In vitro study of adherence, invasion, and persistence of Streptococcus iniae in fibroblastic-like fish cell line SAF-1

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.     

Phenotypic and Genotypic Heterogeneity among Streptococcus iniae Isolates Recovered from Cultured and Wild Fish in North America,Central America and the Caribbean Islands

Abstract

Streptococcus iniae, the etiological agent of streptococcosis in fish, is an important pathogen of cultured and wild fish worldwide. During the last decade outbreaks of streptococcosis have occurred in a wide range of cultured and wild fish in the Americas and Caribbean islands. To gain a better understanding of the epizootiology of S. iniae in the western hemisphere, over 30 S. iniae isolates recovered from different fish species and geographic locations were characterized phenotypically and genetically. Species identities were determined biochemically and confirmed by amplification and sequencing of the 16S rRNA gene. Repetitive-element palindromic PCR fingerprinting as well as biochemical and antimicrobial susceptibility profiles suggest that a single strain of S. iniae was responsible for two different disease outbreaks among reef fishes in the Caribbean, one in 1999 and another in 2008. Interestingly, a majority of the isolates recovered from cultured fish in the Americas were genetically distinct from the Caribbean isolates and exhibited a trend toward higher minimal inhibitory concentration with respect to several antibiotics as well as greater genetic variability. The biological significance of this genetic variability is unclear, but it could have implications for future vaccine development and treatment.

Received April 20, 2014; accepted July 7, 2014     

Laboratory Efficacy of Amoxicillin for the Control of Streptococcus iniae Infection in Blue Tilapia

Abstract

Experimental feeding trials were performed to evaluate the efficacy of amoxicillin (AMX) in controlling Streptococcus iniae infection in blue tilapia Oreochromis aureus. The doses of AMX active ingredient tested were 0, 5, 10, 30, and 80 mg/kg of fish body weight (BW) per day. Administration of medicated feed started within 22–24 h postchallenge by waterborne exposure to S. iniae (after skin scraping) and continued for 12 consecutive days, followed by a 17-d posttreatment observation. Oral administration of AMX-medicated feed for 12 d at a daily rate of 10, 30, and 80 mg/kg BW significantly increased the survival of S. iniae?infected tilapia from 3.8% in the challenged, nonmedicated positive control to 45, 75, and 93.8%, respectively. The survival rate was significantly higher in the 80-mg treatment (93.75%) than the 10-mg treatment (45%) but did not differ significantly between the 10-mg (45%) and 30-mg (75%) treatments. At the conclusion of the experiment, no carriers were detected in any challenged group receiving AMX-medicated diet, whereas the bacterium was recovered from the nonmedicated, challenged survivors of the infection.     

Cell-Surface-Associated Properties of Fish Pathogenic Bacteria

Abstract

Pathogenicity assays showed that 33 of 42 potentially pathogenic strains of bacteria tested were virulent to rainbow trout Oncorhynchus mykiss. Regardless of their degree of virulence to fish, strains of motile Aeromonas, A. salmonicida, and Vibrio anguillarum were moderately hydrophobic. Only 46 and 25°10 of the strains were able to hemagglutinate human and trout erythrocytes, respectively. Hydrophobicity and hemagglutination were practically absent in isolates of Yersinia ruckeri. A notable number of the strains positively adhered to salmonid (51%) and nonsalmonid (55%) fish cells. Whereas the treatment of the bacteria with proteinase K or trypsin did not decrease the hydrophobicity of the isolates, within motile Aeromonas and A. salmonicida species, strains with both protease-sensitive and -resistant hemagglutinating and adhesive abilities occurred. The effects of heat and sugars on hemagglutinating and hydrophobic properties varied within all bacterial groups. Although treatment of strains with D-mannose or L-fucose had distinct effects on adhesiveness according to the bacterial species and the cell system used, none of the heat-treated (80°C for 15 min) bacteria lost their capacity to adhere to cultured fish cells. The results showed that there was no direct relationship between any of the cell surface properties analyzed and the degree of virulence of the strains.     

