Identification and characterization of motile Aeromonas spp. isolated from farmed Nile tilapia (Oreochromis niloticus) in the Philippines.

AIMS: Motile Aeromonas septicaemia (MAS) caused by motile Aeromonas species is an important disease in farmed freshwater fish due to intensification of culture and improper farm practices. This study characterized and profiled motile Aeromonas species recovered from clinically sick tilapia farmed in the Philippines, with a view to identifying targeted disease prevention and control measures against MAS in farmed tilapia species. METHODS AND RESULTS: Sixteen isolates from diseased farmed Nile tilapia were identified as Aeromonas veronii (n = 14), Aeromonas caviae (n = 1), and Aeromonas dhakensis (n = 1). Five biochemical profiles using API 20E were exhibited by the A. veronii strains giving an unreliable identification. A high level of agreement was observed in identifying the Aeromonas strains using 16S rRNA and rpoD gene sequencing, although the latter has a higher discriminatory value. Three or more virulence genes dominated by cytotoxic enterotoxin act and aerolysin aer were detected. Different genotypes based on virulence gene clustering suggested varied mechanisms used by Aeromonas to colonize and infect or to mutualistically co-exist with the fish. Acquired multiple antibiotic resistance was found in a single A. veronii isolate. All were susceptible to enrofloxacin, oxolinic acid, florfenicol, and chloramphenicol. Tetracycline and sulfonamide resistances and class 1 integron were detected in three A. veronii isolates. CONCLUSION: Several strains of motile aeromonads, especially A. veronii, which have varied genotypes based on virulence, biochemical profile, and antibiotic resistance, are involved in MAS in natural disease outbreaks in farmed Nile tilapia in the Philippines.

Probiotic effect of Bacillus spp. isolated from African nightcrawler (Eudrilus eugeniae) on the performance of Nile Tilapia (Oreochromis niloticus L.).

This study evaluated the effects of the probiotic Bacillus spp. isolated from African nightcrawler (Eudrilus eugeniae) on the growth and survival performances of Nile tilapia (Oreochromis niloticus). A total of 400 Nile tilapia fingerlings (1.76 ± 0.07 g) were equally and randomly distributed into 20 glass aquaria and divided into five experimental groups with four replicates. Each experimental group was fed with a commercial diet (control) supplemented (at 108 CFU·g-1) with one of the four Bacillus spp. (ANSCI9, BFAR9, RM3, and RM10) at five percent of their body weight. After 30 days of feeding, the surviving fish were challenged with Aeromonas hydrophila to observe their survival. The treatment groups supplemented with BFAR9 (2.73 ± 0.26 g) and RM10 (3.15 ± 0.30 g) showed significantly higher (P < 0.05) absolute growth than the control (2.20 ± 0.16 g) group. Furthermore, those given with RM10 had better (P < 0.05) specific growth rate (SGR) (1.60 ± 0.10%·day-1) relative growth rate (RGR) (181.39 ± 18.16%) and feed conversion ratio (FCR) (1.99 ± 0.13) compared to the control group (SGR = 1.29 ± 0.07%·day-1; RGR = 129.84 ± 9.77%; FCR = 2.60 ± 0.16). The challenge test revealed that all Bacillus spp.-treated groups showed significantly higher (P < 0.05) survival rates (ANSCI9 = 82.5 ± 8.16%; BFAR9 = 80.0 ± 10.00%; RM3 = 77.5 ± 20.82%; RM10 = 85.0 ± 10.00%) than the control group (55.00 ± 19.15%), with the highest relative level of protection recorded for RM10 (66.67%). These results revealed that the probiotic Bacillus spp. isolated from E. eugeniae improved the growth, feed utilization, waste excretion, and the disease resistance of Nile tilapia.

Isolation and molecular characterization of streptococcal species recovered from clinical infections in farmed Nile tilapia (Oreochromis niloticus) in the Philippines.

