolates have been identified using the Biolog(R) GP microplate panels and Microlog(R) database. The isolates were confirmed as S. iniae by specific PCR methods and have been found to conform to the result obtained with the type strain S. iniae ATCC 29178.     

Duration of protective antibodies and correlation with survival in Nile tilapia Oreochromis niloticus following Streptococcus agalactiae vaccination

Streptococcus agalactiae is a major piscine pathogen that causes significant morbidity and mortality among numerous species of freshwater, estuarine and marine fishes. Considering the economic importance of fishes susceptible to S. agalactiae throughout the world, an efficacious S. agalactiae vaccine was developed using an extracellular product (ECP) fraction and formalin-killed whole cells of S. agalactiae. A vaccine study was conducted by intraperitoneal (i.p.) injection in Nile tilapia Oreochromis niloticus in order to determine the duration of protection and its correlation to antibodies specific for this pathogen. After 47, 90 or 180 d post-vaccination (DPV), the fish were i.p. challenged with approximately 2.0 x 10(4) S. agalactiae colony-forming units (CFU) fish(-1) to determine the duration of protective immunity. The percent survival in control fish i.p.-injected with sterile TSB was 16,16, and 4% on 47, 90 and 180 DPV, respectively, while the percent survival for the vaccinated fish was 67, 62 and 49%, respectively. The specific mean antibody concentration of the vaccinated fish was significantly higher than that of the control fish, with significant correlation between the ELISA optical density (OD) and protection. These results indicate that the specific antibody has a correlation with protection following immunization with the S. agalactiae vaccine and that the vaccine can confer protection against S. agalactiae up to 180 DPV.     

Pathological changes in cultured channel catfish Ictalurus punctatus spontaneously infected with Streptococcus iniae

The pathological changes present in channel catfish Ictalurus punctatus spontaneously infected by Streptococcus iniae are described. The most consistent gross findings were marked petechial hemorrhages of the skin and congestion of internal organs, particularly the liver, spleen and kidneys. Other features included color fading at the edge of fin rays, enteritis and ascites. Histological examination showed oedema, degeneration and necrotic changes in many organs. Further, hepatitis, splenitis, interstitial nephritis, and meningitis with numerous monocyte and neutrocyte infiltrates were evident. Intact S. iniae cells were seen in macrophages. Apparently, spontaneous S. iniae infection caused acute septicaemia in channel catfish. This is the first histopathological report on channel catfish naturally infected with S. iniae.     

Identification of lactoferrin-binding proteins in Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus agalactiae isolated from cows with mastitis

Three strains of Streptococcus dysgalactiae subsp. dysgalactiae (S. dysgalactiae) and five strains of Streptococcus agalactiae were used to identify lactoferrin-binding proteins (LBPs). LBPs from extracted surface proteins were detected by polyacrylamide gel electrophoresis and Western blotting. All strains of S. dysgalactiae evaluated had 52- and 74-kDa protein bands. All strains of S. agalactiae evaluated had 52-, 70- and 110-kDa protein bands. In addition, a 45-kDa band was detected in two of five S. agalactiae strains evaluated. This study demonstrated that S. dysgalactiae and S. agalactiae of bovine origin contain two and three major LBPs, respectively.     

海南罗非鱼无乳链球菌分离鉴定及其特性研究

从海南省患暴发性疾病的罗非鱼(Tilapia)上分离出1株细菌HNLFYL4,对分离菌株进行了鉴定及致病性和药物敏感性研究.通过形态学观察和生理生化鉴定,结果显示,分离菌株为无乳链球菌(Streptococcus agalactiae).对分离菌株的16S rRNA基因进行PCR扩增和测序,所得序列已登录到GenBank,登录号为HQ645983,与GenBank中收录的链球菌16S rRNA 基因进行比对并构建系统进化树,结果表明,分离菌株的16S rRNA基因序列与无乳链球菌同源性高达100%,进一步确定分离菌株为无乳链球菌.人工感染显示分离菌株对小白鼠和罗非鱼均具有致病性,对小白鼠的LD50为1.0×104 CFU/mL,对体重为500g±20g的罗非鱼的LD50为1.729×109CFU/mL.分离菌株对氯霉素、青霉素G、呋喃妥因等敏感,对丁胺卡那、链霉素、卡那霉素等不敏感.     

