In Vitro Study of Adherence,Invasion, and Persistence of Streptococcus iniae in Fibroblastic-Like Fish Cell Line SAF-1

Abstract

Streptococcus iniae is a major fish pathogen producing invasive infections that result in economic losses in aquaculture. Gentamicin protection assays were used to investigate the ability of different S. iniae strains to invade and adhere to fibroblastic-like fish cell line SAF-1. All strains tested were detected intracellularly using both techniques, with variable internalization degrees between strains. The experiments carried out at 4°C demonstrated that active cell metabolism is necessary for bacterial internalization. Intracellular bacteria were detected for up to 3 d with a round morphology and were stained with 4′,6-diamidino-2-phenylindole (DAPI), indicating that some bacterial cells may remain viable inside SAF-1 cells. Our in vitro findings indicate that S. iniae is capable of adhering, entering, and surviving within fibroblastic cells, which may be important for the persistence and establishment of a carrier state.

Received July 8, 2011; accepted February 12, 2012     

Strain-associated virulence factors of Streptococcus iniae in hybrid-striped bass

Streptococcus iniae is a major fish pathogen producing invasive infections that result in economic losses in aquaculture. Development of in vitro models of S. iniae virulence may provide insight to the pathogenesis of infection in vivo. Three S. iniae strains (K288, 94-426, and 29178) were tested for virulence in a hybrid-striped bass (HSB) model using intraperitoneal injection. S. iniae strains K288 and 94-426 caused high levels of mortality in HSB (lethal dose 2x10(5)CFU) while strain 29178 was avirulent even upon IP challenge with 1000-fold higher inocula. In vitro assays were developed to test for the presence of characteristics previously associated with virulence in other species of pathogenic Streptococcus in animals and humans. In vitro differences relevant to virulence were not detected for beta-hemolysin activity, sensitivity to antimicrobial peptides, or adherence and invasion of epithelial cell layers. However, in whole-blood killing assays, the pathogenic strains were resistant to blood clearance, while 29178 was cleared (P<0.001) and more sensitive to complement (P<0.001). The avirulent strain 29178 was most efficiently phagocytosed and was most susceptible to intracellular killing (P<0.01) by the carp leukocyte cell line (CLC). When exposed to reactive oxygen species, strain 29178 was most susceptible. When the oxidative burst of CLC cells was inhibited, intracellular survival of 29178 was rescued fivefold, while no significant enhancement in survival of K288 or 94-426 was detected. Our results indicate that resistance to phagocytosis, oxidative killing, and associated phagocytic clearance is a significant factor in S. iniae virulence.     

Antiproliferative effects and apoptosis induction by probiotic cytoplasmic extracts in fish cell lines

Probiotic bacteria are known to exert a wide range of beneficial effects on their animal hosts. Control of intestinal homeostasis, inflammation suppression and a reduction in the incidence of cancer all rely on the antiproliferative potential of probiotics. In this paper, we assess the antiproliferative activity of probiotics in two teleost fish cell lines SAF-1, a fibroblast cell line and EPC, an epithelioma from carp. Cells were grown in the presence of cytoplasmic extracts obtained from two bacterial strains, Lactobacillus delbrüeckii subsp. lactis (LDL) and 51M6. Proliferation and apoptosis were measured after 4, 24, 48 or 72h in culture by the crystal violet or by double staining flow cytometry assays, respectively. Generally, LDL had stronger effects on cell growth than 51M6. Moreover, SAF-1 cells were more susceptible to growth inhibition than EPC cells. Apoptosis took place following growth inhibition, especially when LDL extracts were used. The results are discussed in terms of the biological significance of probiotic bacteria that naturally occur on the fish mucosal surfaces with an emphasis on how dose and species specificity may be determinant factors.     

