Fermentative Properties of Proteolytic Pediococcus Strains Isolated from Salt Fermented Fish Hydrolysate Prepared Using Freshwater Fish Rohu (Labeo rohita)

Salt fermented fish hydrolysate (SFFH) was prepared from freshwater fish Rohu (Labeo rohita), an Indian major carp, using salt at room temperature for 50 days. Total lactic acid bacteria (LAB) counts of SFFH prepared using 20% and 30% NaCl (w/w) were found to be in the range of 5.83–7.25 log cfu/ml and 3.93–5.46 log cfu/ml, respectively, whereas the log cfu/ml values for the halotolerant LAB counts varied from 2.09 to 7.17 and 2.02 to 4.77, respectively. Out of 96 isolates from SFFH, 21 isolates were screened based on their pH reducing ability, total titratable acidity, and proteolytic properties. Morphological, biochemical, and molecular characterization of four selected LAB strains were carried out. The isolates FSAP3-3 (Pediococcus pentosaceus), FSBP4-40 (Pediococcus pentosaceus), FSBP16-40 (Pediococcus pentosaceus), and FSBP28-50 (Pediococcus acidilactici) showed both halotolerant and proteolytic properties. The identified LAB isolates showed antagonistic properties against several human pathogens and exhibited sensitivity toward a broad spectrum of antibiotics. All the identified LAB isolates showed excellent proteolytic activity and phosphohydrolase activity. These halotolerant LAB isolates that had both proteolytic properties and acidifying ability have the potential for application in an acceleration of fermentation to produce salted fish products (i.e., fish sauce, fish paste, fish mince, etc.).     

Evaluation of the INNO‐LiPA mycobacteria v2 assay for identification of aquatic mycobacteria

Fifty‐seven isolates of mycobacteria comprising 10 reference strains, 47 field isolates and one non‐Mycobacterium isolate were screened using commercial INNO‐LiPA v2 assay kits. All mycobacteria isolates tested hybridized with the Mycobacterium genus probe on the LiPA strip. All M. marinum, M. fortuitum and M. chelonae reference and field strains and three out of the four M. gordonae isolates hybridized to their corresponding species‐ or complex‐specific probes. Two cultures (a type strain and a field isolate) yielded mixed growth of two mycobacterial species, i.e. M. chelonae and M. fortuitum. A Mycobacterium isolate from one of these cultures was subsequently purified and correctly identified with the kit. However, sequence analysis of the 16S–23S rRNA internal transcribed spacer (ITS) region of various mycobacteria isolates revealed a misidentification of M. shottsii and M. pseudoshottsii with the kit because these isolates reacted with the M. marinum/M. ulcerans probe. Moreover, nine of the 13 field isolates presumed to be M. fortuitum from the results of the kit had closer ITS sequence homology with M. conceptionense, a species which, to our knowledge, has never been reported in fish. These findings highlight the need to redesign the M. fortuitum–M. peregrinum probe included in the INNO‐LiPA assay and to introduce additional complex‐specific probes into the kit. Nevertheless, the kit proved to be a rapid and reliable method for identifying mycobacteria in the aquatic environment and would be particularly useful in laboratories without sequencing facilities.     

Selection of lactic acid bacteria as candidate probiotics for Vibrio parahaemolyticus depuration in pacific oysters (Crassostrea gigas)

The application of probiotics in food is now widespread and is widely accepted by consumers. Lactic Acid Bacteria (LAB) were isolated from traditional salted fish and then characterized by its ability to inhibit Vibrio parahaemolyticus growth using minimal inhibitory concentration tests. Five out of these strains were identified as Lactobacillus delbrueckii, Lactobacillus casei and three Lactobacillus gasseri by PCR using 16S and 23S rRNA genes. Antibiofilm activity of Lactobacillus spp. extracts were also tested in 96 polystyrene plates. A potential antibiofilm effect was demonstrated as most LAB. Although most LAB extract were able to eradicate pre‐formed biofilm, results demonstrated that five Lactobacillus spp. exhibited a stronger inhibitory effect against V. parahaemolyticus in infected oysters. Vibrio parahaemolyticus viable cells number declined from 10⁶ UFC to 10⁴ UFC after 3 days of incubation with Lactobacillus spp. Probiotic applications, in biological control of seafood associated pathogens can be an alternative solution, providing consumer with a product of good quality owing to the use of non‐toxic compounds. Based on our results, LAB could be used as a bioprotective culture in oyster's depuration to prevent V. parahaemolyticus growth.     