Streptococcosis cause severe losses for global tilapia farming, especially in developing countries. The aim of this study was to identify and characterize streptococci recovered from Nile tilapia farmed in the Philippines. Moribund and apparently healthy fish were sampled from grow-out cages, ponds and hatcheries. Clinical signs observed included exophthalmia, eye opacity, ascites, lethargy, erratic swimming and haemorrhages. Results showed that both Streptococcus iniae and Streptococcus agalactiae were associated with disease in these sites. Consistent with global reports, including those from South-East Asia, S. agalactiae was more widespread than S. iniae. Molecular serotyping of the S. agalactiae isolates identified the serotype Ia and serotype Ib. Histopathological findings were meningitis, meningoencephalitis and septicaemia. Identical virulence profiles were found for all strains of S. iniae, while S. agalactiae strains were separated into virulence profile I and profile II. All strains were susceptible to the tested antibiotics and resistant to oxolinic acid. Only S. agalactiae serotype Ib showed resistance to sulphamethoxazole-trimethoprim. This is the first study from the Philippines to characterize the streptococci involved in disease outbreaks in tilapia aquaculture. Outputs from this study will promote the development of efficacious disease control strategies in tilapia farming for the Philippines and South-East Asia.

Selection and screening of bacteria from African nightcrawler, Eudrilus eugeniae (Kinberg, 1867) as potential probiotics in aquaculture.

Earthworms are used as an alternative protein source in aquaculture. These organisms serve as an ideal and favorable site for bacterial growth and activity. Hence, in our present study, we isolated and screened potential probiotic bacteria from African nightcrawler (Eudrilus eugeniae). Among 45 bacterial isolates, four (ANSCI9, BFAR9, RM3, and RM10) were selected based on their hydrophobicity, hydrolytic enzyme production, pH and fish bile tolerance, aggregation, and antimicrobial properties. The selected isolates showed good hydrophobicity (≥ 30%) and enzyme production (≥ 10 mm clearing zones), tolerance to pH and fish bile, and inhibitory properties against pathogenic microorganisms. The isolates were identified as Bacillus sp. RM3 (MH919306), Bacillus sp. RM10 (MH919308), Bacillus sp. ANSCI9 (MH919310) and Bacillus sp. BFAR9 (MH919302). These isolates were individually incorporated in the diets of Nile tilapia (Oreochromis niloticus) fingerlings for 14 days to assess their biosafety. The results showed that the survival rates in all treated groups (98.75 ± 2.5 to 100.00 ± 0.0%) were not significantly different (P < 0.05) from the control group (commercial diet) (96.25 ± 2.5%), suggesting that isolates have no adverse effect on the host. This study revealed the presence of potential probiotic microorganisms in E. eugeniae that are beneficial to the aquaculture industry.

Isolation and characterization of a Myoviridae bacteriophage against Staphylococcus aureus isolated from dairy cows with mastitis.

A lytic bacteriophage (phage), designated SAH-1, was isolated from sewage effluent near a dairy cow farm in Gwacheon, South Korea to search for biocontrol agents against Staphylococcus aureus infections. The SAH-1 was morphologically classified as Myoviridae and possessed an approximate 144 kb double-stranded genomic DNA. The phage showed broad host ranges within S. aureus strains including methicillin-resistant strains, and its latent period and burst size were approximately 20 min and 100 PFU/cell, respectively. Moreover, morphologic and genomic analysis of SAH-1 revealed that the phage was closely related to other Myoviridae phages infecting Staphylococcus species. The bacteriolytic activity of phage SAH-1 at a multiplicity of infection (MOI) 1 and 100 indicated its efficiency for reducing bacterial growth. Based on these results, phage SAH-1 could be considered a potential therapeutic or prophylactic candidate against S. aureus infections.

Molecular identification and phylogenetic characterisation of Thelohanellus kitauei - short communication.

Thelohanellus kitauei was isolated from the koi Cyprinus carpio haematopterus, and the 18S rRNA gene of T. kitauei was amplified by optimised nested-PCR. The PCR product was sequenced and compared with other 18S rRNA genes of Thelohanellus species to investigate the relationships between their host specificities and infection sites. Based on the 18S rRNA sequences, T. kitauei is most closely related to T. hovorkai (which can infect the intestine). Phylogenetic analysis revealed that T. kitauei was clustered with other Thelohanellus spp. infecting Cyprininae. The present study suggests that the infection site and the host specificity (subfamily level) are reflected in the genetic relationships among Thelohanellus species.

