Biological characteristics of marine Streptomyces SK3 and optimization of cultivation conditions for production of compounds against Vibiriosis pathogen isolated from cultured white shrimp (Litopenaeus vannamei).

Antibiotic resistance in shrimp farms has emerged as an extremely serious situation worldwide. The main aim of this study was to optimize the cultural conditions for producing new antibiotic agents from marine Streptomyces species. Streptomyces SK3 was isolated from marine sediment and was identified by its 16S rDNA as well as biochemical characteristics. This microbe produced the highest concentration of bioactive secondary metabolites (BSMs) when cultured in YM medium (YM/2). It produced the maximum total protein (41.8 ± 6.36 mg/ml) during the late lag phase period. The optimum incubation temperature was recorded at 30 °C; BSMs were not produced at ≤10 °C within an incubation period of 3-4 days. The suitable agitation speed was found to be 200 rpm with pH 7.00. The proper carbon, nitrogen, and trace elements supplementation consisted of starch, malt extract, calcium carbonate (CaCO3), and magnesium sulfate (MgSO4). The ethyl acetate extract was found to act strongly against three vibriosis pathogens, Vibrio harveyi, Vibrio parahaemolyticus, and Vibrio vunificus, as indicated by the inhibition zones at 34.5, 35.4, and 34.3 mm, respectively. The extract showed the strongest anti-V. harveyi activity, as indicated by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of 0.101 ± 0.02 and 0.610 ± 0.04 mg/ml, respectively. Basic chemical investigation of the crude extract using thin layer chromatography (TLC), bioautography, liquid chromatography tandem mass spectrometry (LC‒MS/MS), Fourier transform infrared spectroscopy (FTIR), and proton nuclear magnetic resonance (1H-NMR) revealed that the active components were the terpenoid and steroid groups of compounds. They showed carboxylic acid and ester functions in their molecules.

Characterization and Genome Analysis of Vibrio campbellii Lytic Bacteriophage OPA17.

Vibrio campbellii is a marine bacterium that is associated with luminous vibriosis, especially in the hatchery and nursery stages of penaeid shrimp cultivation worldwide, which has led to low survival rates of shrimp during aquaculture. Phage therapy has been reported as an alternative biocontrol agent which can reduce or replace the use of antibiotics and other chemicals. This study characterized a lytic V. campbellii bacteriophage, OPA17, originally isolated from bloody clams and investigated its biocontrol efficacy against V. campbellii infection in a model system, Artemia franciscana. Phage OPA17 lysed 83.89% of V. campbellii strains tested (n = 118) with clear plaque morphology. Some strains of Vibrio parahaemolyticus and Vibrio vulnificus were also infected by phage OPA17. Transmission electron microscopy and genetic features indicated that OPA17 belongs to the Siphoviridae family. The latent period and burst size of OPA17 were approximately 50 min and 123 PFU/cell, respectively. Moreover, it survived in artificial seawater throughout the 2-month study period and effectively destroyed Vibrio campbellii biofilms after 4 h of incubation. The addition of OPA17 significantly increased the survival of A. franciscana nauplii infected with V. campbellii. The genome sequence of OPA17 showed that it does not carry genes unsuitable for phage therapy. The phylogenetic tree analysis showed that OPA17 was closely related to the V. vulnificus lytic phage SSP002 (98.90% similarity), which was previously reported as a potential biocontrol agent. Accordingly, the results of this study provide valuable information regarding the potential biocontrol application of phage OPA17 against V. campbellii. IMPORTANCE V. campbellii is an emerging luminous pathogen associated with vibriosis, especially in marine shrimp hatcheries. Several strategies, including pond management and use of natural antimicrobials and probiotics, have been studied for control of this organism. Phage therapy is considered one of the effective biocontrol strategies against bacterial infections in aquaculture. However, there has been limited study of V. campbellii bacteriophages. In this study, V. campbellii-specific bacteriophage OPA17 was isolated, characterized, and investigated for its biocontrol efficacy against V. campbellii infection in an Artemia nauplii model. Phage OPA17 belongs to the Siphoviridae family and shares significant genome similarity to phage SSP002, a potential biocontrol agent against V. vulnificus infection in a murine model. However, the host range of OPA17 was broader than that of SSP002. Overall, we discuss the potential of OPA17 for phage therapy application in shrimp hatcheries.