Prevalence and different characteristics of two serotypes of Streptococcus parauberis isolated from the farmed olive flounder, Paralichthys olivaceus (Temminck and Schlegel), in Korea

The prevalence of two serotypes of Streptococcus parauberis isolated from the olive flounder, Paralichthys olivaceus, was evaluated in a total of 29 isolates between 2003 and 2010 in Korea. Streptococcus parauberis isolates were divided into two serologically distinct types (serotype 1 and serotype 2), except for one strain (S1091), using an agglutination assay with rabbit antiserum, and serotype 1 was identified as the dominant type (24 of 29 isolates) in this study. To identify the characteristics of the two serotypes of S. parauberis, we conducted a biochemical test using the API 20 Strep kit, a transmission electron microscopy (TEM) assay, sequence analysis of 16S‐23S rRNA intergenic spacer region (ISR) and a pathogenicity test. In TEM, both serotypes possessed polysaccharide capsule layers around the cell surface when bacterial cells were treated with a homologous serotype of rabbit antiserum. However, we were unable to discriminate serotype‐specific biochemical characteristics and genetic characteristics of 16S‐23S rRNA ISR between the two serotypes. In the pathogenicity test, the serotype 1 strains induced significantly higher mortality than the serotype 2 strains in olive flounder when experimentally inoculated via the intraperitoneal route.     

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 .     

Selection of lactic acid bacteria as candidate probiotics and in vivo test on Artemia nauplii

Lactic acid bacteria (LAB) were isolated from rotifer cultures in a marine hatchery to search for potential probiotics for marine animals. Fifteen strains were first selected among a total of 55, according to antibacterial activity against Vibrio sp. Among eight strains identified as Lactobacillus casei, four were highly adhesive, suggesting some ability for surface colonization. The other strains were identified as Lactobacillus plantarum, Lactobacillus dextrinicus, and Leuconostoc sp. To validate probiotic potential, Artemia were challenged against pathogenic Vibrio alginolyticus, with or without one of six selected LAB strains. The six strains protected Artemia against the pathogen, to some extent on condition that nutrient enrichment was provided. La. casei BR51 and X2 were preferred, as they were efficient even in the absence of nutrient supply. La. casei X2 was finally selected as candidate probiotic, due to the best growth performances of Artemia, with or without the pathogen.     

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.     

The administration of Lactobacillus plantarum directly to food: An effective way to improve the survival and growth of juvenile lined seahorses (Hippocampus erectus)

Previously, we found Lactobacillus plantarum had probiotic effects on lined seahorse (Hippocampus erectus) juveniles by the addition Lactobacillus directly into rearing seawater containing juveniles and their food (i.e. copepods). However, how the juveniles consumed Lactobacillus, directly from water or from copepods in whose guts Lactobacillus may have accumulated by ingestion, is still unclear. To clarify this, three experiments were successively carried out in this study. In the first experiment, the manner by which juveniles consumed Lactobacillus, namely, directly from food, was discovered via a 2 × 2 factorial design with two factors of water and food, and two levels with and without L. plantarum. In the second experiment, a 5 × 4 factorial design with five levels of L. plantarum doses (D: 5, 10, 20, 40 and 80 × 10⁸ cfu/L) and four levels of enrichment time (T: 0.5, 1.0, 1.5 and 2.0 hr) was conducted to analyse lactic acid bacteria (LAB) contents in the enriched copepods under different treatments. In the third experiment, six kinds of Lactobacillus‐enriched copepods chosen from the second experiment that carried significantly different LAB contents were fed to juveniles, and it was found that the copepods containing LAB greater than 5 log₁₀colonies/g had the most improved effects for survival and growth. In conclusion, the effective manner of L. plantarum administration in seahorses is by the addition to their food. Additionally, D40 * T1.0, whose LAB content was more than 5 log₁₀colonies/g, is generally the most economic copepod treatment for improving survival and growth in H. erectus rearing.     