Streptococcus iniae: an aquatic pathogen of global veterinary significance and a challenging candidate for reliable vaccination

Streptococcus iniae has become one the most serious aquatic pathogens in the last decade causing high losses in farmed marine and freshwater finfish in warmer regions. Although first identified in 1976 from a captive Amazon freshwater dolphin, from which it derives its name, disease outbreaks had most likely been occurring for several decades in marine aquaculture in Japan. S. iniae is globally distributed throughout warm water finfish aquaculture. In common with other encapsulated beta-haemolytic streptococci and in direct contradiction to the phenomenal success story of bacterial vaccines in finfish aquaculture, control of S. iniae by vaccination has met with limited success. Thus, antibiotic usage is the current practice for reducing mortality and consequent economic loss. Vaccine failure appears to result in part from serotypic variation and, whilst 2 serotypes have been named, variation would appear to be more complex. S. iniae also has zoonotic potential, with human infections identified in the USA, Canada, and throughout Asia. In humans, infection is clearly opportunistic with all cases to date associated with direct infection of puncture wounds during preparation of contaminated fish, and generally in elderly or immunocompromised individuals. Significant progress has been made in terms of research into pathogenic mechanisms of S. iniae, with recent research elucidating the role of capsule in virulence for fish through antiopsonic activity. In light of this recent coverage in the literature, the present review centres on areas of direct veterinary interest including identification, epidemiology, therapy and prevention in farmed finfish. Clearly as the prevalence of S. iniae and associated economic losses continue to increase, further work towards developing a reliable vaccine is essential. This would appear to require a much better understanding of cell-surface variability amongst S. iniae isolates.     

Immunoproteomic analysis of the antibody response obtained in Nile tilapia following vaccination with a Streptococcus iniae vaccine

Streptococcus iniae is one of the most economically important Gram-positive pathogens in cultured fish species worldwide. The USDA-ARS Aquatic Animal Health Research Unit developed a modified (contains concentrated culture supernatant) S. iniae bacterin that has been demonstrated to be efficacious, and protection is mediated by specific anti-S. iniae antibodies. Although effective, the specific vaccine components important for efficacy are not known. In the present study, an immunoproteomic approach was utilized to identify whole-cell lysate proteins of S. iniae that stimulated specific antibody production in Nile tilapia (Oreochromis niloticus) following vaccination. Groups of tilapia were vaccinated by intraperitoneal injection with the modified S. iniae bacterin or were mock-vaccinated, and at 30 d post-vaccination sera samples were obtained from individual fish. Vaccination of tilapia with the S. iniae vaccine stimulated significantly elevated specific antibody responses against proteins of the bacterium and passive immunization of tilapia with this serum demonstrated the antibodies were highly protective. Whole-cell lysate proteins of S. iniae were separated by 2D-PAGE and were probed with a pooled serum sample from vaccinated tilapia. A total of eleven unique immunogenic proteins were positively identified by mass spectrometry. Based on research conducted on homologous proteins in other Streptococcus spp., antibodies specific for three of the identified proteins, enolase, glyceraldehyde-3-phosphate dehydrogenase, and fructose-bisphosphate aldolase, are likely involved in protection from streptococcosis caused by S. iniae.     

PCR detection and PFGE genotype analyses of streptococcal clinical isolates from tilapia in China

Large-scale streptococcal outbreaks occurred continuously in tilapia farms of China from 2009 to 2011. The objective of this study was to characterize the prevalent strains of tilapia streptococci from the main cultured areas of China through species specific PCR and pulse field gel electrophoresis (PFGE). A total of 105 prevalent strains were isolated from Guangdong, Guangxi, Hainan and Fujian provinces between 2006 and 2011, 85 of which were identified as Streptococcus agalactiae while the rest were all identified as Streptococcus iniae. The prevalent stains in 2006 and 2007 were S. iniae (94.7%, 18/19), with S. agalactiae account for only 5.3% (1/19); The prevalent strains in 2009 and 2011 however changed to S. agalactiae (97.7%, 84/86), with only 2.3% (2/86) was S. iniae. Of these 105 strains, a total of 13 PFGE types (A-M) were characterized, among which D, F, G and K genotypes were predominant, accounting for 81.90% (86/105). The cluster analysis of PFGE electropherograms separated S. iniae and S. agalactiae to two distinctive branches, 20 strains of S. iniae exhibiting 3 types of PFGE band patterns with a similarity of 94.8-100%, and the 85 strains of S. agalactiae producing 10 types of PFGE band patterns with a similarity between 48.4% and 100%. Data suggested that the prevalent strains of tilapia streptococci in China have shifted from the former (before 2008) dominant strains of S. iniae to the current (2009-2011) dominant strains of S. agalactiae. Moreover, PFGE genotypes of the prevalent strains demonstrated geographic differences and temporal changes.     

