Characterization of a Probiotic Lactobacillus fermentum Isolated from Snakehead,Channa striatus,Stomach

This study sought to isolate and characterize lactic acid bacteria (LAB) from stomach of adult snakehead fish, Channa striatus, to be used as probiotics for freshwater fish. A total of 13 strains were isolated from the stomach of 10 fish, and 4 of these belonged to LAB. Strain LAB‐3 showing highest in vitro growth inhibition of Aeromonas hydrophila in a disk diffusion test was identified as Lactobacillus fermentum by conventional and molecular techniques and evaluated in vitro through various tests. The bacterium could grow at pH 3–8; but the optimum growth was observed at pH 6. Moreover, LAB‐3 grew at 0.15 and 0.3% bile salt concentrations, from 15 to 45 C, and at 4% NaCl. L. fermentum showed in vitro inhibitory activity against three fish pathogens, A. hydrophila, Pseudomonas aeruginosa, and Shewanella putrefaciens, tested by disk diffusion and well diffusion methods. Antibiotic sensitivity tests indicated that L. fermentum was resistant to streptomycin, gentamycin, and kanamycin, intermediate to tetracycline, but sensitive to chloramphenicol, amoxicillin, and ampicillin. Challenge test by using A. hydrophila showed that survival of snakehead was significantly (P < 0.05) improved when 2 × 10⁶ LAB‐3/g was supplemented to the diet. Therefore, this study suggests that L. fermentum might be a promising probiotic in snakehead aquaculture .     

Disease Problems Affecting Fish in Tropical Environments

ABSTRACT

Aquaculture produce in Asian countries contributes a major share to total global fish production. However, disease due to biotic and abiotic factors results in considerable losses to the industry. The most frequently encountered bacterial agents associated with fish diseases in tropical environments are Vibrio sp. in marine and brackish-water systems and motile aeromonads in freshwater systems. Virulence mechanisms of these bacterial species have been widely studied. Though a few viral agents such as rhabdovirus, reo-like virus, infectious pancreatic necrosis virus, picornavirus, and irido-like virus have been recorded in different parts of Asia, such reports are scanty. Among fungal pathogens, Aphanomyces invaderis is the most virulent and has been reported to be associated with epizootic ulcerative syndrome. Other fungal agents such as Achlya sp. and Saprolegnia sp. have been recorded in various disease conditions. In addition to various conventional microbiological and serological methods, nucleic acid-based methods are being widely adopted for diagnosis of different fish diseases. Few successful vaccines have been developed to protect against disease conditions, and some of the better known ones are against vibriosis and furunculosis. Many other vaccines are still in the experimental stages. Early diagnosis of disease and development of successful vaccines are important for the future development of aquaculture.     

Isolation and Antimicrobial Resistance of Staphylococcus spp. in Freshwater Fish and Greek Marketplaces

The presence and antibiotic resistance of Staphylococcus spp. in freshwater fish and the environment of fish markets of Northern Greece were investigated. A total of 269 samples were examined, consisting of 71 rainbow trout (Oncorhynchus mykiss), 65 gibel carp (Carassius gibelio), and 133 environmental samples swabbed from various surfaces at fish markets.Staphylococcus spp. was isolated from 27% of the samples, and 16%, 9%, 0.7%, 0.4% and 0.4% were found to be positive for S. aureus, S. epidermidis, S. warneri, S. hominis-hominis, and S. haemolyticus, respectively. All isolates were examined for their susceptibility to 20 antibiotics. None of the S. aureus isolates were resistant to oxacillin and glycopeptides. Three S. epidermidis, two S. Warneri, and one S. haemolyticus isolates were resistant to oxacillin (methicillin resistant Staphylococcus, MRS). All isolates, except one, showed resistance to as many as 15 antibiotics. The population of Staphylococcus spp. in fish did not exceed 1.0 log CFU/g. Freshwater fish and the environments of retail fish markets were found to harbor multi-drug resistant staphylococci. Whether these findings present a real health risk for humans and to what extent needs to be evaluated.     

