Biofilms are aggregates of attached microbial organisms whose existence on tissues is often recognised as a mechanism for the establishment of most chronic diseases. Herein we investigated the ability of piscine Streptococcus agalactiae, an important aquatic pathogen, for adaptation to this sessile lifestyle in vitro and in the brain of a tilapia fish model. Piscine S. agalactiae exhibited a weak attachment to polystyrene plates and expressed a low biofilm phenotype under the study conditions. Furthermore, fluorescent in situ hybridization and confocal laser scanning microscopy revealed discrete aggregates of attached S. agalactiae within brain tissues and around meningeal surfaces. They were embedded in an exopolysaccharide containing matrix, intractable to inflammatory response and showed some level of resistance to penicillin despite proven susceptibility on sensitivity test. Intracellular bacterial aggregates were also observed, moreover, antibody mediated response was not demonstrated during infection. Nucleated erythrocytes appear to facilitate brain invasion possibly via the Trojan horse mechanism leading to a granulomatous inflammation. We have demonstrated that biofilm is associated with persistence of S. agalactiae and the development of chronic meningoencephalitis in fish.
The current experiment was conducted to evaluate and compare the efficacy of two different probiotics Bacillus subtilis WB60 and Lactobacillus plantarum KCTC3928 in diet of Japanese eel, Anguilla japonica. Seven experimental diets were formulated to contain no probiotics (CON), three graded levels of B. subtilis at 106 (BS1), 107 (BS2), 108 (BS3) and L. plantarum at 106 (LP1), 107 (LP2), 108 (LP3) CFU/g diet. Twenty fish averaging 8.29 ± 0.06 g were distributed in to 21 aquaria and were randomly assigned to one of the experimental diets in triplicate groups. Average weight gain (WG), feed efficiency (FE), and protein efficiency ratio (PER) of fish fed B. subtilis at 107 (BS2) and 108 (BS3) CFU/g diet were significantly higher than those of fish fed other experimental diets (P fish fed B. subtilis at 107 (BS2) and 108 (BS3) CFU/g diet were detected to be significantly higher than that from fish fed CON diet (P fish fed B. subtilis at 107 and 108 CFU/g diet were recorded to be significantly higher than those of fish fed other experimental diets (P fish fed108 (BS3) CFU/g diet as compared to other treatment groups. Whereas, results from the disease challenge test with bacteria Vibrio angulillarum showed significantly lower survival rate for fish fed CON diet than those of fish fed other experimental diets. Therefore, these results indicated that oral supplement of B. subtilis at 108 (BS3) CFU/g diet could be a more effective source of probiotic compared to L. plantarum in Japanese eel.
Use of antibiotics for the control of bacterial diseases in shrimp culture has caused several adverse impacts to the industry. This has resulted in the search for alternative environment friendly approaches to overcome bacterial infections. This study was conducted to investigate the use of beneficial bacteria as an alternative to antibiotics. Ten pathogenic bacterial species isolated from shrimp, Penaeus monodon, and Artemia cysts were tested for susceptibility to indigenous marine Bacillus subtilis AB65, Bacillus pumilus AB58, Bacillus licheniformis AB69 and compared with oxytetracycline, chloramphenicol, gentamicin and bacitracin, which are common antibiotics used in Asian aquaculture. The Bacillus spp. were isolated from the local marine environment for bioremediation use in shrimp hatcheries and were proven to reduce total ammonium nitrogen. The pathogenic bacterial isolates were 90% susceptible to B. subtilis AB65, 70% susceptible to B. pumilus AB58 and B. licheniformis AB69 and 100% susceptible to oxytetracycline, chloramphenicol and gentamicin but only 40% to bacitracin. Two representative isolates of the vibrio group, Vibrio alginolyticus VaM11 and Vibrio parahaemolyticus VpM1, when tested for competitive exclusion by a common broth method using the marine Bacillus spp., showed decreased viable counts from 10(8) to 10(2) cfu mL(-1). The results suggest that the action of the marine bacteria appears to be significant in protecting the host shrimp against pathogenic bacteria. In addition to the alternative use of antibiotics, the selected marine bacteria had additional bioremediation properties of reducing ammonia.