Recovery of Hafnia alvei from diseased brown trout,Salmo trutta L., and healthy noble crayfish,Astacus astacus (L.), in Bulgaria

Hafnia alvei was isolated in Bulgaria from healthy noble crayfish, Astacus astacus (L.), and then from farmed diseased brown trout, Salmo trutta L., with signs of haemorrhagic septicaemia. The isolates were identified initially with conventional phenotyping and commercial Merlin Micronaut and API 20E rapid identification systems, followed by sequencing of the 16S rRNA gene. Hafnia alvei Bt1, Bt2 and Aa4 were of low virulence to rainbow trout and brown trout, although cytotoxicity was demonstrated by Bt1 and Bt2, but not by Aa4.     

Microbial and chemical properties of <Emphasis Type="Italic">aji-no-susu</Emphasis>, a traditional fermented fish with rice product in the Noto Peninsula,Japan

Aji-no-susu is a Japanese fermented fish product prepared from salted horse mackerel Trachurus japonicus, and cooked rice. We studied the organic acid and free amino acid contents and microflora in 12 aji-no-susu products to clarify their features as a lactic-acid-fermented food. Salinity of the samples was approximately 7.0% (rice portion) and 6.0% (fish portion) (w/w). Water activity was approximately 0.9, and pH was approximately 4.4 and lower. In the rice portions, lactic acid content was very high (57 mg/g sample). The predominant amino acids were alanine (2.3 mg/g rice portion) and lysine (2.1 mg/g). In the case of long-fermented (4 and 12 months) aji-no-susu, a high content of gamma-aminobutyric acid (GABA, 1.5 and 1.4 mg/g) was detected. Total viable counts in rice and fish portions were 7.7 and 7.4 log colony-forming units (cfu)/g, respectively. The number of lactobacilli in the rice and fish portions was 7.3 and 7.1 log cfu/g, respectively. Yeasts were detected in eight samples. Furthermore, acid-tolerant lactic acid bacteria (LAB) (Lactobacillus plantarum), GABA-producing LAB (Lactobacillus sp.), and halophilic or halo-tolerant yeast (Debaryomyces hansenii) were isolated and identified. Results in this study indicate that aji-no-susu is a typical traditional lactic-acid-fermented fish product.     

Impacts of Lactobacillus plantarum in Depuration for Reducing Vibrio parahaemolyticus in Pacific Oysters (Crassostrea gigas)

This study investigated potential application of lactic acid bacteria (LAB) in depuration for reducing Vibrio parahaemolyticus in oysters. Lactobacillus plantarum ATCC 8014, which exhibited strong bactericidal effects against V. parahaemolyticus in vitro, was added to artificial seawater for depuration of Pacific oysters (Crassostrea gigas) inoculated with V. parahaemolyticus BE 98-2029 (O3:K6) to levels of about 10⁴ MPN/g at 15 ± 1 and 10 ± 1°C. Application of L. plantarum ATCC 8014 treatment (10⁷ CFU/mL) in oyster depuration did not enhance reductions of V. parahaemolyticus in oysters depurated at 15 ± 1°C but significantly decreased (p < 0.05) levels of V. parahaemolyticus in oysters depurated at 10 ± 1°C after 5 days (3.40 log reductions) when compared with controls (2.75 log reductions). It is not clear if a competitive exclusion by LABs to compete with V. parahaemolyticus binding sites in oyster tissues plays a role in the reduction of V. parahaemolyticus in the oysters. Further studies utilizing different types of LABs in oyster depuration might provide additional knowledge for application of LAB in depuration for decontaminating V. parahaemolyticus in oysters.     