Draft Genome Sequence of Vibrio parahaemolyticus SNUVpS-1 Isolated from Korean Seafood.

Vibrio parahaemolyticus is the leading cause of food-borne diseases, and several pathogenic strains cause global gastroenteritis outbreaks. Here, we report a draft genome sequence of V. parahaemolyticus SNUVpS-1, which was isolated from seafood in a fishery market in the Republic of Korea and contained TL, toxR, and toxRS(old) genes. The current draft genome sequence will contribute to the effort to monitor the spread of V. parahaemolyticus seafood isolates and clinical isolates.

Complete genome sequence of bacteriophage phiAC-1 infecting Acinetobacter soli strain KZ-1.

To date, a number of bacteriophages (phages) infecting Acinetobacter species have been reported and characterized. However, Acinetobacter phages which infect A. soli have not been investigated yet. Here, we report the complete genome sequence of Acinetobacter phage phiAC-1, which belongs to the Myoviridae, infecting Acinetobacter soli strain KZ-1.

Complete genome sequence of a novel marine siphovirus, pVp-1, infecting Vibrio parahaemolyticus.

Among the abundant bacteriophages that belong to the order Caudovirales in the ocean, the genome sequences of marine siphoviruses are poorly investigated in comparison to those of myo- or podoviruses. Here we report the complete genome sequence of Vibrio phage pVP-1, which belongs to the family Siphoviridae and infects Vibrio parahaemolyticus ATCC 33844.

Complete genome sequence of bacteriophage phiAS7, a T7-like virus that infects Aeromonas salmonicida subsp. salmonicida.

To date, a number of Myoviridae bacteriophages that infect Aeromonadaceae have been identified and characterized. However, the genome sequences of Aeromonas phages that not belong to the Myoviridae have not been investigated yet. Herein, we report the complete genome sequence of Aeromonas phage phiAS7, which belongs to the Podoviridae and infects Aeromonas salmonicida subsp. salmonicida.

Isolation, molecular characterization, and antibiotic susceptibility of Vibrio parahaemolyticus in Korean seafood.

The principal objective of this study was to investigate the incidence, risk assessment, antibiotic resistance, and genotyping of Vibrio parahaemolyticus in Korean seafood. The incidence of V. parahaemolyticus in seafood obtained from several fish markets in Korea was investigated from May to December of 2009, except between July and September. Two selective mediums (TCBS [thiosulfate, citrate, bile salts, and sucrose] agar and CHROMagar™ Vibrio) were used, and the V. parahaemolyticus strains were identified via polymerase chain reaction (PCR) amplification (Vp. flaE, tl, and toxR). 16S rRNA gene sequencing and their virulence were analyzed via the detection of tdh, trh, ORF8, toxRS/old, and toxRS/new genes. We collected 24 strains of V. parahaemolyticus: 19 seafood isolates, three environmental isolates, and two clinical (human) isolates. Among these strains, two tdh+ strains, two ORF8+ strains, 16 toxRS/old+ strains, and one toxRS/new+ strain were isolated. Twenty-two commercial antibiotics were used to assess the antibiotic susceptibility of isolates, and all the strains evidenced resistance to more than four antibiotics. The strains harboring antibiotic-resistant genes such as TetA (25%) and strB (4.16%) were detected via PCR. Repetitive extragenic palindromic sequence (REP)-PCR analysis revealed differences in the V. parahaemolyticus strains from other species and intraspecific strains.

Complete genome sequence and characterization of a broad-host range T4-like bacteriophage phiAS5 infecting Aeromonas salmonicida subsp. salmonicida.