Characterization of spaC‐type Erysipelothrix sp. isolates causing systemic disease in ornamental fish

Since 2012, low‐to‐moderate mortality associated with an Erysipelothrix sp. bacterium has been reported in ornamental fish. Histological findings have included facial cellulitis, necrotizing dermatitis and myositis, and disseminated coelomitis with abundant intralesional Gram‐positive bacterial colonies. Sixteen Erysipelothrix sp. isolates identified phenotypically as E. rhusiopathiae were recovered from diseased cyprinid and characid fish. Similar clinical and histological changes were also observed in zebrafish, Danio rerio, challenged by intracoelomic injection. The Erysipelothrix sp. isolates from ornamental fish were compared phenotypically and genetically to E. rhusiopathiae and E. tonsillarum isolates recovered from aquatic and terrestrial animals from multiple facilities. Results demonstrated that isolates from diseased fish were largely clonal and divergent from E. rhusiopathiae and E. tonsillarum isolates from normal fish skin, marine mammals and terrestrial animals. All ornamental fish isolates were PCR positive for spaC, with marked genetic divergence (<92% similarity at gyrB, <60% similarity by rep‐PCR) between the ornamental fish isolates and other Erysipelothrix spp. isolates. This study supports previous work citing the genetic variability of Erysipelothrix spp. spa types and suggests isolates from diseased ornamental fish may represent a genetically distinct species.     

Genotypic and phenotypic characterization of Edwardsiella isolates from different fish species and geographical areas in Asia: Implications for vaccine development

The genus Edwardsiella is one of the major causes of fish diseases globally. Herein, we examined 37 isolates from ten different fish species from India, South Korea and Taiwan to gain insight into their phenotypic and genotypic properties, of which 30 were characterized as E. tarda with phenotypic homology estimated at 85.71% based on API‐20E biochemical tests. Genotyping using 16S rRNA put all isolates together with E. anguillarum, E. hoshinae, E. tarda, E. piscicida and E. ictaluri reference strains in a monophyletic group. In contrast, the gyrB phylogenetic tree clearly separated E. ictaluri, E. tarda and E. hoshinae reference strains from our isolates and put our isolates into two groups with group I being homologous with the E. anguillarum reference strain while group II was homologous with the E. piscicida reference strain. Hence, our findings point to E. piscicida and E. anguillarum as species infecting different fish species in Asia. Homology of the ompW protein suggested that strains with broad protective coverage could be identified as vaccine candidates. This study underscores the importance of combining genotyping with phenotyping for valid species classification. In addition, it accentuates the importance of phylogenetic comparison of bacterial antigens for identification of potential vaccine candidates.     

Susceptibility of a number of Australian freshwater fishes to dwarf gourami iridovirus (Infectious spleen and kidney necrosis virus)

Megalocytiviruses cause high mortality diseases that have seriously impacted aquaculture, with the most frequent outbreaks occurring in East and South‐East Asia. The international trade of juvenile fish for food and ornamental aquaculture has aided the spread of these viruses, which have spread to Europe and Australia and other regions. Australian freshwater fishes were examined for susceptibility to infection with the exotic megalocytivirus, dwarf gourami iridovirus (DGIV), which belongs to a group with the type species, Infectious spleen and kidney necrosis virus (ISKNV). Fish were held at 23 ± 1 °C and challenged by intraperitoneal (IP) injection or by cohabitation with Murray cod, Maccullochella peelii (Mitchell) infected with DGIV. A species was deemed to be susceptible to DGIV based on evidence of viral replication, as determined by qPCR, and megalocytic inclusion bodies observed histologically. Horizontal transmission occurred between infected Murray cod and golden perch, Macquaria ambigua (Richardson), Macquarie perch, Macquaria australasica (Cuvier) and Murray cod. This indicated that DGIV shed from infected fish held at 23 °C can survive in fresh water and subsequently infect these naïve fish. Further, DGIV administered IP was highly pathogenic to golden perch, Macquarie perch and Murray cod. Compared to these species, the susceptibility of southern pygmy perch, Nannoperca australis (Gunther) was lower. Freshwater catfish (dewfish), Tandanus tandanus (Mitchell), were not susceptible under the experimental conditions based on the absence of clinical disease, mortality and virus replication. This study showed the potential risks associated with naïve and DGIV‐infected fish sharing a common water source.     