Red drum Sciaenops ocellatus mortalities associated with Streptococcus iniae infection.

We isolated for the first time Streptococcus iniae strains associated with diseased marine fish. Diseased red drum Sciaenops ocellatus were lethargic, and presented external signs (exophthalmia and loss of orientation) resembling those of freshwater fish infected by S. iniae. Skin lesions, extending to a necrotizing myositis, were typical of S. iniae infection of red drum. Histopathological findings indicate that S. iniae infection in red drum produces a chronic disease with systemic involvement characterized by multiple necrotic foci. Molecular epidemiology (RFLP [restriction fragment length polymorphism] ribotyping) revealed that 2 different ribotypes were involved in a single outbreak. The first is the EcoRI 'Israeli' trout and tilapine ribotype (Hind III type a strains), while the second is the EcoRI 'American' ribotype (Hind III type b strains), typical of tilapines farmed in Texas and Idaho.     

Electrotransformation of Streptococcus agalactiae with plasmid DNA

Abstract A protocol for efficient electrotransformation of Streptococcus agalactiae (group B streptococcus) Lancefield's strain O90R (NTCT 9993) (an unencapsulated derivative of type Ia strain O90) was developed. The Escherichia coli - Streptococcus shuttle vector pDP28 (7.8 kb) carrying the ermB gene for resistance to erythromycin was used as donor DNA. Frozen 'electrocompetent' cells were prepared by repeated washes in 10% glycerol. A 50-μl aliquot containing about 5×10⁹ colony forming units of bacteria was subjected to the electric pulse. Optimal conditions for electrotransformation were determined using different media, harvesting cells at different points of the growth curve, and using different field strengths. The dose-response curve for transformation of S. agalactiae with pDP28 showed one-hit kinetics as donor DNA varied between 0.01 and 3 μg. The efficiency of electrotransformation for this range of amounts of donor DNA was 1.2×10⁴ cfu μg⁻¹. The transformation frequencies obtained with this electroporation protocol are high enough to allow both subcloning and shotgun cloning of streptococcal DNA in S. agalactiae .     

Streptococcus iniae type II infections in rainbow trout Oncorhynchus mykiss

Clinical and pathological findings (anorexia, hemorrhage, lethargy, loss of orientation and exophthalmia) indicated that Streptococcus iniae type II is responsible for a fatal disease in rainbow trout. Histopathological findings revealed that S. iniae type II produces a systemic disease, including a diffuse necrotizing myositis. The distribution of viable bacteria in infected tissues substantiated the pathological findings, confirming that S. iniae type II is responsible for a generalized septic disease of rainbow trout.     

Cloning and analysis of the L-lactate utilization genes from Streptococcus iniae.

The presence of lactate oxidase was examined in eight Streptococcus species and some related species of bacteria. A clone (pGR002) was isolated from a genomic library of Streptococcus iniae generated in Escherichia coli, containing a DNA fragment spanning two genes designated lctO and lctP. We show that these genes are likely to be involved in the L-lactic acid aerobic metabolism of this organism. This DNA fragment has been sequenced and characterized. A comparison of the deduced amino acid sequence of LctP protein demonstrated that the protein had significant homology with the L-lactate permeases of other bacteria. The amino acid sequence of the LctO protein of S. iniae also showed a strong homology to L-lactate oxidase from Aerococcus viridans and some NAD-independent lactate dehydrogenases, all belonging to the family of flavin mononucleotide-dependent alpha-hydroxyacid-oxidizing enzymes. Biochemical assays of the gene products confirm the identity of the genes from the isolated DNA fragment and reveal a possible role for the lactate oxidase from S. iniae. This lactate oxidase is discussed in relation to the growth of the organism in response to carbon source availability.     