The approaches of transcriptomic and proteomic have been widely used to study host-pathogen interactions in fish diseases, and this is comparable to the recently emerging application of metabolomic in elucidating disease-resistant mechanisms in fish that gives new insight into potential therapeutic strategies to improve fish health. Metabolomic is defined as the large-scale study of all metabolites within an organism and represents the frontline in the 'omics' approaches, providing direct information on the metabolic responses and perturbations in metabolic pathways. In this review, the current research in infectious fish diseases using metabolomic approach will be summarized. The metabolomic approach in economically important fish infected with viruses, bacteria and nematodes will also be discussed. The potential of the metabolomic approach for management of these infectious diseases as well as the challenges and the limitations of metabolomic in fish disease studies will be explored. Current review highlights the impacts of metabolomic studies in infectious fish diseases, which proposed the potential of new therapeutic strategies to enhance disease resistance in fish.
This study was carried out to determine the antibody responses and protective capacity of an inactivated recombinant vaccine expressing the cell wall surface anchor family protein of Streptococcus agalactiae following oral vaccination against streptococcosis in tilapia. Tilapia were vaccinated orally with 10(6) CFU/mL of the recombinant vaccine incorporated in feed (feed-based recombinant vaccine) (vaccinated group or Group 1), 10(6) CFU/mL of pET-32 Ek/LIC vector without cell wall surface anchor family protein (control group or Group 2), 10(6) CFU/mL of formalin-killed cells of S. agalactiae vaccine incorporated in feed was also prepared (feed-based vaccine) (vaccinated group or Group 3), and unvaccinated control group or Group 4 (fed with commercial pellets). During the course of study, serum, mucus and gut lavage fluid were collected to evaluate the antibody levels via enzyme-linked immunosorbent assay (ELISA). The results showed that tilapia immunized with the feed-based recombinant vaccine developed a strong and significantly (P
The Lancefield group C alpha-hemolytic Streptococcus dysgalactiae ssp. dysgalactiae (GCSD) causes systemic granulomatous inflammatory disease and high mortality rates in infected fish. Superantigen and streptolysin S genes are the most important virulence factors contributing to an invasive streptococcal infection. PCR amplification revealed that all strains isolated from moribund fish harbored the streptolysin S structural gene (sagA). GCSD fish isolates were PCR negative for emm, speA, speB, speC, speM, smeZ, and ssa. However, the size of the streptococcal pyrogenic exotoxin G (spegg) locus, a superantigen, in positive S. dysgalactiae fish and pig strains was variable. The ORF of the spegg locus of 26 GCSD fish strains and one GCSD pig strain was inserted with IS981SC. Interestingly, the ORF of the spegg locus of two fish strains of GCSD collected in Malaysia was inserted with an IS981SC-IS1161 hybrid IS element. The hybrid IS element was found in all of the GCSD fish isolates and one GCSD pig through PCR screening. Although no insertion sequence (IS) was detected in the spegg locus of S. dysgalactiae ssp. equisimilis (GCSE) strains, a five-nucleotide deletion mutation was detected in the ORF of the spegg locus of one GCSE strain at the supposed site of IS981SC insertion, resulting in a frameshift mutation.
The current study was designed to evaluate the effects of Excoecaria agallocha leaf extracts on immune mechanisms and resistance of tilapia, Oreochromis niloticus, after challenge with Streptococcus agalactiae. Fish were divided into 6 groups; groups 1-5 fed with E. agallocha leaf extracts at 10, 20, 30, 40 and 50mgkg(-1) level, respectively. Group 6 were fed without extract addition and acted as control. E. agallocha extracts were administered as feed supplement in fish diet for 28days and the hematological, immunological, and growth performance studies were conducted. Fish were infected with S. agalactiae at a dose of 15×105CFUmL(-1) and the total white blood cell (WBC), phagocytosis and respiratory burst activities of leukocytes, serum bactericidal activity, lysozyme, total protein, albumin, and globulin levels were monitored and mortalities recorded for 15days post infection. Results revealed that feeding O. niloticus with 50mgkg(-1) of E. agallocha enhanced WBC, phagocytic, respiratory burst, serum bactericidal and lysozyme activities on day 28 pre-challenge and on 3rd, 6th, 9th, 12th and 15th day post-challenge as compared to control. Total protein and albumin were not enhanced by E. agallocha diet. E. agallocha increased the survival of fish after challenge with S. agalactiae. The highest mortality rate (97%) was observed in control fish and the lowest mortality (27%) was observed with group fed with 50mgkg(-1) extract. The results indicate that dietary intake of E. agallocha methanolic leaf extract in O. niloticus enhances the non-specific immunity and disease resistance against S. agalactiae pathogen.