Identification of bacteria from the normal flora of perch, Perca fluviatilis L., and evaluation of their inhibitory potential towards Aeromonas species

Pathogenic Aeromonas sobria has been identified as a causative agent of ulcerative disease in farmed European perch, Perca fluviatilis L. To study the effect of the normal intestinal bacterial flora of perch against A. sobria, we sampled 193 bacterial isolates from the perch digestive tract. The isolates were identified by sequence analysis of the 16S rRNA gene and their inhibitory potential against A. sobria was evaluated in vitro. Nineteen of the strains isolated showed inhibition and were also tested against other aeromonad and non-aeromonad fish pathogens including Yersinia ruckeri and Vibrio anguillarum. Isolates showing inhibition were primarily Pseudomonas spp.; however, inhibitory Shewanella spp., and Delftia sp. were also identified. A Pseudomonas chlororaphis isolate showed inhibition against all fish pathogens tested.     

Effect of lipid levels on the reproductive performance of Snakehead murrel,Channa striatus

Present study aimed to determine the optimum dietary lipid level in snakehead murrel channa striatus broodstocks. Triplicate groups of fish were fed for 240 days with isonitrogenous experimental diets with increasing lipid levels (100, 140, and 180 g kg^?1), using fish oil and soybean oil as the lipid sources with the ratio of (1:1). Weight gain, GSI, fecundity, oocyte diameter and number of mature oocyte were found to be significantly higher in the group fed with diet containing 180 g kg^?1 lipid level. Muscle fatty acid profile showed a significant increase in LA (18:2n‐6), LNA (18:3n3), total PUFA, n‐6 and ArA (20:4n‐6) in fish fed with diet containing 180 g kg^?1 lipid. Increasing lipid level up to 180 g kg^?1 resulted in significant increase in PUFA (LA & LNA), lc‐PUFA (EPA, DHA, ArA), total PUFA, n‐3 and n‐6 series in ovary and liver of female C. striatus.     

First isolation of hirame rhabdovirus from freshwater fish in Europe

A rhabdovirus was isolated in cell culture inoculated with tissue material from diseased grayling, Thymallus thymallus (L.), originating from a fish farm affected by a mortality episode in Poland. Diagnostics tests showed that the virus was not related to novirhabdoviruses known in Europe, nor to vesiculovirus‐like species, except perch rhabdovirus (PRhV) with which it shared moderate serological relations. However, RT‐PCR with PRhV probes gave negative results. To identify the virus, a random‐priming sequence‐independent single primer amplification was adopted. Surprisingly, two of the obtained sequences exhibited a high identity (>99%) with hirame rhabdovirus (HIRRV), a novirhabdovirus usually found in fish in marine Asiatic countries, for instance Japan, China and Korea. The full‐length sequence of the phosphoprotein gene (P) demonstrated a higher identity of the present isolate with HIRRV from China compared with the Korean isolate. An identical viral sequence was also found in brown trout, Salmo trutta trutta L., affected by mortalities in a second farm in the same region, after a likely contamination from the grayling farm. To our knowledge, this is the first report of HIRRV in Europe, and in two hosts from fresh water that have not been described before as susceptible species.     

Novel non-motile phenotypes of Yersinia ruckeri suggest expansion of the current clonal complex theory

The biochemical and cell surface characteristics of 63 non‐motile isolates of Yersinia ruckeri from various sources were compared using the API 20E rapid identification system and conventional phenotypic methods. Eight individual phenotypic groups from a variety of fish species were observed from the data set. Non‐motile isolates were not exclusively observed from serogroup O1; membership of biotype 2 was recorded for representatives from serogroups O2–O7. Variations in phenotypes highlights that new clonal groups are arising and that the current typing scheme requires expansion. Previously, it was hypothesized that disease was caused by a few virulent clones; data in this paper suggests that this assumption is not the case. The lipopolysaccharide (O antigen) type in the non‐motile biotype was different from other isolates of Y. ruckeri.     