In this study, we report one lytic Myoviridae bacteriophage (phage) infecting Aeromonas salmonicida subsp. salmonicida. The phage (named as phiAS5) was isolated from environmental river waters in Korea, and showed broad infectivity to other bacterial species in the family Aeromonadaceae as well as antibiotic-resistant A. salmonicida subsp. salmonicida strains. The biological properties and complete genome of phiAS5 were simultaneously investigated. The complete genome of phiAS5 composed of linear double-stranded DNA of 225,268 bp with G+C content of 43.0%, and encoded 343 putative ORFs, 69 putative promoters, 33 transcriptional terminator regions and 24 tRNA-encoding genes. A high degree of similarity to other T4-like Aeromonas phage was found in most ORFs of phiAS5. Therefore, the genome of phiAS5 was further compared with T4 phage and the closest relative, Aeromonas phage Aeh1, and the result demonstrated that it could be classified as a new member of the T4-like group. The bacteriolytic activity of phiAS5 against A. salmonicida subsp. salmonicida was evaluated at different doses of multiplicity of infection using one each of virulent strain that possesses the ascV gene and multi-drug resistant strain, and the results proved to be efficient for the reduction of bacterial growth. Based on these results, phiAS5 may have the potential for reducing the impacts of virulent or antibiotic-resistant A. salmonicida subsp. salmonicida in aquaculture and may also advance our understanding of the biodiversity of T4-like Aeromonas phages.

Cryopreservation of goldfish caudal fin explants using glycerol as a cryoprotecant.

The objective of this study was to investigate the effect of glycerol on the cryopreservation fin explants of goldfish, Carassius auratus. Four different concentrations, 5, 10, 15, and 20% (v/v) of glycerol and a control were tested. These were prepared in Dulbecco's modified Eagle's medium with 20% (v/v) Fetal Bovine Serum. Attachment and outgrowing rates were monitored from day 3 to day 14. Results showed that fin explants cryopreserved in 20% concentration of glycerol was significantly higher (P < 0.05) with a 100% attachment rate compared to 5, 10, and 15% concentrations with 36.67, 84.19 and 86.51% attachment rate, respectively. Fin explants cryopreserved in 20% glycerol concentration also had significantly higher (P < 0.05) outgrowth of cells (73%) than the other three concentrations on day 3. Moreover, a 100% outgrowth of cells in all concentrations was achieved after 14 days of culture. No attachment and out growth of cells were observed in control group. Goldfish caudal fin explants cryopreserved in glycerol can produce live cells efficiently, regardless of concentration.

Surgical removal of an anal cyst caused by a protozoan parasite (Thelohanellus kitauei) from a koi (Cyprinus carpio).

CASE DESCRIPTION: An 8-month-old koi (Cyprinus carpio) fish was examined at the animal hospital at Seoul National University for anal obstruction. CLINICAL FINDINGS: The affected fish was lethargic and anorexic, appeared depressed, and had a nodular obstruction at the anus. A biopsy specimen from the anal mass was submitted for histologic examination, which revealed a number of protozoa. On the basis of the morphological characteristics of the spores and the location of the plasmodia (ie, vegetative form of the parasite), a diagnosis of a cyst containing Thelohanellus kitauei was made. Thelohanellus kitauei is a protozoan parasite that affects freshwater fish by producing cyst-like tumors that may cause intestinal obstruction. Thelohanellus kitauei infection with cystic disease has been reported to affect Cyprinus spp worldwide. TREATMENT AND OUTCOME: The cyst was removed surgically. After surgery, low-concentration tricaine methanesulfonate immersion was used for sedation and antimicrobial treatment was administered. The surgical wound healed completely, and the fish was clinically normal 14 months after surgery. CLINICAL RELEVANCE: The successful outcome in this fish suggested that surgical removal may be a viable option for treatment of T kitauei infection in koi fish. The results of morphological analyses provided basic information on the relationships between tissue tropism and Thelohanellus spp.

Establishment of glass catfish (Kryptopterus bicirrhis) fin-derived cells.

Genetically manipulated transparent animals were already explored in many species for in vivo study of gene expression, transplantation analysis and cancer biology. However, there are no reports about transparent animals as in vitro genetic resources. In the present study, fin-derived cells from glass catfish (Krytopterus bicirrhis), naturally transparent fish with a visible skeleton and internal organs, were isolated after culturing fin explants and characterized using cryopreservation and cell cycle analysis. The cells grew well in DMEM (Dulbecco's modified Eagle's medium) containing 1% (v/v) P/S (penicillin-streptomycin) and 10% (v/v) fetal bovine serum at 26°C and showed increased cryopreservation efficiency with the slow-freezing method in the presence of 15% dimethyl sulfoxide. In addition, cell cycle analysis was evaluated based on flow cytometric analysis, and culturing to confluence (>85%) was more effective for synchronizing cells at the G(0)/G(1) stages than roscovitine treatment (<75%). This is the first report about cell isolation from transparent animals. The results from testing the cell's viability following cryopreservation and subjecting the cells to cycle analysis can be useful tools for genetic resource management.