An initial evaluation of spray vaccination was carried out with 60 hybrid Red Tilapia Oreochromis spp., divided into three groups that consisted of 10 fish per group with duplicates. The formalin-killed cells (FKCs) of Streptococcus agalactiae were administered once to group 1 by spray and once daily for five consecutive days to group 2. Group 3 remained as the untreated control group and was sprayed with normal saline. A booster was given twice to all the groups, once at the second week and again at the fourth week after the first vaccination. After this initial evaluation, a challenge study was conducted with 40 tilapia divided into two groups that consisted of 10 fish per group with duplicates. Group 1 was vaccinated with FKCs of S. agalactiae by a single spray administration while group 2 remained as the untreated control group. A booster was given twice using the same protocol as in the initial evaluation. After 6 weeks, fish from one of the duplicate tanks from each of groups 1 and 2 were challenged with pathogenic S. agalactiae by intraperitoneal (IP) injection, while fish in another tank were challenged through immersion. Based on the observations, serum immunoglobulin M (IgM) levels were significantly higher (P < 0.05) in the challenged fish than in the either the preexposed fish or the control group 1 week after the initial exposure. However, no significant differences (P > 0.05) were noted between challenged groups 1 and 2. In addition, no significant differences (P > 0.05) were observed between the frequencies of exposure. The mucus IgM level, however, remained high after each booster until the end of the 8-week study period. Meanwhile, serum IgM levels decreased after the challenge. A higher percentage of survival was noted for fish challenged through immersion (80%) compared with IP injection (70%). These results suggested that single spray exposure was able to induce IgM, which gave moderate to high protection during the challenge study.
This study investigated the environmental factors associated with the presence of Vibrionaceae in economically important cage-cultured tropical marine fishes: the Asian Seabass Lates calcarifer, snapper Lutjanus sp., and hybrid grouper Epinephelus sp. Fish sampling was conducted at monthly intervals between December 2016 and August 2017. The body weight and length of individual fish were measured, and the skin, eye, liver, and kidney were sampled for bacterial isolation and identification. Water physicochemical parameters during the sampling activities were determined, and the enumeration of total Vibrionaceae count was also conducted from water and sediment samples. Nine species of Vibrio were identified, including V. alginolyticus, V. diabolicus, V. harveyi, V. campbellii, V. parahaemolyticus, V. rotiferianus, V. furnissii, V. fluvialis, and V. vulnificus. Photobacterium damselae subsp. damselae was also identified. A total of 73% of the isolated Vibrio belonged to the Harveyi clade, followed by the Vulnificus clade (5.5%) and Cholera clade (0.6%). Highest occurrence of Vibrio spp. and P. damselae subsp. damselae was found in hybrid grouper (72%), followed by Asian Seabass (48%) and snapper (36%). The associations of Vibrio spp. and P. damselae subsp. damselae with the host fish were not species specific. However, fish mortality and fish size showed strong associations with the presence of some Vibrio spp. On average, 60% of the infected cultured fish exhibited at least one clinical sign. Nevertheless, inconsistent associations were observed between the pathogens and water quality. The yearlong occurrence and abundance of Vibrionaceae in the environmental components indicate that they might serve as reservoirs of these pathogens.