First report on the autochthonous gut microbiota of brown trout (Salmo trutta Linnaeus)

A study was conducted to characterize the autochthonous gut microbiota present in the pyloric caeca (PC), anterior mucosa (AM) and posterior mucosa (PM) of brown trout (Salmo trutta). Total viable counts (TVC) bacterial populations were enumerated using tryptone soy agar, lactic acid bacteria (LAB) levels were enumerated on de Man, Rogosa & Sharpe agar and PCR‐DGGE was employed as a culture‐independent method to assess the total communities. No significant differences were observed between the different gut regions for TVC or LAB levels. 16S rRNA sequencing identified all LAB isolates as Carnobacterium maltaromaticum. In contrast, the TVC community was more diverse; Firmicutes and Bacteroidetes were present but all gut regions were dominated by Proteobacteria, accounting for 88.4–92.6% of the communities. Citrobacter freundii was the dominant species and accounted for 51.0–57.8% of the isolates. Complex bacterial communities were observed using PCR‐DGGE and a trend towards the reduction in the number of operational taxonomic units (OTUs), microbial richness and microbial diversity was observed from the PC to the PM. The similarity between regions was low (52–68%) and cluster analysis revealed that the communities grouped into two distinct clusters; one dominated by the PM samples and the other contained the AM and PC samples. OTUs from the DGGE were identified as members of the phyla Proteobacteria and Firmicutes. Many OTUs were detected in all gastrointestinal regions, however, some OTUs showed regional specialization. Further studies are required to elucidate the activity of these genera in situ and how their actions impact the host.     

Effects of Lactobacillus pentosus on the growth performance,digestive enzyme and disease resistance of white shrimp,Litopenaeus vannamei (Boone, 1931)

The objective of this study was to evaluate the probiotic properties of lactic acid bacteria (LAB) strains isolated from digestive tract of white shrimp Litopenaeus vannamei. Eighteen LAB colonies were isolated and one bacterium was found capable of producing three extracellular enzymes (protease, cellulose and lipase) simultaneously and exhibited antagonistic activity against shrimp pathogens (Vibrio vulnificus, V. rotiferianus and V. campbellii). The putative probiotic strain AS13 was identified as Lactobacillus pentosus based on 16S rRNA sequencing. The L. vannamei were fed diet containing 0 (control), 10⁶, 10⁷ and 10⁸ CFU g^?1 bacterial cells of AS13 for 28 days. The results showed that supplementation of L. pentosus significantly improved the growth performance and feed utilization in the treated groups over the control. Similarly, digestive enzyme activities were elevated in the intestines of treated groups. Moreover, feeding of supplemented diets containing AS13 significantly reduced the mortality rate caused by pathogenic Vibrio species (V. vulnificus, V. rotiferianus and V. campbellii). Our results indicated L. pentosus AS13 addition at 10⁷ CFU g^?1 can effectively enhance the growth performance, feed utilization, digestive enzymes and disease resistance of L. vannamei in the laboratory condition.     

Recovery of Chitin from Antarctic Krill (Euphausia superba) Shell Waste by Microbial Deproteinization and Demineralization

A two-stage fermentation process of deproteinization and demineralization was used to purify chitin from Antarctic krill shell waste. Two bacterial cultures were isolated and tested for the degradation of krill shell waste. According to the morphological examination, physiological tests, and applied molecular techniques conducted, isolates were identified as Bacillus subtilis and Lactobacillus plantarum. At a temperature of 37°C, both strains were cultivated separately in flasks containing krill shell waste. At determined periods of time, deproteinization and demineralization of residuals were measured. For deproteinization tests, the liquid phase fermentation of krill shell waste showed protein removal of 88.9% after 60 h. For demineralization tests, the liquid phase fermentation of krill shell waste showed calcium carbonate removal of 84.6% after 72 h. Fourier transform infrared spectroscopy (FTIR) analysis of chitin prepared by the process was carried out and compared with spectra of samples treated by strong acids and bases.     