Optimal Replacement of Soybean Meal with Fermented Palm Kernel Meal as Protein Source in a Fish Meal-Soybean Meal-Based Diet of Sex Reversed Red Tilapia (Oreochromis niloticus × O. mossambicus).

The solid-state fermentation by effective microorganisms (containing photosynthetic bacteria, lactic acid bacteria, nitrogen-fixing bacteria, yeast and Bacillus sp.) improved the nutritive values of palm kernel meal (PKM). Increased crude protein (20.79%), nitrogen-free extract (40.07%) and gross energy (19.58%) were observed in fermented PKM (FPKM) relative to raw PKM while crude lipid (15.65%), crude fiber (36.45%) and ash (29.54%) were decreased. Replacement of soybean meal (SBM) with FPKM as a protein source was investigated for its effects in sex-reversed red tilapia (Oreochromis niloticus × O. mossambicus). The two-month-old fish (14.85 ± 0.28 g initial weight) were fed fish meal-SBM-based diets with replacement by FPKM at 25% (25FPKM), 50% (50FPKM), 75% (75FPKM) and 100% (100FPKM), while an FPKM-free diet (0FPKM) was used as a control. The five treatments, comprising triplicate cement ponds and forty fish each, were conducted in a recirculating system over 12 weeks. At the end of the feeding trial, fish fed the 50FPKM diet were superior in growth performance, while the feed utilization parameters were similar across all five treatments. Physiological adaptation of the protein-digesting (pepsin and trypsin) and lipid-digesting (lipase) enzymes was detected at all protein replacement levels (except for 25FPKM), as well as of the enzyme for cellulose digestion (cellulase), but not of the carbohydrate-digesting enzymes (amylase). Protein synthesis capacity in flesh was improved in fish fed the 50FPKM diet, while the quality of the main flesh proteins, actin and myosin, showed no significant differences across the five treatments. No differences in carcass composition and no negative effects on hematological parameters or liver histoarchitecture at the 50% replacement level of SBM by FPKM also support this alternative. Findings from the current study indicate the low-cost FPKM-containing diet for tilapia in comparison with control diet.

In vitro and in vivo assessments of Rhodomyrtus tomentosa leaf extract as an alternative anti-streptococcal agent in Nile tilapia (Oreochromis niloticus L.).

PURPOSE: Rhodomyrtustomentosa is a Thai medicinal plant that has been attracting attention for its remarkable antibacterial properties against Gram-positive pathogenic bacteria. The purpose of this study was to evaluate the antibacterial properties of R. tomentosa leaf extract against Streptococcus agalactiae and Streptococcus iniae isolated from infected tilapia. METHODOLOGY: The anti-streptococcal activity of R. tomentosa was determined using broth microdilution assays. RESULTS: The extract demonstrated strong antibacterial activity against the fish pathogens, with minimum inhibitory concentrations (MICs) ranging from 7.8‒62.5 µg ml-1. It was found to possess a dose-dependent bacteriostatic effect on this organism. Scanning electron microscopy revealed irregular and long chains of swollen cells, as well as corkscrew shapes andincomplete separation of cell division of S. agalactiae cells following the treatment at sub-MIC. Moreover, S. agalactiae cells pre-treated with the extract became more sensitive to oxidative stress induced by H2O2 than the untreated cells. Based on the mortality of Nile tilapia after intraperitoneal infection of S. agalactiae at median lethal dose (LD50), the pre-treated cells caused a significant (P<0.01) reduction in mortality of S. agalactiae-infected Nile tilapia. CONCLUSION: The results suggested that R. tomentosa could be further developed as a simple and effective agent for the treatment of streptococcosis in Nile tilapia.