Group C Streptococcus dysgalactiae (GCSD) is a pathogen of farmed fish. Almost all GCSD isolates from Asian countries, including Japan, Taiwan, Malaysia, and China, have a serum opacity factor (SOF-FD). Although the SOF-FD sequences in different GCSD isolates are identical, different opacification activities are observed. Three types of variations were observed in the upstream sequence of the sof-FD gene in GCSD isolates with different SOF-FD activities. Type 1 was characterized by insertion of an IS981-like element into the upstream region of the sof-FD gene. In Type 2, an IS981-like element was inserted into the upstream region in a direction opposite to that in Type 1. In Type 3, no IS element was inserted. Type 1 was predominant among Japanese isolates (129 of 133). Isolates from other Asian countries were generally Type 3 (13 of 16). Except for 1 strain, Type 1 strains exhibited opacification activities with optical densities (ODs)>0.6, while Type 2 and Type 3 strains have low opacification activities (ODs >0.2). Only Type 1 strains have putative -10 and -35 promoter regions upstream of the sof-FD gene, and the expression level of the sof-FD gene was higher in Type 1 strains than in Type 2 and Type 3 strains.
In September 2014, a freshwater oomycete was first isolated from Asian Seabass Lates calcarifer fry that were reared in freshwater at a fish hatchery in Sabah, Malaysia. A fungal strain was isolated from infected fry by using glucose yeast extract (GY) agar. From morphological identification, the strain belonged to the genus Achlya based on the mode of zoospore release. Molecular phylogenetic analysis of the internal transcribed spacer region sequences from the strain showed high similarity (99-100%) to Achlya oblongata. The isolate was able to grow on GY agar incubated at 15-35°C, in GY broth adjusted to pH 3.0-11.0, and in up to 1.0% NaCl. This is the first report of Achlya infection in freshwater-reared Asian Seabass in Malaysia.
Swabs from the brain, eyes and kidneys of tilapia from 11 farms were collected for a period of 2 years. They were grown on blood agar before cultures of suspected Staphylococcus aureus were subjected to ABI STAPH Detection Kit and PCR for identification. They were then grown on oxacillin resistance screening agar base (ORSAB) and subjected to PCR using the MRSA 17 kb forward and reverse primers to identify the methicillin-resistant S. aureus (MRSA). A total of 559 isolates of Staphylococcus spp. were obtained, from which 198 (35%) isolates were identified as S. aureus. Of the 198 S. aureus isolated from tilapias, 98 (50%) were identified as methicillin-resistant S. aureus (MRSA). Since global spread of multi-drug-resistant bacteria has increased in the past decade, this new finding in fish should be of concern.
Vibrio parahaemolyticus has long been known pathogenic to shrimp but only recently it is also reported pathogenic to tropical cultured marine finfish. Traditionally, bacterial diseases in aquaculture are often treated using synthetic antibiotics but concern due to side effects of these chemicals is elevating hence, new control strategies which are both environmental and consumer friendly, are urgently needed. One promising control strategy is the bacteriophage therapy. In this study, we report the isolation and characterization of a novel vibriophage (VpKK5), belonging to the family Siphoviridae that was specific and capable of complete lysing the fish pathogenic strain of V. parahaemolyticus. The VpKK5 exhibited short eclipse and latent periods of 24 and 36 min, respectively, but with a large burst size of 180 pfu/cell. The genome analysis revealed that the VpKK5 is a novel bacteriophage with the estimated genome size of 56,637 bp and has 53.1% G + C content. The vibriophage has about 80 predicted open reading frames consisted of 37 complete coding sequences which did not match to any protein databases. The analysis also found no lysogeny and virulence genes in the genome of VpKK5. With such genome features, we suspected the vibriophage is novel and could be explored for phage therapy against fish pathogenic strains of V. parahaemolyticus in the near future.