Prevalence of Streptococcus iniae in tilapia, hybrid striped bass, and channel catfish on commercial fish farms in the United States

OBJECTIVE: To determine the prevalence of Streptococcus iniae in tilapia (Oreochromis spp), hybrid striped bass (Morone chrysops X M saxatilis), and channel catfish (Ictalurus punctatus) on commercial fish farms in the United States. ANIMALS: 1,543 fish (970 tilapia, 415 hybrid striped bass, and 158 channel catfish). PROCEDURES: The dry-swab technique was used for collection of specimens for streptococcal isolation. Specimens were shipped by overnight delivery and processed by use of standard bacteriologic techniques. RESULTS: Streptococcus iniae was not isolated from market-size channel catfish. Prevalence in tilapia and hybrid striped bass was 37 of 970 (3.81%) and 30 of 415 (7.23%), respectively. Prevalence by farm ranged from 0.0 to 27.4% for tilapia and 0.0 to 21.6% for hybrid striped bass. In tilapia, prevalence was lowest in market-size and nursery fish (4 of 239 [1.67%] and 3 of 339 [0.88%], respectively), with an increase in prevalence for fish in the grow-out stage (30 of 337 [7.96%]). For hybrid striped bass, prevalence was lowest in nursery and market-size fish (3 of 96 [3.12%] and 1 of 47 [2.12%], respectively) and highest in fish in the grow-out stage (26 of 272 [9.56%]). Prevalence in market-size tilapia and hybrid striped bass was 5 of 286 (1.75%). CONCLUSIONS AND CLINICAL RELEVANCE: Results of this study do not support the contention that S iniae is a serious public health threat associated with commercially raised fish; rather, it represents a limited risk for older or immunocompromised people who incur puncture wounds while handling and preparing fish.     

Effectiveness of aquaflor (50% florfenicol) to control mortality associated with Streptococcus iniae in freshwater-reared subadult sunshine bass

We conducted a field trial to evaluate the effectiveness of Aquaflor (50% florfenicol) for controlling mortality associated with Streptococcus iniae in freshwater-reared subadult sunshine bass (female white bass Morone chrysops X male striped bass M. saxatilis). Bacterial samples collected from moribund fish representing a reference population were presumptively identified microbiologically and were later confirmed to be S. iniae by biochemical characterization and polymerase chain reaction. The trial comprised a 1-d acclimation period, 10-d treatment period, and 14-d posttreatment period. During the treatment period, Aquaflor-medicated feed was administered to treated tanks (N = 3) at a target dose of 10 mg of florfenicol x kg of fish(-1) x d(-1), and nonmedicated feed was administered to control tanks (N = 3). At the end of the posttreatment period, mean (+/- SD) cumulative mortality in treated tanks (9 +/- 11%) was significantly (P = 0.040) less than that in control tanks (52 +/- 13%). Analysis of medicated feed samples revealed that treated tanks had received an actual dose of 8.3 mg florfenicol x kg fish(-1) x d(-1) (83% of target). No florfenicol was detected in control feed samples. Although the actual florfenicol dose administered to treated tanks was less than the target dose, the trial was accepted by the U.S. Food and Drug Administration Center for Veterinary Medicine as demonstrating the efficacy of Aquaflor to control mortality associated with S. iniae in cultured sunshine bass populations.     

Development of a PCR assay for Streptococcus iniae based on the lactate oxidase (lctO) gene with potential diagnostic value

Streptococcus iniae is a well-known pathogen of both fish and humans that is difficult to identify by conventional biochemical tests. A PCR assay based on the lactate oxidase (lctO) gene of S. iniae was developed for the rapid and specific detection and identification of this pathogen from different sources. The PCR assay had a detection limit of 62-31 cells, and 25 pg of DNA per PCR reaction mixture. The PCR was also effective in detecting the bacterium from inoculated tissue homogenates, suggesting its potential use for a rapid and accurate diagnosis of S. iniae infections.     