Epidemiological study on <Emphasis Type="Italic">Lactococcus garvieae</Emphasis> isolates from fish in Japan

In Japan, Lactococcus garvieae infection has been the main fish disease in aquaculture. Although commercial oral and injectable vaccines have been used to prevent L. garvieae infection in Japan, L. garvieae has been isolated not only from unvaccinated fish but also from vaccinated fish in which immunity induced by vaccination had diminished. In order to obtain epidemiological information on this fish pathogen, we conducted biased sinusoidal field gel electrophoresis (BSFGE) pattern analysis and phage typing of L. garvieae isolates (n = 427) from fish in Japan. These isolates were obtained from 13 different fish species between 1980 and 2007. In the BSFGE analysis, L. garvieae isolates were classified into 17 groups (S1–S17) based on the SmaI digestion patterns and into four groups (A1–A4) based on the ApaI digestion patterns. Phage typing revealed five different phage susceptibility profiles (A–E) in L. garvieae isolates. Since 2005, comparisons of the results of phage typing and BSFGE have indicated the presence of a novel genotype (S16/A4) with phage type E. All the strains belonging to this type showed lincomycin sensitivity.     

Weaning methods using formulated feeds for snakehead (Channa striata and Channa micropeltes) larvae

The culture of snakehead fish (Channa striata and Channa micropeltes) in Vietnam is limited, and snakehead culture has been banned in Cambodia, because traditional practices include capture of fingerlings from the wild as seed, as well as capture of small‐size (also known as trash fish or low‐value) fish. As hatchery breeding technology has improved, we investigated the optimal weaning practices for these two species. Both laboratory experiments and farm trials were conducted. For C. striata, the optimal weaning procedure is to begin at 17 days after hatch (dah) and wean the fish at 10% replacement of live feed with formulated feed per day. However, for C. micropeltes, the optimal procedure is to wait until 40 dah to begin weaning and then to wean the fish with a 10% replacement of live feed with formulated feed every 3 days. These results should enable farmers to domesticate snakehead culture in Vietnam and Cambodia and eliminate reliance on fish captured from the wild as both seed and feed.     

Prey selection and growth in 0+ Eurasian perch Perca fluviatilis L. in littoral zones of seven temperate lakes

We studied the relationships between the planktonic food base and feeding patterns of juvenile mid-summer/early autumn Eurasian perch Perca fluviatilis L., a common predatory freshwater fish in large parts of Europe and Asia. The feeding of 0+ perch was studied during summer and autumn in littoral habitats of seven lakes with different environmental conditions – four Latvian (Auciema, Riebinu, Vārzgūnes, Laukezers) and three Estonian (Kaiavere, Prossa and Akste) lakes. Simultaneously, the abundance, biomass and structure of zooplankton communities were examined. We focused on the littoral areas because many studies in lakes suggest that littoral habitats are particularly important for 0+ fish growth and survival. We were interested in the question: can the diet and growth of 0+ perch be explained by zooplankton community structure? We also presumed that if the amount of zooplankton is low, more benthic invertebrates will be consumed by 0+ perch. Opposite to expectations, we found that zooplankton always counted for over 90% of diet biomass in perch. There were also clear correlations between the zooplankton biomass in a given lake, the zooplankton biomass in 0+ perch stomachs, and the fish growth rate. The study also suggested that nutrient enrichment can positively impact the 0+ perch feeding conditions in lakes.     

Comparison of rhabdoviruses associated with epizootic ulcerative syndrome (EUS) with respect to their structural proteins, cytopathology and serology

Abstract. Seven rhabdoviruses isolated from fish suffering from epizootic ulcerative syndrome (EUS) were compared in terms of their morphology, cytopathogenicity, antigenic relatedness and structural polypeptide composition. All strains exhibited a bullet-shaped morphology, but the T9204 isolate was found to be longer and more variable in length than the other strains. Sixteen fish cell lines investigated showed some variation in susceptibility to each isolate, but the cytopathic changes induced by T9204 in SSN-2, RSN, GCP, ONP, FHM, AS and MUL lines were significantly different from the other isolates. Polyclonal antisera raised against the BPV, 20E and SL11 strains neutralized six isolates (BPV, 02, 19, 20E, A4 and SL11), but not T9204. Conversely, anti-T9204 serum only neutralized homologous virus. Polyacrylamide gel electrophoresis demonstrated that BPV, 02, 19, 20E, A4 and SL11 had virtually identical protein profiles, whereas T9204 differed both in the number of protein bands and in their migration pattern. Western blots of these gels identified the proteins specific to T9204 that reacted with anti-T9204 serum. Therefore, the isolates represent two distinct species of fish rhabdoviruses, but as yet, no causal relationship with EUS, or any other disease condition, has been established.     