Strain variation and geographic endemism in Streptococcus iniae

Twenty-six Israeli isolates of Streptococcus iniae from both marine and fresh/brackish water sources were compared with each other and with 9 foreign isolates. All the isolates were tentatively identified according to their biochemical profile. Direct sequencing of approximately 600 bp PCR products of the 16S rDNA confirmed their identification as S. iniae at the molecular level and revealed a new (one-nucleotide) variant among Israeli isolates, in addition to 2 variants that had been previously reported. Strain variation was further examined by subjecting the isolates to randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analyses. The RAPD method allowed separation of the isolates into only 2 groups, one including 5 Israeli fresh/brackish water isolates and one including all the other isolates. The AFLP method grouped the Israeli marine isolates into one homogeneous cluster, although they had been obtained in different years (1995 to 2001) from different species of fish, and from wild (Red Sea) as well as cultured (both Mediterranean and Red Sea) sources. The Israeli fresh/brackish water isolates and foreign isolates separated into distinct entities that clustered at generally high degrees of similarity. The distance between the clusters of the Israeli marine and fresh/brackish water isolates indicates that the S. iniae streptococcosis that has been afflicting the aquaculture industries in the 2 environments in recent years was caused by distinct strains. AFLP showed superior discriminative properties over RAPD in detecting intraspecific variation and proved to be an important tool for the characterization of S. iniae. A correlation between strain variation and geographic endemism was established.     

Streptococcus iniae infections in Red Sea cage-cultured and wild fishes

Streptococcus iniae was isolated from 2 moribund wild Red Sea fishes, Pomadasys stridens (Pomadasyidae) and Synodus variegatus (Synodontidae), both collected in shallow waters along the Israeli coast of the Gulf of Eilat. The site is approximately 2 km from a mariculture cage farm in which streptococcal infections were diagnosed in previous years in the red drum Sciaenops ocellatus. This is the first report of S. iniae in Red Sea fishes. Biochemical and molecular similarities between the isolates from cultured fishes and those from the wild specimens suggest that a single strain is involved, and that 'amplification' and dispersal of this pathogen from captive to feral fishes have occurred. At the molecular level, the pathogen is different from the S. iniae isolates that have been afflicting the Israeli freshwater aquaculture in recent years. Although S. iniae prevalence in the wild fish populations of the area remains to be determined, the northernmost region of the Gulf of Eilat, virtually landlocked and with generally calm seas and weak currents, seems to be particularly vulnerable to the impact of diseases that develop in this mariculture system.     

Streptococcus iniae, a bacterial infection in barramundi Lates calcarifer.

The cause of ongoing mortality in barramundi Lates calcarifer (Bloch) in seawater culture was identified as Streptococcus iniae by biochemical and physiological tests. This is the first published record of this bacterial species in Australia and the first confirmed report of S. iniae causing mortality in barramundi. The bacterium was highly pathogenic for barramundi when challenged by bath exposure. The pathogen was found to have a LD50 of 2.5 x 10(5) and 3.2 x 10(4) colony-forming units at 48 h and 10 d respectively. Experimental challenge of barramundi resulted in high levels of mortality (> 40%) within a 48 h period. Ten days after the challenge, S. iniae could not be isolated from kidney, spleen, liver or eye of surviving fish. However, the organism was easily isolated from the brain of both moribund and healthy fish, indicating that barramundi can carry the bacterium asymptomatically.     