Roles of phagocytosis activating protein (PAP) in Aeromonas hydrophila infected Cyprinus carpio.

Cyprinus carpio (koi) is one of the most popular ornamental fish. A major problem for C. carpio farming is bacterial infections especially by Aeromonas hydrophila. Previously studies had shown that the Phagocytosis Activating Protein (PAP) gene was involved in the innate immune response of animals. Therefore, we attempted to identify a role for the PAP gene in the immunology of C. carpio. The expression of the PAP was found in C. carpio whole blood and increased when the fish were stimulated by inactivated A. hydrophila. In addition, PAP-phMGFP DNA was injected as an immunostimulant. The survival rate and the phagocytic index were significantly increased in the A. hydrophila infected fish that received the PAP-phMGFP DNA immunostimulant. A chitosan-PAP-phMGFP nanoparticle was then developed and feeded into fish which infected with A. hydrophila. These fish had a significantly lower mortality rate than the control. Therefore, this research confirmed a key role for PAP in protection fish from bacterial infection and the chitosan-PAP-phMGFP nanoparticle could be a good prototype for fish immunostimulant in the future.

Serotype distribution and antimicrobial susceptibilities of Streptococcus agalactiae isolated from infected cultured tilapia (Oreochromis niloticus) in Thailand: Nine-year perspective.

Streptococcus agalactiae (group B Streptococcus, GBS) infection remains a major problem associated with high mortality of cultured tilapia worldwide. The present study reports the serotype distribution and antimicrobial susceptibilities of GBS isolated from infected tilapia cultured in Thailand. One hundred and forty-four GBS isolates were identified by biochemical, serological and molecular analyses. Of these 144 GBS isolates, 126 were serotype Ia and 18 were serotype III. Antimicrobial susceptibilities of the 144 GBS isolates were determined by the disc diffusion method. Most GBS isolates were susceptible to lincomycin, norfloxacin, oxytetracycline, ampicillin, erythromycin and chloramphenicol, but resistant to oxolinic acid, gentamicin, sulfamethoxazole and trimethoprim. However, 17 isolates displayed an oxytetracycline-resistant phenotype and harboured the tet(M) gene. The broth microdilution method was used to determine the minimal inhibitory concentrations (MICs) of 17 oxytetracycline-resistant GBS isolates, and then minimal bactericidal concentrations (MBCs) of these isolates were evaluated. Oxytetracyline-resistant isolates were found to be susceptible to ampicillin, lincomycin, norfloxacin, erythromycin and chloramphenicol, with the MIC and MBC ranging from ≤ 0.125 to 0.5 µg ml- 1 and ≤ 0.125 to 2 µg ml- 1, respectively. Moreover, all 17 oxytetracycline-resistant isolates demonstrated resistance to trimethoprim, oxolinic acid, gentamicin, sulfamethoxazole and oxytetracycline, with the MIC and MBC ranging from 16 to ≥ 128 µg ml- 1 and ≥ 128 µg ml- 1, respectively. These findings are useful information for antibiotic usage in fish aquaculture.

Euclinostomum heterostomum infection in guppies Poecilia reticulata cultured in Southern Thailand.

During April to June 2009 and February 2010 to February 2011, numerous digenetic trematode metacercariae were observed embedded in the muscles of guppies Poecilia reticulata cultured at Kidchakan Supamattaya Aquatic Animal Health Research Center, Songkhla, southern Thailand. A total of 424 guppies were examined to identify the parasite and to study its pathogenicity. Based on conventional parasitological techniques, the parasitic digenean found in the guppies was identified as Euclinostomum heterostomum Rudolphi, 1809. Histological analysis revealed numerous metacercariae embedded in the fish muscle. A life cycle study indicated that the snail Indoplanorbis exustus was the first intermediate host, with the guppies serving as the second intermediate host. No E. heterostomum metacercariae were found in cohabited fish species, giant sailfin molly Poecilia velifera or platy Xiphophorus maculatus, which indicated that the guppy was the only suitable fish host present.