Genotypic diversity among Shewanella spp. collected from freshwater fish

Different Shewanella species are isolated both from healthy and from diseased fish. To date, contemporary methods do not provide sufficient insight to determine species and detail differentiation between tested strains. Bacteria isolated from cultured (n = 33), wild (n = 12) and ornamental (n = 6) fish, as well as several reference strains, were tested by 16S rRNA gene sequencing, ERIC‐PCR and pulsed‐field gel electrophoresis (PFGE) assays. Our study indicates that isolates collected from freshwater fish were genetically diverse. Based on 16S rRNA gene sequences, bacteria were clustered into groups S. putrefaciens, S. xiamenensis and S. oneidensis. Some isolates were classified only to genus Shewanella; thus, 16S rRNA gene analyses were not enough to determine the species. ERIC‐PCR revealed 49 different genotype profiles indicating that the method might be useful for differentiation of Shewanella isolates irrespectively to species identification, contrary to PFGE which is not suitable for Shewanella typing.     

Use of formalin and copper to control ichthyophthiriosis in the Australian freshwater fish silver perch (Bidyanus bidyanus Mitchell)

Infestations of the protozoan parasite, Ichthyophthirius multifiliis, cause the serious disease ichthyophthiriosis in freshwater fish throughout the world. Formalin is a recommended treatment for ichthyophthiriosis in the Australian fish silver perch (Bidyanus bidyanus Mitchell), but the disease is difficult to control in ponds, particularly at low water temperatures. Experiments were carried out to develop an improved treatment regime for formalin and to evaluate copper as a therapeutant. Silver perch fingerlings infested with I. multifiliis were stocked into 55 L aquaria at temperatures of 14.8–17.6 °C and alkalinities of 70–110 mg L^?1. Formalin (34–38% formaldehyde) or copper (24.5% copper sulphate) were added to the aquaria and then monitored and readjusted to nominal concentrations daily. A concentration of 30 mg L^?1 formalin controlled ichthyophthiriosis, but fish treated with 20 mg L^?1 remained infested with theronts and trophonts on day 17; survival at both concentrations was 100%. A concentration of 10 mg L^?1 formalin did not control ichthyophthiriosis and all fish were dead from the infestation by day 17. Fish treated with 0.1 or 0.2 mg L^?1 copper were free of theronts and trophonts by days 17 and 14, respectively, and survival was 100%. Survival at 0.05 mg L^?1 copper was 100%, but fish remained infested. At 0.25 mg L^?1 copper, survival was 82.5% and there were no theronts or trophonts on gill and skin tissues of fingerlings by day 14. There was total mortality of fish treated with 0.5 or 1.0 mg L^?1 copper suggesting these concentrations are toxic to silver perch. All fish in infested‐control treatments died. In earthen ponds containing silver perch, 0.2 mg L^?1 copper was depleted to below 0.1 mg L^?1 within 24 h, and concentrations of 25–38 mg L^?1 formalin were depleted to below 15 mg L^?1 within 48 h. Treatment regimes involving daily applications of formalin or copper controlled ichthyophthiriosis in silver perch in earthen ponds at costs of $US466.37 and $US65.58 hectare^?1 day^?1 respectively. This study has developed a new formalin‐treatment regime for the control of ichthyophthiriosis, and demonstrated that copper sulphate is a potential therapeutant for this serious disease of silver perch.     