Aerobic Metabolism of Streptococcus agalactiae

Streptococcus agalactiae cultures possess an aerobic pathway for glucose oxidation that is strongly inhibited by cyanide. The products of glucose oxidation by aerobically grown cells of S. agalactiae 50 are lactic and acetic acids, acetylmethylcarbinol, and carbon dioxide. Glucose degradation products by aerobically grown cells, as percentage of glucose carbon, were 52 to 61% lactic acid, 20 to 23% acetic acid, 5.5 to 6.5% acetylmethylcarbinol, and 14 to 16% carbon dioxide. There was no evidence for a pentose cycle or a tricarboxylic acid cycle. Crude cell-free extracts of S. agalactiae 50 possessed a strong reduced nicotinamide adenine dinucleotide (NADH(2)) oxidase that is also cyanide-sensitive. Dialysis or ultrafiltration of the crude, cell-free extract resulted in loss of NADH(2) oxidase activity. Oxidase activity was restored to the inactive extract by addition of the ultrafiltrate or by addition of menadione or K(3)Fe(CN)(6). Noncytochrome iron-containing pigments were present in cell-free extracts of S. agalactiae. The possible participation of these pigments in the respiration of S. agalactiae is presently being studied.     

Selective agars for the isolation of Streptococcus iniae from Japanese flounder, Paralichthys olivaceus, and its cultural environment

Two kinds of selective agar were developed for the isolation of Streptococcus iniae, the causal agent of streptococcosis, from Japanese flounder (Paralichthys olivaceus) and from culture tanks in flounder farms. The selective agars were heart infusion agar with added thallium acetate and oxlinic acid (TAOA), and colistin sulphate and oxolinic acid (CSOA). For samples containing various bacterial flora, selective agars were supplemented with defibrinated horse blood in order to distinguish beta-haemolytic colonies of Strep. iniae. Streptococcus iniae was quantitatively isolated from the brain and kidney of diseased flounders in pure culture. Two-thirds of isolates picked up from selective blood agars inoculated with intestinal samples were identified as Strep. iniae. The bacterial colony numbers of deposits and water from culture tanks on selective blood agars were about 10-10(5) times smaller than those on control heart infusion agar; Strep. iniae was isolated from few deposit and water samples.     

Partial two-dimensional gel electrophoresis (2-DE) maps of Streptococcus iniae ATCC29178 and Lactococcus garvieae KG9408

To examine the proteomes of 2 important causative agents of fish streptococcosis, Streptococcus iniae ATCC29178 and Lactococcus garvieae KG9408, we used 2-dimensional gel electrophoresis (2-DE) followed by mass spectrometry to generate 2-DE maps of these type strains. Silver-stained 2-DE gels of S. iniae ATCC29178 and L. garvieae KG9408 revealed approximately 320 and 300 spots, respectively, and immobilized pH gradient strips (13 cm, pH 4 to 7) revealed that the majority of the detected spots were concentrated in the pH range of 4.5 to 5.5. The spots were randomly selected from the 2-DE profiles and identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time of flight mass spectrometry. The majority of the identified proteins were functionally related to energy and carbohydrate metabolism (e.g. enolase ATPase, glyceraldehyde-3-phosphate dehydrogenase) or translation and translocation (e.g. elongation factor G, elongation factor Tu, DNA-directed RNA polymerase alpha chain). These data, along with our partial 2-DE maps of S. iniae ATCC29178 and L. garvieae KG9408, may help suggest antigenic proteins for the development of effective diagnostic tools and vaccines against S. iniae and L. garvieae.     

Identification of Streptococcus Agalactiae by the Fluorescent Antibody

A fluorescent antibody technic for identification of Streptococcus agalactiae is described. Smears from colonies on blood agar plates were tested. A pool of conjugates to four different Streptococcus agalactiae antisera stained all the 80 Streptococcus agalactiae strains investigated. The pool proved superior to individual conjugates. Also strains of groups C and G were stained by the Streptococcus agalactiae conjugates. These, however, could be differentiated from Streptococcus agalactiae strains by examination of the controls because the conjugates of antisera to some group C strains stained group C and G strains but not Streptococcus agalactiae strains.