Effects of dietary Lactobicillus plantarum on the growth,carcass quality,and immune response of African catfish (Clarias gariepinus) challenged with Salmonella typhi

An experiment was conducted to investigate the effects of Lactobacillus plantarum on the production of African catfish (Clarias gariepinus). Five experimental diets containing 0, 10³, 10⁵, 10⁷, and 10⁹ CFU of Lactobacillus plantarum g^?1 diet (T₁–T₅ treatments respectively) were fed to African catfish (Clarias gariepinus) (9.20 ± 0.1 g initial body mass), for 70 days. Results indicated significantly lower growth performance in the fish fed diet without probiotics and in those fed diet with the least probiotic level. Treatments with probiotics significantly improved the blood profile and carcass protein but significantly reduced the carcass fat (P < 0.05); these treatments also marginally improved the carcass minerals in comparison with the treatment without probiotic (P < 0.05). Challenging the fish fed the experimental diets with Salmonella typhi showed higher immunity of fish fed the probiotic diets than those fed the nonprobiotic diet. Duncan’s multiple range test showed that the best fish performance was observed with 10³ CFU g^?1 L. plantarum for very parameter measured. However, regression analyses showed the optimum level of the bacteria to be 10^4.13–10⁵ CFU g^?1     

Effects of synbiotic containing Lactobacillus plantarum 7–40 and galactooligosaccharide on the growth performance of white shrimp,Litopenaeus vannamei

This study aimed to develop a synbiotic combination with probiotic, Lactobacillus plantarum 7–40 and one of three prebiotics, fructooligosaccharide (FOS), galactooligosaccharide (GOS) and mannan oligosaccharide (MOS). The best in vitro growth was observed when probiotic was cultured in the medium containing either FOS or GOS as the sole of carbon source. The analysis of enzyme activity revealed that GOS induced the highest activities of protease and β‐galactosidase of probiotic. Based on the findings, probiotic + GOS were selected as synbiotic to evaluate if it could promote the growth of white shrimp, Litopenaeus vannamei. For this, four diets, including a basal diet with no GOS or probiotic (control), 0.4% GOS (PRE), 10⁸ CFU probiotic kg^?1 (PRO) and 0.4% GOS in combination with 10⁸ CFU probiotic kg^?1 (SYN), were fed to shrimp for 60 days, and then the growth performance, intestinal microbiota (including total Vibrio counts, VBCs; and lactic acid bacteria, LAB) and digestive enzyme (including protease, leu‐aminopeptidase and β‐galactosidase) were evaluated. The weight gain (WG) of shrimp fed the PRO did not significantly differ from those of control (p > .05). Shrimp fed the SYN had significantly higher WG compared with the other treatments (p < .05). In addition, the SYN‐fed shrimp had significantly higher LAB and protease, leu‐aminopeptidase and β‐galactosidase activity (p < .05). The lowest presumptive Vibrio count (VBC) was also observed in intestines of SYN‐fed shrimp. Therefore, we suggested that Lac. plantarum 7–40+ GOS can be used as a synergistic synbiotic for shrimp culture.     

Isolation and characterization of bacteria causing mortality in early stage larvae of captive‐bred Japanese eels (Anguilla japonica Temminck & Schlegel)

Bacteria with lethal effect on eel larvae were isolated from moribund captive‐bred eel larvae and their 16S rRNA gene sequences were analysed. Nucleotide sequence of 10 isolates showed highest similarity with Lacinutrix algicola, Crocinitomix catalasitica and Pseudoalteromonas rubra. Age‐dependent changes in the susceptibility of eel larvae were observed in response to challenge by a highly lethal isolate. Compared with 10 and 11 days after hatching (DAH), larval susceptibility to the highly lethal isolate was lower at 18 DAH. We found that the bacterial isolates have lethal effect on the captive‐bred eel larvae, especially at the early developmental stage. These results will be useful to establish appropriate culture practices for eel larvae that will improve the success of mass production of glass eels for aquaculture.     