In view of the epidemiological success of CTX-M-15-producing lineages of Escherichia coli and particularly of sequence type 131 (ST131), it is of significant interest to explore its prevalence in countries such as India and to determine if antibiotic resistance, virulence, metabolic potential, and/or the genetic architecture of the ST131 isolates differ from those of non-ST131 isolates. A collection of 126 E. coli isolates comprising 43 ST131 E. coli, 40 non-ST131 E. coli, and 43 fecal E. coli isolates collected from a tertiary care hospital in India was analyzed. These isolates were subjected to enterobacterial repetitive intergenic consensus (ERIC)-based fingerprinting, O typing, phylogenetic grouping, antibiotic sensitivity testing, and virulence and antimicrobial resistance gene (VAG) detection. Representative isolates from this collection were also analyzed by multilocus sequence typing (MLST), conjugation, metabolic profiling, biofilm production assay, and zebra fish lethality assay. All of the 43 ST131 E. coli isolates were exclusively associated with phylogenetic group B2 (100%), while most of the clinical non-ST131 and stool non-ST131 E. coli isolates were affiliated with the B2 (38%) and A (58%) phylogenetic groups, respectively. Significantly greater proportions of ST131 isolates (58%) than non-ST131 isolates (clinical and stool E. coli isolates, 5% each) were technically identified to be extraintestinal pathogenic E. coli (ExPEC). The clinical ST131, clinical non-ST131, and stool non-ST131 E. coli isolates exhibited high rates of multidrug resistance (95%, 91%, and 91%, respectively), extended-spectrum-β-lactamase (ESBL) production (86%, 83%, and 91%, respectively), and metallo-β-lactamase (MBL) production (28%, 33%, and 0%, respectively). CTX-M-15 was strongly linked with ESBL production in ST131 isolates (93%), whereas CTX-M-15 plus TEM were present in clinical and stool non-ST131 E. coli isolates. Using MLST, we confirmed the presence of two NDM-1-positive ST131 E. coli isolates. The aggregate bioscores (metabolite utilization) for ST131, clinical non-ST131, and stool non-ST131 E. coli isolates were 53%, 52%, and 49%, respectively. The ST131 isolates were moderate biofilm producers and were more highly virulent in zebra fish than non-ST131 isolates. According to ERIC-based fingerprinting, the ST131 strains were more genetically similar, and this was subsequently followed by the genetic similarity of clinical non-ST131 and stool non-ST131 E. coli strains. In conclusion, our data provide novel insights into aspects of the fitness advantage of E. coli lineage ST131 and suggest that a number of factors are likely involved in the worldwide dissemination of and infections due to ST131 E. coli isolates.
Epizootic ulcerative syndrome (EUS) is a devastating fish disease caused by the fungus, Aphanomyces invadans. Rapid diagnosis of EUS is needed to control and treat this highly invasive disease. The current diagnostic methods for EUS are labor intensive. We have developed a highly sensitive and specific electrochemical genosensor towards the 18S rRNA and internal transcribed spacer regions of A. invadans. Multiple layers of latex were synthesized with the help of polyelectrolytes, and labeled with gold nanoparticles to enhance sensitivity. The gold-latex spheres were functionalized with specific DNA probes. We describe here the novel application of this improved platform for detection of PCR product from real sample of A. invadans using a premix sandwich hybridization assay. The premix assay was easier, more specific and gave higher sensitivity of one log unit when compared to the conventional method of step-by-step hybridization. The limit of detection was 0.5 fM (4.99 zmol) of linear target DNA and 1 fM (10 amol) of PCR product. The binding positions of the probes to the PCR amplicons were optimized for efficient hybridization. Probes that hybridized close to the 5' or 3' terminus of the PCR amplicons gave the highest signal due to minimal steric hindrance for hybridization. The genosensor is highly suitable as a surveillance and diagnostic tool for EUS in the aquaculture industry.