Euclinostomum heterostomum and E. ardeolae in tilapia species of Aswan Governorate,Egypt: morphological,molecular, and histopathological characterization

Euclinostomum heterostomum and Euclinostomum ardeolae, both encysted metacercariae (EMC), were found to infect farmed Oreochromis niloticus in the Sahary fish hatchery and wild O. niloticus, Sarotherodon galilaeus, and Tilapia zillii in Lake Nasser, Aswan Governorate, Egypt, at a prevalence of 25.25% and an infection intensity of 1–14 EMC/fish. Macroscopic and microscopic examinations were used to identify them morphologically. PCR amplification, sequencing of the 28S large ribosomal RNA region, and phylogenetic analysis were used for molecular characterization. E. heterostomum and E. ardeolae were isolated from farmed O. niloticus kidneys, peritoneum, abdominal cavity, and under the gills attached to the head bone at prevalence of 13.5% and 6%, respectively, and from wild O. niloticus, S. galilaeus, and T. zillii at a prevalence of 15% and 4.5%, 18% and 10%, and 22.5% and 11.5% respectively. The molecular analyses of rRNA-28S marker revealed that the two Euclinostomum species examined are related yet distinct. This is the first molecular and phylogenetic evidence linking E. ardeolae to Euclinostomum. The present study added two new sequences to the GenBank databases for the genus Eulinostomum from Egypt, with accession numbers MW604803 and MW604806. Euclinostomum spp. have been shown to have detrimental and destructive effects on the kidneys of infected fish; these effects may eventually result in the parasites’ cysts replacing the kidney’s tissues. The kidneys of tilapia spp. infected with Euclinostomum spp. displayed degenerative alterations, with dilated renal tubules associated with migratory tunnels composed primarily of leucocytic infiltrations. Euclinostomum spp. exhibited clear hemophagic characteristics.

     

Comparative genomic insights into the taxonomy of Edwardsiella tarda isolated from different hosts: Marine,freshwater and migratory fish

Edwardsiella tarda, a Gram‐negative member of the family Enterobacteriaceae, has been isolated from many animal species worldwide, especially fish species. Its broad host range indicates the diversity in taxonomy, which attracted the attention of many researchers. Here, we added genome of E. tarda strain isolated from freshwater fish to comparative genomics study for the first time. We sequenced and assembled the genome of E. tarda ASE201307 which was isolated from freshwater Asian swamp eel. ASE201307 genome contained a single circular chromosome of 3.68M with G+C 57.09% content. Comparative genomics including SNP calling, synteny block, Core/Pan genes analysis and phylogeny analysis was conducted among ASE201307 and other Edwardsiella strains isolated from different fish species. Results of SNP analysis and synteny block demonstrated the close relative of ASE201307, FL95.01 and DT which were all isolated from freshwater fish. In further analysis heat map of dispensable genes and phylogenetic tree, all E. tarda strains were divided into two groups. One was isolated from freshwater fish and the other was isolated from marine/migratory fish. Based on all studies above, we proposed the living environment of hosts as a new taxonomic character and divided E. tarda isolated from diseased fish into freshwater group and marine/migratory group.     

Identification and partial characterization of potential probiotic lactic acid bacteria in freshwater Labeo rohita and Cirrhinus mrigala

Carps are the most diversified freshwater fish belonging to family Cyprinidae. Numerous probiotic and pathogenic lactic acid bacteria (LAB) have been characterized from carps. However, the diversity of these ecologically important bacteria is entirely unknown in freshwater fish of Pakistan. The present study aimed to characterize and identify the lactic acid bacteria from two carps viz. Laboe rohita and Cirrhinus mrigala and determine their antagonistic activity. Seventeen bacterial isolates were purified from the gastrointestinal tract and gills of these fish and characterized morphologically. Initially, seven isolates were screened as LAB using agar supplemented with CaCO₃. Subsequently, only two isolates CILB2 and RIL10 were selected as LAB after high‐performance liquid chromatography analysis for lactic acid production. Isolates CILB2 and RIL10 were genetically identified as Enterococcus faecalis and Weissella sp., respectively after 16S rRNA gene sequence analysis. Both strains exhibited significant antagonistic activity against common fish pathogens Streptococcus agalactiae, Staphylococcus aureus and Escherichia coli. Enterococcus faecalis CILB2 and Weissella sp. RIL10 were also found negative for haemolysis and gelatinase activities and were sensitive to ampicillin, amoxicillin, doxycycline, erythromycin, chloramphenicol and co‐trimoxazole antibiotics. The identified LAB strains may further be investigated for their potential probiotic application in fish feed and food preservation techniques.