Isolation and Pathogenicity of Streptococcus iniae in Cultured Red Hybrid Tilapia in Malaysia

Abstract

This study describes the isolation and pathogenicity of Streptococcus iniae in cultured red hybrid tilapia (Nile Tilapia Oreochromis niloticus × Mozambique Tilapia O. mossambicus) in Malaysia. The isolated gram-positive S. iniae appeared punctiform, transparently white, catalase and oxidase negative and produced complete β-hemolysis on blood agar, while a PCR assay resulted in the amplification of the 16 S rRNA gene and lactate oxidase encoded genes. The isolate was sensitive to tetracycline, vancomycin, and bacitracin but was resistant to streptomycin, ampicillin, penicillin, and erythromycin. Pathogenicity trials conducted in local red hybrid tilapia (mean ± SE = 20.00 ± 0.45 g) showed 90.0, 96.7, and 100.0% mortality within 14 d postinfection following intraperitoneal exposure to 10⁴, 10⁶, and 10⁸ CFU/mL of the pathogen, respectively. The clinical signs included erratic swimming, lethargy, and inappetance at 6 h postinfection, while mortality was recorded at less than 24 h postinfection in all infected groups. The LD_{50-336 h} of S. iniae against the red hybrid tilapia was 10² CFU/mL. The post mortem examinations revealed congested livers, kidneys, and spleens of the infected fish. This is the first report of S. iniae experimental infection in cultured red hybrid tilapia in Malaysia.

Received January 20, 2017; accepted July 16, 2017 Published online October 11, 2017     

Comparative single‐dose pharmacokinetics of orally administered florfenicol in rainbow trout (Oncorhynchus mykiss,Walbaum, 1792) at health and experimental infection with Streptococcus iniae or Lactococcus garvieae

This study evaluates changes in the pharmacokinetic behavior of a single oral dose of florfenicol in rainbow trouts experimentally infected with Lactococcus garvieae or Streptococcus iniae. One hundred and fifty fish were randomly divided into three equal groups: 1—healthy fish, 2—fish inoculated with S. iniae (2.87 × 10⁷ CFU/ml, i.p.), and 3—fish inoculated with L. garvieae (6.8 × 10⁵ CFU/ml, i.p.). Florfenicol was administered to all groups at 15 mg/kg by oral gavage. Blood sampling was performed at 0, 2, 3, 6, 8, 12, 24, 48, 72, and 120 hr after drug administration to each group, and plasma concentration of florfenicol was assayed by HPLC method. The MICs of florfenicol were 1.2 μg/ml and 5 μg/ml against L. garviae and S. iniae, respectively. Healthy fish showed higher values for most of the PK/PD parameters as compared to fish infected with L. garvieae which was reversed in fish infected with S. iniae. Fish infected with L. garvieae showed decreased relative bioavailability accompanied by increased volume of distribution at steady‐state (Vd_ss) and total body clearance (Cl_B)_. Infection with S. iniae increased the peak concentration of drug after administration (Cmax) and decreased elimination half‐life (T_{1/2 β})_, central compartment volume (V_c), and Vd_ss. In conclusion, infection with these bacteria can affect the pharmacokinetic behavior of florfenicol in rainbow trouts as shown by decreased bioavailability and increased total body clearance and volume of distribution in L. garvieae infection and decreased volume of distribution accompanied by increased Cmax in S. iniae‐infected fish.     

Development of a multiplex PCR assay to detect Edwardsiella tarda,Streptococcus parauberis,and Streptococcus iniae in olive flounder (Paralichthys olivaceus)

A multiplex PCR protocol was established to simultaneously detect major bacterial pathogens in olive flounder (Paralichthys olivaceus) including Edwardsiella (E.) tarda, Streptococcus (S.) parauberis, and S. iniae. The PCR assay was able to detect 0.01 ng of E. tarda, 0.1 ng of S. parauberis, and 1 ng of S. iniae genomic DNA. Furthermore, this technique was found to have high specificity when tested with related bacterial species. This method represents a cheaper, faster, and reliable alternative for identifying major bacterial pathogens in olive flounder, the most important farmed fish in Korea.     

