Isolation and Infectious Temperature Optimization of Genetically Similar VHSV Isolates in Farmed Olive Flounder, Paralichthys olivaceus

Viral hemorrhagic septicemia virus (VHSV) was isolated from farmed olive flounder (Paralichthys olivaceus). The viral N-gene was amplified by reverse transcriptase PCR, cloned, and sequenced for phylogenetic analysis to identify the genotype (I-IV). Virus isolates were cultured on Epithelioma papulosum cyprini cell line and, after completion of the cytopathic effects, the supernatant was collected and used to challenge virus-free flounder. The infected founder were reared in 16°C, 21°C, or 25°C, and compared to an unchallenged control. Virus titration was measured in the head kidneys, spleens, livers, brains, muscles, and gills of challenged fish using real-time quantification of the VHSV G-gene. Phylogenetic analysis confirmed that the isolates were VHSV Genogroup IV. The VHSV-challenged fish in the 16°C group showed 100% mortality with significantly increased expression of viral G-gene mRNA in the spleen, compared to fish reared in other temperatures and the control fish, suggesting that fish reared in 16°C are more susceptible to VHSV infection

Dietary Effect of Lonicera japonica on Immune Expression in Olive Flounder Paralichthys olivaceus, Challenged with Vibrio anguillarum

Respiratory burst, lysozyme, phagocytic activities, and immune gene expression levels were assessed in olive flounder (Paralichthys olivaceus, 10.52±2.5 g) fed with 0%, 0.025%, 0.05%, 0.1%, 0.2%, and 0.4% Lonicera japonica leaf powder (LjLP) for four weeks. All flounder fish fed LjLP showed significantly increased respiratory burst, lysozyme and phagocytic activity. Total immunoglobulin levels in LjLP diet fed fish groups were higher than in fish fed the basal diet (BD). In flounder fed LjLP, immune gene expression and antioxidant activity were significantly enhanced after 4 weeks of culture. One week following a challenge with Vibrio anguillarum, fish fed with different concentrations of LjLP showed decreased cumulative mortality and enhanced immunity all compared to the BD diet group. Our findings have shown that Lonicera japonica leaf powder enhanced immune response and resistance to V. anguillarum infection in olive flounder

Isolation, Characterization, Antioxidant, Antimicrobial and Cytotoxic Effect of Marine Actinomycete, Streptomyces Carpaticus MK-01, against Fish Pathogens

ABSTRACT Present study aim to evaluate the antimicrobial, antioxidant and cytotoxic potential of crude extract of Marine Streptomyces carpaticus MK-01 isolated from seawater collected from Daejeong-cost of Jeju Island. About 24 actinomycetes strains were isolated and subjected to morphological and molecular analysis that confirmed the isolate as S. carpaticus MK-01. Crude ethyl acetate extract of MK-01 strain showed extensive antibacterial activity against Gram-positive fish pathogenic bacteria namely Streptococcus iniae and S. parauberis with a maximum zone of inhibition (0.92±0.03mm) was recorded against S. parauberis at the minimum extract concentration (3.12µg/ml). The MK-01 ethyl acetate extract shows dose dependant significant increase in antioxidant activity. The 50% cytotoxic concentration (CC50) of MK-01 ethyl acetate extract was attained at 53.71 μg/ml and the effective concentration 50 (EC50) against virus-infected Epithelioma papulosum cyprini cell lines was 8.72 μg/ml of S. carpaticus MK-01 crude ethyl acetate extract

Meta-analysis Reveals That the Genus Pseudomonas Can Be a Better Choice of Biological Control Agent against Bacterial Wilt Disease Caused by Ralstonia solanacearum