The grow out of Asian seabass Lates calcarifer in marine net-cages is a popular aquaculture activity in Malaysia. Production of this species is greatly affected by the occurrence of vibriosis, which causes heavy mortality. Generally, young fish are more susceptible; they exhibit anorexia and skin darkening, followed by heavy mortality. The acutely affected older fish may also exhibit bloody lesions around the anus and the base of the fins. Twenty-one bacterial isolates obtained from internal organs (kidney, heart, spleen and liver) of the affected specimens were subjected to phenotypic characterization, testing for antibiotic susceptibility, and 16S ribosomal DNA sequencing. The sequencing result showed that all of the bacterial isolates belonged to Vibrio harveyi. The phenotypic characterization, however, identified 4 of the bacterial isolates as V. harveyi, 16 as V. parahaemolyticus, and 1 as V. alginolyticus. These findings suggest that biochemical features alone cannot be reliably used to identify bacterial pathogens, including V. harveyi, in aquaculture. Antibiotic susceptibility assays showed that some antibiotics, including oxytetracycline, nitrofurantoin, furazolidone, streptomycin, sulfamethoxazole, chloramphenicol, nalidixic acid, and oxolinic acid were effective against V. harveyi. Considering the side effects of these antibiotics, however, their use is not recommended in the aquaculture of Asian seabass.
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 LD50-336 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.
In this study, we report the starvation effect and vibriosis infection on a tropical fish, the tiger grouper (Epinephelus fuscoguttatus). The tiger groupers were infected with Vibrio vulnificus for 21 days. Gas chromatography-mass spectrometry combined with multivariate analysis was used to assess the variation in metabolite profiles of E. fuscoguttatus. Metabolite productions in infected fishes were significantly influenced by fatty acid production. The Omega 9 (ω-9) was abundant under the challenged conditions compared to Omega 3 (ω-3) and Omega 6 (ω-6). A total of six fatty acids from the ω-9 group were detected in high concentration in the infected fishes compared to the control groupers. These metabolites are Oleic acid, Palmitoleic acid, 6,9-Octadecenoic acid, 8,11-Eicosadienoic acid, cis-Erucic acid and 5,8,11-Eicosatrienoic acid. The production of ω-9 differed significantly (p ≤ 0.001) in the challenged samples. The detected ω-9 compounds were quantified based on three different extraction techniques with Supelco 37-component FAME mix (Supelco, USA). The highest concentration of ω-9 groups compared to the other fatty acids detected is 1320.79 mg/4 g and the lowest is 939 mg/4 g in challenged-starved; meanwhile, in challenged-fed, the highest concentration detected is 1220.87 mg/4 g and the lowest is 917.25 mg/4 g. These changes demonstrate that ω-9 can be used as a biomarker of infection in fish.
In this study, we reported a molecular characterization of a novel proto-type galectin-1 from the striped murrel Channa striatus (named as CsGal-1). The full length CsGal-1 was identified from an established striped murrel cDNA library and further we confirmed the sequence by cloning. The complete cDNA sequence of CsGal-1 is 590 base pairs (bp) in length and its coding region encoded a poly peptide of 135 amino acids. The polypeptide contains a galactoside binding lectin domain at 4-135. The domain carries a sugar binding site at 45-74 along with its signatures (H(45)-X-Asn(47)-X-Arg(49) and Trp(69)-X-X-Glu(72)-X-Arg(74)). CsGal-1 shares a highly conserved carbohydrate recognition domain (CRD) with galectin-1 from other proto-type galectin of teleosts. The mRNA expressions of CsGal-1 in healthy and various immune stimulants including Aphanomyces invadans, Aeromonas hydrophila, Escherchia coli lipopolysaccharide and poly I:C injected tissues of C. striatus were examined using qRT-PCR. CsGal-1 mRNA is highly expressed in kidney and is up-regulated with different immune stimulants at various time points. To understand its biological activity, the coding region of CsGal-1 gene was expressed in an E. coli BL21 (DE3) cloning system and its recombinant protein was purified. The recombinant CsGal-1 protein was agglutinated with mouse erythrocytes at a concentration of 4μg/mL in a calcium independent manner. CsGal-1 activity was inhibited by d-galactose at 25mM(-1) and d-glucose and d-fructose at 100mM(-1). The results of microbial binding assay showed that the recombinant CsGal-1 protein agglutinated only with the Gram-negative bacteria. Interestingly, we observed no agglutination against Gram-positive bacteria. Overall, the study showed that CsGal-1 is an important immune gene involved in the recognition and elimination of pathogens in C. striatus.