The efficacy of amoxicillin sodium against streptococcosis in cultured olive flounder Paralichthys olivaceus and its pharmacokinetics

The efficacy of amoxicillin sodium for controlling field and experimental Streptococcus iniae and S. parauberis infections in olive flounder (Paralichthys olivaceus) was evaluated after a single intramuscular administration. Furthermore, the minimal inhibitory concentrations (MIC) against 21 Streptococcus strains were determined. In addition, the pharmacokinetics and residue depletion in olive flounder were investigated. Single intramuscular doses of amoxicillin sodium at 20, 40, 80, and 160 mg/kg b.w. fish significantly reduced cumulative mortality rates to 18.8–31.3% (P < 0.05) for S. iniae and to 5.0–15.0% (P < 0.01) for S. parauberis, whereas the S. iniae‐ and S. parauberis‐infected positive control groups showed cumulative mortality rates of 68.8% and 60.0%, respectively. In a S. parauberis outbreak, amoxicillin sodium reduced the cumulative mortality rate to 7.5% and 4.8% at 20 and 40 mg/kg b.w. fish, respectively, whereas that of the untreated control group was 35.2%. Peak plasma concentrations (C_max) following a single intramuscular dose of 40 and 80 mg/kg b.w. fish were 62.64 (T_max, 1.59 h) and 87.61 (T_max, 3.02 h) μg/mL, respectively, with large AUC_0?t/MIC and C_max/MIC ratios, and sufficient T > MIC (time for maintaining plasma drug concentration greater than MICs) for S. iniae and S. parauberis. The estimated withdrawal period of amoxicillin sodium from muscle of olive flounder was about 8 days at 40 mg/kg b.w. fish (at 22 ± 1 °C). These results demonstrated a single intramuscular administration of amoxicillin sodium to be effective against streptococcosis in olive flounder.     

Effect of Vitamin E on the Immune Response of Channel Catfish to Edwardsiella ictaluri

Abstract

Three-month-old fingerling channel catfish Ictalurus punctatus were fed purified diets supplemented with ∝-tocopherol acetate to provide 0, 60, and 2,500 mg vitamin E/kg for 180 d. A 30-s immersion bath and an oral booster were used to deliver a bacterin of formalin-killed Edwardsiella ictaluri to half of the fish from each dietary treatment. Resistance of red blood cells to peroxidation was used as an index of antioxidant status. The susceptibility of red blood cells to oxidative hemolysis decreased with increasing levels of dietary vitamin E. Vaccinated and nonvaccinated fish were evaluated for agglutinating antibody titers and macrophage activity. Humoral antibody titers in response to E. ictaluri were significantly (P ≤ 0.05) higher in vaccinated fish than in nonvaccinated fish; however, no such differences in agglutinating antibody titers were detected among any of the dietary treatment groups. Both vaccination and vitamin E significantly enhanced the ability of macrophages to phagocytize virulent E. ictaluri. Results of this study indicate that elevated levels of dietary vitamin E may affect the ability of channel catfish to respond immunologically to bacterial pathogens.     

Streptococcus iniae Associated with Mortality of Tilapia nilotica × T. aurea Hybrids

Abstract

The cause of an ongoing mortality of hybrid tilapias Tilapia nilotica × T. aurea in a Texas fish farm was a biotype of Streptococcus iniae. Identification was based upon classical biochemical and physiological analysis as well as ribosomal RNA gene sequencing. The causative agent, a β-hemolytic Streptococcus species, grew better at 37°C than at 10, 25, or 40°C, and its growth was inhibited at pH 9.6, in 6.5% NaCl, and in 40% bile. The bacterium was resistant to ampicillin and furazolidone but was susceptible to several antibiotics including tetracycline, oxytetracycline, and sulfadimethoxine-ormetoprim (5:1). This is the first record of this bacterial species affecting fish in the USA. External signs of disease in tilapia were loss of orientation, exophthalmia, corneal opacity, and petechia around the mouth, anus, and proximal margins of the pectoral fins. Internally, fluid accumulation in the peritoneal cavity and enlargement of the liver, spleen, and kidney were observed.     