Isolation of a zoonotic pathogen Kluyvera ascorbata from Egyptian fruit-bat Rousettus aegyptiacus.

The Egyptian fruit-bat Rousettus aegyptiacus which had been raised at the private commercial aquarium in Seoul, Korea for indoor exhibition was found dead and submitted to College of Veterinary Medicine, Seoul National University for postmortem examination. A pure bacterium of Kluyvera ascorbata was isolated from the blood specimen. The isolation of K. ascorbata from fruit bat is very important, because it is the most infectious agent of the genus Kluyvera that cause serious diseases to animals and human. Fruit-bats which are distributed in pet shops through black-market in Korea although unproven become popular pet nowadays. This situation enhances chance of zoonosis. This paper describes the first isolation of K. ascorbata from the Egyptian fruit-bat.

Different culture conditions used for arresting the G0/G1 phase of the cell cycle in goldfish (Carassius auratus) caudal fin-derived fibroblasts.

One of the most important factors determining the success of the development of cloned embryos is the cell cycle stage of the donor cells. We investigated the effects of serum starvation, culturing to confluence and roscovitine treatment on the cell cycle synchronization of goldfish caudal fin-derived fibroblasts by flow cytometric analysis. The results show that culturing the cells to confluence (85.5%) and roscovitine treatment (82.71%) yield a significantly higher percentage of cells arrested in the G0/G1 (P<0.05) phase than serum starvation (62.85%). Different concentrations of roscovitine (5, 10, or 15 microM) induce cell cycle arrest at the G0/G1 phase.

Genetic analysis of betanodaviruses in subclinically infected aquarium fish and invertebrates.

Betanodaviruses causing viral nervous necrosis (VNN) have been detected and isolated from several species of cultured marine fish worldwide. In Korea, VNN was identified in several species of cultured marine fish. This study presents data on the amplified nested PCR product (420 bp) of 11 nodavirus strains from different species of apparently healthy aquarium fish and invertebrates collected in one private commercial aquarium in Korea. Phylogenetic analyses based on the partial nucleotide sequence (177 bases) of the RNA2 coat protein gene were identical to the redspotted grouper nervous necrosis virus (RGNNV) genotype (96%-100%). The presence of the RGNNV type of betanodaviruses in these subclinically infected aquarium fish and invertebrates imported from different countries probably indicates that the samples were contaminated inside the aquarium and represents a serious challenge for its management of viral nervous necrosis. These positive samples can be an inoculum source of betanodavirus infection to other susceptible fish species inside the aquarium.

Molecular detection of betanodaviruses from apparently healthy wild marine invertebrates.

One hundred eighteen samples (21 species) of wild marine invertebrates were collected from western and southern coastal area of Korean Peninsula. Four of 78 (18 species) samples collected at Namhae (South) area were positive for nodavirus in nested PCR test. Of the 40 samples (5 species) collected at Hwanghae (West) areas, all samples were negative for nodavirus in both RT-PCR and nested PCR tests. Positive nested PCR results were obtained from the following species: Charybdis bimaculata Charybdid crab; Pandalus hypsinotus Southern humpback shrimp and Mytilus galloprovincialis Mediterranean mussel. Phylogenetic analysis based on the partial nucleotide sequence (177 bases) of the RNA2 coat protein gene showed that the four strains were highly homologous (100%) and closely related to that of the known betanodaviruses, redspotted grouper nervous necrosis virus (RGNNV). These results indicate that nodavirus is present from wild marine invertebrates in the southern coastal areas of Korean Peninsula. These subclinically infected marine invertebrates may constitute an inoculum source for betanodavirus infection and cause mortality in cultured fishes in Korea.