Effect of Lactobacillus plantarum and Jerusalem artichoke (Helianthus tuberosus) on growth performance,immunity and disease resistance of Pangasius catfish (Pangasius bocourti,Sauvage 1880)

This study evaluated the effects of Jerusalem artichoke (JA) and Lactobacillus plantarum singly or combined on growth, immunity and disease resistance of Pangasius bocourti. In the first experiment, different concentrations of JA (0, 5, 10, 20, 40, 80 and 160 g kg^?1) were administered to determine an optimal concentration on growth of P. bocourti. In the second experiment, the optimal concentration of JA (5 g kg^?1) was combined with 10⁸ cfu g^?1 L. plantarum. In the final experiment, five randomly selected fish from the second experiment were challenged with Aeromonas hydrophila. Treatments for second and third experiments were 0 g kg^?1 JA (Diet 1), 5 g kg^?1 JA (Diet 2), 10⁸ cfu g^?1 L. plantarum (Diet 3) and 5 g kg^?1 JA + 10⁸ cfu g^?1 L. plantarum (Diet 4). Fish fed 5 g kg^?1 JA or 10⁸ cfu g^?1 of L. plantarum significantly improved specific growth rate (SGR), feed conversion ratio (FCR), serum lysozyme activity and postchallenge survival rate (PCSR). Dietary in the combination of JA and L. plantarum showed significantly enhanced SGR, FCR, serum lysozyme, phagocytosis, respiratory burst activities and PCSR compared with control and individual applications. Dietary JA and L. plantarum significantly stimulated growth, immunity and disease resistance of P. bocourti.     

Evaluations of lactic acid bacteria as probiotics for juvenile seabass Lates calcarifer

Lactic acid bacteria (LAB) were isolated from adult, wild‐caught and farmed seabass (Lates calcarifer) intestines for evaluation as possible probiotics using the well agar diffusion method. Five LAB isolates (designated as LAB‐1–5) were found to inhibit Aeromonas hydrophila, a known seabass pathogen. Median lethal concentrations (LC₅₀) of A. hydrophila on juvenile seabass were measured in aquaria. Median lethal concentration values of 7.76, 7.47 and 7.26 log₁₀ CFU mL^?1 for 72, 96 and 120 h, respectively, were found. Juvenile seabass (0.6±0.2 g) were cultured in aquaria and fed individual LAB‐1–5 fortified feeds with 7 log₁₀ CFU g^?1 LAB. Seabass fed LAB‐4 fortified feed had significantly greater growth (P<0.05) than fish fed other feeds. Seabass fed LAB‐4 also had greater survival, but this was non‐significant (P<0.05). Challenge tests of LAB‐4 fed seabass with A. hydrophila at ～7 log₁₀CFU mL^?1 yielded significantly greater survival compared with control seabass (P<0.05). Aeromonas hydrophila infections in seabass were confirmed by observing disease manifestation and by immunohistochemistry techniques. LAB‐4 was preliminarily identified using lactic acid analysis, biochemical and physical characteristics. It was further identified using 16S rDNA sequencing. LAB‐4 was identified as Weissella confusa (identity of 99%). GenBank accession number for the 16S rDNA sequence for LAB‐4 was AB023241.     

Molecular characterization and pathogenicity of Francisella noatunensis subsp. orientalis isolated from cultured tilapia (Oreochromis sp.) in Taiwan

Francisella noatunensis subsp. orientalis is a causative agent of systemic granulomatous disease in tilapia. The present study was designed to understand the genetic and phenotypic diversities among Taiwanese Fno isolates obtained from tilapia (n = 17) and green Texas cichlid (Herichthys cyanoguttatus) (n = 1). The enzymatic profiles of the isolates were studied using the API ZYM system. Phylogenetic tree analysis of the 16S rRNA and housekeeping gene and pulsed‐field gel electrophoresis (PFGE) were carried out to determine the genotypic characters of all isolates. The phylogenetic tree showed similarity of 99%–100% nucleotide sequences of 16S rRNA and housekeeping genes compared to the Fno references genes from GenBank database. Comparatively, the results revealed an identical profile of enzymatic and PFGE pattern which was distincted from that of F. philomiragia. To understand the pathogenicity, the isolates were intraperitoneal injected to tilapia the gross lesions were observed concomitant with natural outbreak. Median lethal dose upon Nile tilapia and red tilapia were 9.06 × 10³ CFU/fish and 2.08 × 10² CFU/fish, respectively. Thus, our data provide understanding the epidemiology of Taiwanese Fno isolates, and help in development of future control and prevention.