The conserved surface M-protein SiMA of Streptococcus iniae is not effective as a cross-protective vaccine against differing capsular serotypes in farmed fish

Streptococcus iniae causes invasive infections in fresh and saltwater fish and occasional zoonoses. Vaccination against S. iniae is complicated by serotypic variation determined by capsular polysaccharide. A potential target for serologically cross-protective vaccines is the M-like protein SiMA, an essential virulence factor in S. iniae that is highly conserved amongst virulent strains. The present study determined how SiMA is regulated and investigated potential as a cross-protective vaccine for fish. Electrophoretic mobility shift suggested that SiMA is regulated by the multigene regulator Mgx via a binding site in the −35 region of the simA promoter. Moreover, expression of simA and mgx was highly correlated, with the highest level of simA and mgx expression during exponential growth under iron limitation (20-fold increase in relative expression compared to growth in Todd-Hewitt broth). Based on these results, a vaccination and challenge experiment was conducted in barramundi (Lates calcarifer) to determine whether SiMA is protective against S. iniae infection and cross-protective against a different capsular serotype. The challenge resulted in 60% mortality in control fish. Formalin-killed bacterins prepared from the challenge strain resulted in 100% protection, whereas bacterins prepared from a serotypically heterologous strain resulted in significantly reduced protection, even when culture conditions were manipulated to optimise SiMA expression. Moreover, recombinant SiMA protein was not protective against the challenge strain in spite of eliciting specific antibody response in vaccinated fish. Specific antibody did not increase oxidative activity or phagocytosis by barramundi macrophages. Indeed incubating S. iniae with antisera significantly reduced phagocytosis. Lack of specific-antibody mediated opsonisation in spite of 100% protection against challenge with the homologous vaccine suggests that other immune parameters result in protection of challenged fish.     

Comparison of Methods Used to Detect Renibacterium salmoninarum,the Causative Agent of Bacterial Kidney Disease

Abstract

Various diagnostic methods used to detect Renibacterum salmoninarum, the causative agent of bacterial kidney disease (BKD), were compared. The most sensitive method was the enzyme immunoassay (the indirect dot blot assay), which could detect 10² bacterial cells per gram of kidney tissue. The minimum bacteria concentrations showing positive reactions to the indirect fluorescent antibody test (IFAT) and the coagglutination test were 10³ and 10⁴ cells/g kidney, respectively. The sensitivities of the Gram stain, immunodiffusion procedure, and latex agglutination test were low, and these methods could only be applied to fish with overt BKD symptoms. Altogether, 656 coho salmon Oncorhynchus kisutch were examined for R. salmoninarum antigen with the direct and indirect dot blot assays (DDBA and IDBA) and the IFAT. Among the fish sampled, positive reactions were obtained in 11.8% by the DDBA, 28.2% by the IDBA, and 12.9% by the IFAT.     

Identification of some main <Emphasis Type="Italic">Streptococcus iniae</Emphasis> associated proteins: relationship

The surface-associated proteins play a key role in bacterial physiology and pathogenesis, and are the major targets in the development of new vaccines. These proteins contribute to the adaptation of bacteria to different hosts and environments. To study differences at the genomic level, we first sequenced the whole genome of Streptococcus iniae from fish (IUSA-1 strain) and compared it to Streptococcus iniae from human (9117 strain), revealing a high similitude between both strains. To gain further insights into host- and environment-specific differences, we then studied proteins in silico and by High Performance Liquid Chromatography. This approach successfully identified 54 secreted and surface proteins, including several proteins involved in cell wall synthesis and transport of solutes, as well as proteins with yet unknown function. These proteins highlight as interesting targets for further investigation in the interaction between Streptococcus iniae and its environment. Results reported in this study have shown a first analysis about the predicted and experimental associated proteins of Streptococcus iniae isolated from two different hosts: human and fish.     

Screening for the Fish Disease Agent Aeromonas salmonicida with an Enzyme-Linked Immunosorbent Assay (ELISA)

Abstract

Serological detection of bacterial pathogens in fish tissue is an important tool for surveying epidemiological situations. Whenever antibacterial treatment of fish is recommended, it becomes necessary, however, to culture the pathogen for sensitivity testing. An enzyme-linked immunosorbent assay (ELISA) was developed to identify Aeromonas salmonicida in culture. This serological identification can partly substitute for biochemical characterization of the organism and thus decrease the time between isolation and sensitivity testing by at least 3 d. The ELISA works with only one bacterial colony and yields results within 4 h. During this time, a bacterial suspension can be prepared for the resistance test. The specificity of an antiserum, raised in rabbits, against whole cells of A. salmonicida can be increased by adsorption with strains of cross-reacting species. However, difficulties arise when serologically heterogeneous species (e.g., A. hydrophila) are used as the cross-reacting bacterium. In the present study, severalfold adsorption with four isolates did not totally rule out cross-reactivity against additional strains. Therefore, the strain in question should also be checked for colony morphology, production of pigment, or presence of cytochrome oxidase to validate the serologically obtained result.     