Binding of host glycosaminoglycans and milk proteins: possible role in the pathogenesis of Streptococcus uberis mastitis

The role of indirect binding of host proteins through glycosaminoglycans (GAGs) on adherence and internalization of Streptococcus uberis to bovine mammary epithelial cells was evaluated. Preincubation of S. uberis with GAGs followed by incubation with fetal bovine serum (FBS), bovine milk or milk proteins resulted in greater adherence to and internalization of S. uberis into mammary epithelial cells than observed in untreated controls. Highest values were detected, when final incubation was done with milk. Greater adherence to and internalization into mammary epithelial cells were observed when heparin sulfate (HEP) and milk were used compared with any other GAG and FBS. When individual milk proteins were used, greatest adherence and internalization were observed when S. uberis strains were pretreated with HEP followed by treatment with beta-casein. The findings of this study illustrate a pathogenic strategy of S. uberis that may occur during the very early stages of infection.     

Comparison of growth phase on Salmonella enterica serovar Typhimurium invasion in an epithelial cell line (IPEC J2) and mucosal explants from porcine small intestine

Salmonella Typhimurium DT104 is a zoonotic enteropathogen of increasing concern for human health. In this study, the influence of growth phase on invasiveness of a S. Typhimurium DT104 field isolate and two reference strains (SL1344 and ATCC 14028) was compared in IPEC J2 cells and mucosal explants from porcine ileum. Internalized bacteria were quantified by a gentamicin resistance assay. After 90 min of exposure to the apical aspect of epithelial monolayers or luminal surface of explants, internalization of all S. Typhimurium strains in mid-logarithmic phase of bacterial growth was comparable. Internalization of stationary phase bacteria was reduced relative to log phase bacteria, with DT104 exhibiting the greatest decrease. Growth phase-related differences in S. Typhimurium invasion are similar in porcine intestinal epithelial cells and mucosal explants, but may be greater in multidrug-resistant strains.     

Identification and expression profiles of multiple genes in Nile tilapia in response to bacterial infections

To understand the molecular mechanisms involved in response of Nile tilapia (Oreochromis niloticus) to bacterial infection, suppression subtractive cDNA hybridization technique was used to identify upregulated genes in the posterior kidney of Nile tilapia at 6h post infection with Aeromonas hydrophila. A total of 31 unique expressed sequence tags (ESTs) were identified from 192 clones of the subtractive cDNA library. Quantitative PCR revealed that nine of the 31 ESTs were significantly (p<0.05) upregulated in Nile tilapia at 6h post infection with A. hydrophila at an injection dose of 10(5)CFU per fish (≈ 20% mortality). Of the nine upregulated genes, four were also significantly (p<0.05) induced in Nile tilapia at 6h post infection with A. hydrophila at an injection dose of 10(6)CFU per fish (≈ 60% mortality). Of the four genes induced by A. hydrophila at both injection doses, three were also significantly (p<0.05) upregulated in Nile tilapia at 6h post infection with Streptococcus iniae at doses of 10(6) and at 10(5)CFU per fish (≈ 70% and ≈ 30% mortality, respectively). The three genes induced by both bacteria included EST 2A05 (similar to adenylate kinase domain containing protein 1), EST 2G11 (unknown protein, shared similarity with Salmo salar IgH locus B genomic sequence with e value of 0.02), and EST 2H04 (unknown protein). Significant upregulation of these genes in Nile tilapia following bacterial infections suggested that they might play important roles in host response to infections of A. hydrophila and S. iniae.     