Biological control agents (BCAs) from different microbial taxa are increasingly used to control bacterial wilt caused by Ralstonia solanacearum. However, a quantitative research synthesis has not been conducted on the role of BCAs in disease suppression. Therefore, the present study aimed to meta-analyze the impacts of BCAs on both Ralstonia wilt disease suppression and plant (host) growth promotion. The analysis showed that the extent of disease suppression by BCAs varied widely among studies, with effect size (log response ratio) ranging from −2.84 to 2.13. The disease incidence and severity were significantly decreased on average by 53.7% and 49.3%, respectively. BCAs inoculation also significantly increased fresh and dry weight by 34.4% and 36.1%, respectively on average. Also, BCAs inoculation significantly increased plant yield by 66%. Mean effect sizes for genus Pseudomonas sp. as BCAs were higher than for genus Bacillus spp. Among antagonists tested, P. fluorescens, P. putida, B. cereus, B. subtilis and B. amyloliquefaciens were found to be more effective in general for disease reduction. Across studies, highest disease control was found for P. fluorescens, annual plants, co-inoculation with more than one BCA, soil drench and greenhouse condition were found to be essential in understanding plant responses to R. solanacearum. Our results suggest that more efforts should be devoted to harnessing the potential beneficial effects of these antagonists, not just for plant growth promoting traits but also in mode of applications, BCAs formulations and their field studies should be considered in the future for R. solanacearum wilt disease suppression

In vitro effect of two commercial anti-coccidial drugs against myxospores of Kudoa septempunctata genotype ST3 (Myxozoa, Multivalvulida)

Kudoa septempunctata (Myxozoa: Multivalvulida) myxospores infect the trunk muscles of olive flounder (Paralichthys olivaceus). In this study, two popular commercially formulated anti-coccidial drugs (amprolium hydrochloride and toltrazuril) were serially diluted and incubated with purified mature Kudoa septempunctata myxospores. The viability of K. septempunctata spores was determined after a 2-day incubation followed by Hoechst 33342 and propidium iodide staining, and scanning electron microscopy. Amprolium hydrochloride significantly decreased spore viability (18% of control) at a concentration of 920 μg/mL, whereas toltrazuril showed almost no effect (83% of control). Viability of the control (untreated spores) was 90%. In vivo studies are required to confirm the efficacy of amprolium hydrochloride in fish infected with K. septempunctata myxospores on their growth and immune system performance

MOLECULAR CHARACTERIZATION OF FIBROBLAST GROWTH FACTOR 5 (FGF5) GENE IN ALPACA

Two coat phenotypes have been described in alpaca, Huacaya and Suri. The most common Huacaya type, is characterized by a crimped, dense fleeces, while the Suri type is characterized by a non-crimped, straight fleece. The genetic background of the two coat phenotypes has not been yet clearly defined. Results from segregation analysis provide statistical evidences that the Suri trait is possible inherited as a single dominant gene. The aim of our study was to molecularly characterize the FGF5 as a possible candidate gene for hair length phenotype in Suri type because of its role in the regulation of the hair follicle growth cycle. Currently, we have isolated and characterized two different cDNA clones encoding for FGF5 obtained from total RNA purified from skin biopsies of Peruvian Suri type alpaca. Sequence analysis revealed two types of transcripts: a long form (FGF5L) containing an ORF of 498 bp encoding for a putative 166 amino-acid polypeptide, and a a short form (FGF5S) of 375 bp encoding for a putative 125 amino-acid polypeptide. On the basis of the partial FGF5 genomic sequence data retrieved from the low-coverage 2.51X assembly of alpaca genome at the Ensembl database, the two transcript are produced by the alternative splicing of exon 5, which results in the loss of a fragment of 104 bases. Furthermore, RACE approaches specifically devised to characterize the 3’ and 5’ UTRs of FGF5 transcripts, show that the FGF5S isoform possesses two 3’-UTRs of 713 and 542 bases respectively.Meanwhile a single 3’UTR has been so far identified for the FGF5L isoform. Work is in progress in our laboratory to better characterize the FGF5 isoforms and their expression in the skin of Suri and Huacaya alpacas, and to identify mutations potentially involved in the fleece variations of alpaca