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.     

Microbial Pollution in Water and Its Effect on Fish

Abstract

A systematic study of microbial contamination of fish grown in ponds in and around Calcutta was initiated as part of the wetland management project in the city. Ponds were either sewage-fed or conventional (i.e., fed by rain and groundwater). Fish from these ponds are used for human consumption, and therefore a quantitative analysis of their microbial content is necessary. When concentrations of Escherichia coli, Salmonelleae (unidentified species), and Staphylococcus aureus in water and different organs of fish from sewage-fed and conventional ponds were compared, water and fish organs from conventional ponds contained about two orders of magnitude more bacterial cells. We tested for Salmonelleae and S. aureus, in addition to E. coli, because of persistent reports from Calcutta hospitals that most enteric and other infectious diseases in this region are caused by these bacteria. Significant linear correlations were found between concentrations of these bacteria in pond water and their recovery from several tissues of the fish.     

Isolation and Characterization of Large Marine Bacteriophage (Myoviridae), VhKM4 Infecting Vibrio harveyi

Abstract

The causative agent responsible for vibriosis in tropical fish aquaculture, Vibrio harveyi, has become a major bacterial pathogen. Studies suggest that this bacterium has developed resistance to antibiotics commonly used in aquaculture. In view of this situation and the requirement for the proposed postantibiotic era, bacteriophage therapy seems to be a promising control strategy for fish vibriosis. In this study, a lytic Vibrio phage VhKM4 belonging to a member of large, marine Myoviridae was successfully isolated. It exhibited bacteriolysis to both V. harveyi VHJR7 and V. parahaemolyticus ATCC 17802. The latent period of the VhKM4 phage was recorded at 60 min. It also recorded average burst size of approximately 52 plaque-forming units per infected cell. A strong bacteriolytic activity at low multiplicity of infection of 0.01 indicates the effectiveness of this large marine myovirid against fish pathogenic strain of V. harveyi VHJR7.

Received June 16, 2016; accepted October 7, 2016 Published online February 6, 2017     

Dietary Supplementation with Allspice Pimenta dioica Reduces the Occurrence of Streptococcal Disease during First Feeding of Mozambique Tilapia Fry

Abstract

Allspice Pimenta dioica as a feed additive was studied for its effects on growth performance and disease resistance in Mozambique Tilapia Oreochromis mossambicus. Five isonitrogenous (36% crude protein) and isocaloric (18.5 kJ/g) diets were formulated to contain 0 (control), 5, 10, 15, or 20 g of allspice/kg of fish feed. In a 50-d feeding trial, 15 plastic tanks (21 L) were stocked with 35 fish fry (0.012 g) each. After the feeding trial, fish were exposed to Streptococcus iniae and mortalities were recorded. The second-order polynomial regression indicated that the dietary allspice level of 10 g/kg provided the best growth performance and feed utilization. The greatest survival after pathogen challenge was also obtained from the diet supplemented with allspice at 10 g/kg. Therefore, allspice acts as a growth promoter to improve feed utilization and weight gain in Mozambique Tilapia fry and acts an antimicrobial agent to enhance disease resistance during first feeding of fry. These results suggest that allspice can be used as an alternative to antibiotics in controlling streptococcal disease in tilapia culture.

Received October 19, 2012; accepted January 20, 2014     

Histopathology of Hybrid Tilapias Infected witha Biotype of Streptococcus iniae

Abstract

A systemic streptococcal disease caused by a biotype of Streptococcus iniae affecting hybrids of Nile tilapia Tilapia nilotica × blue tilapia T. aurea at a commercial aquaculture facility in central Texas was investigated. Histological analysis revealed a general septicemia with the occurrence of cellular infiltration and numerous cocci in most organ systems. Observations included cellular infiltration of the eye, meningitis, meningeal granuloma, numerous foci of infection, and massive cellular infiltration in the kidneys. Bacteria were seen in the subcapsular capillaries of the liver, and some liver tissue was granulomatous. Splenic sinuses contained numerous cocci, and both epicarditis and myocarditis was observed in the heart tissue of some specimens. Cocci were free in the plasma of infected fish and often phagocytized by macrophages.