广西罗非鱼链球菌病病原的生化鉴定及药敏试验

从来自南宁、北海等地的十几个暴发疾病的罗非鱼养殖场发病罗非鱼中,分离得到了8株链球菌。通过生化鉴定及药敏试验,结果表明,分离到的8株致病性链球菌,有6株为海豚链球菌（streptococcus iniae）,2株为无乳链球菌（streptococcus agalactiae）。     

Identification and expression profile of multiple genes in Nile tilapia in response to formalin killed Streptococcus iniae vaccination

Twenty-eight expressed sequence tags (ESTs) were isolated from a Nile tilapia (Oreochromis niloticus) vaccinated vs non-vaccinated subtractive library at 12-h post injection of a formalin killed Streptococcus iniae ARS-98-60 vaccine. The 28 ESTs were classified in terms of their putative functions. Half of the ESTs identified were unknown proteins. Of the remaining half ESTs, 17% have putative functions in protein biosynthesis and 11% have putative functions in immunity, energy production, and signal transduction, respectively. Immunity-related ESTs identified included high density lipoprotein-binding protein vigilin, immunoglobulin heavy chain, and QM-like protein. Quantitative PCR revealed that one EST (cytochrome c oxidase subunit II) was highly upregulated (1825 ± 336 fold) in vaccinated fish compared to that in non-vaccinated fish. Of the remaining 27 ESTs, nine were significantly (P<0.05) upregulated (<20 fold) in vaccinated fish. The nine significantly upregulated genes included five unknown or hypothetical proteins and four known proteins (high density lipoprotein-binding protein vigilin, QM-like protein, ribosomal protein S13, and ribosomal protein L5). The upregulation of these genes induced by killed S. iniae vaccines suggest that they might play important role in Nile tilapia defense against S. iniae infection.     

团头鲂肠道细菌的分离鉴定与药敏试验

从团头鲂(Megalobrama amblycepnala)肠道中分离纯化到4株细菌,菌株编号为MA1～4,通过细菌镜检及培养结果表明,均为革兰氏阴性直杆状菌,在LB培养基上生长;4株细菌氧化酶试验为阴性、过氧化氢酶试验为阳性、半固体动力阳性,不能利用棉子糖、山梨醇、木糖等多种糖类,产H2S、VP试验和葡萄糖酸盐利用试验为阴性;采用数值编码鉴定,MA1为鲶鱼爱德华氏菌(Edwardsiella ictaluri),MA2为摩氏摩根氏菌(Morganella morganii),MA3为嗜线虫致病杆菌(Xenorhab-dus nematophilua),MA4为弗氏志贺菌(Shigella flexneri)。进一步进行药敏试验分析显示,4株菌耐药率分别为52.2%、69.6%、95.7%、100%,均对阿奇霉素、红霉素、洁霉素、氯洁霉素、灭滴灵、头孢噻吩、先锋霉素Ⅳ、复方新诺明和甲氧咔啶具有耐药性;对利福平、氯霉素、头孢孟多、先锋必素、氧哌嗪青霉素、氨苄青霉素、庆大霉素、卡那霉素和四环素等药物敏感性不同。该研究结果对探明鱼类肠道菌群、养殖水体微生态和疾病防治具有重要参考价值。     

Antigen recognition by rainbow trout (Oncorhynchus mykiss) of whole cell proteins expressed by Lactococcus garvieae when obtained directly from fish and under iron limited culture conditions

Infection by Lactococcus garvieae has become a widely recognised problem associated with intensively cultured fish. Long-term control of fish infections may be possible by vaccination providing a suitable and efficacious epitope is expressed during production of cells used for vaccine preparation. The identification of novel vaccine candidates must, therefore, consider how the host species recognises and responds to bacterial cell components. L. garvieae was cultured in iron deficient, limited and haem iron enriched media and the whole cell proteins expressed under these conditions were compared with those expressed in bacteria extracted with Percoll gradients directly from spleen tissue of infected rainbow trout (Oncorhynchus mykiss). SDS-PAGE of the cell proteins showed the existence of several different electropherotypes according to the iron status of the culture media. Only minor differences in cell protein profile were detected in bacteria obtained directly from fish spleens, but when the electropherograms were analysed by Western blots using L. garvieae hyperimmune fish sera, several proteins could be identified that were expressed only when L. garvieae was growing in vivo. Siderophore could be detected in culture supernatant of iron deficient, limited and haem iron enriched media but not in media with higher nutrient concentrations. The siderophore could not be identified as a type of catechol or hydroxymate. Rainbow trout recognise proteins in the range of approximately 50-80 kDa for bacterial cells obtained without subculture from infected fish and culture conditions can influence protein profiles for this pathogen.     

Determination of florfenicol dose rate in feed for control of mortality in Nile tilapia infected with Streptococcus iniae

A dose titration study was conducted to determine the dosage of florfenicol (FFC) in feed to control Streptococcus iniae-associated mortality in Nile tilapia Oreochromis niloticus. Six tanks were assigned to each of five treatments: (1) not challenged with S. iniae and fed unmedicated feed; (2) challenged with S. iniae by injection and fed unmedicated feed; (3) challenged with S. iniae and given FFC at 5 mg/kg of body weight (bw) in medicated feed; (4) challenged with S. iniae and given 10 mg FFC/kg bw; and (5) challenged with S. iniae and given 15 mg FFC/kg bw. Treatment was initiated the day after inoculation, and feed was administered for 10 d. Cumulative mortality was 0% in the unchallenged, untreated group; 35.8 +/- 4.4% (mean +/- SE) in the challenged, unmedicated group; 19.2 +/- 2.7% in the 5-mg/kg treated group, 12.5 +/- 3.8% in the 10-mg/kg group, and 2.5 +/- 1.1% in the 15-mg/kg group. The cumulative mortality was significantly less in each challenged, FFC-treated group than in the challenged, unmedicated controls (5 mg/ kg: P = 0.0156; 10 mg/kg: P = 0.0007; 15 mg/kg: P < 0.0001). The efficacy of the 10- and 15-mg/kg FFC dosages was studied in a separate dose confirmation study. Fish in all tanks were injected with S. iniae. At 4 h postinoculation, 10 tanks were assigned to each of three feed treatments: (1) unmedicated feed; (2) 10 mg FFC/kg bw; and (3) 15 mg FFC/kg bw. Cumulative mortality was 20.5 +/- 2.0% in the challenged, unmedicated group; 11.0 +/- 2.1% in the 10-mg/kg group; and 5.5 +/- 2.4% in the 15-mg/kg group. Mortality was significantly less in the medicated groups than in the challenged, unmedicated control group (10 mg/kg: P = 0.0270; 15 mg/kg: P = 0.0007). Fish in both studies were necropsied, cultured for bacteria, and examined for gross lesions. The minimum inhibitory concentration of FFC against S. iniae in both studies ranged from 0.5 to 1.0 microg/mL. Florfenicol was palatable, safe, and efficacious for control of Nile tilapia mortality due to S. iniae infection.     

Short chain fatty acids (propionic and hexanoic) decrease Staphylococcus aureus internalization into bovine mammary epithelial cells and modulate antimicrobial peptide expression

Short chain fatty acids (SCFAs) are critical nutrients for ruminants and are mainly obtained from bacterial fermentation of carbohydrates. In addition to their nutrimental function, SCFAs have antimicrobial and anti-inflammatory properties, as well as immunomodulatory roles. It has been reported that sodium butyrate reduces Staphylococcus aureus internalization into bovine mammary epithelial cells (bMEC) and modulates antimicrobial peptide mRNA expression. Nevertheless, it has not been evaluated if sodium propionate (NaP) and sodium hexanoate (NaH) have similar actions. Since they are present in milk, the aim of this study was to determinate the effect of both SCFAs on S. aureus internalization into bMEC and to evaluate their effects on modulation of innate immunity elements. Our data showed that both SCFAs (0.25-5mM) did not affect S. aureus growth and bMEC viability. By gentamicin protection assay (MOI 30:1) we showed that NaP and NaH reduced bacterial internalization into bMEC, which ranged 27-55% and 39-65%, respectively, in relation to non treated controls. Also, both SCFAs up-regulate tracheal antimicrobial peptide (TAP) mRNA expression; however, bovine neutrophil β-defensin 5 (BNBD5) mRNA expression was not modified or was down-regulated. In addition, TAP and BNBD5 expression was up-regulated by S. aureus. Finally, the decrease in bacterial internalization under SCFA treatments is not related to nitric oxide production. In conclusion, NaP and NaH decrease S. aureus internalization into bMEC and modulate TAP gene expression, which may be related to the reduction in bacterial internalization.