Identification of Bacterial Fish Pathogens in Brazil by Direct Colony PCR and 16S rRNA Gene Sequencing

Intensive fish farming systems in Brazil have increased the disease incidence, mainly of bacterial origin, due to higher stocking density, high organic matter levels and poor quality of the aquatic environment that causes high mortality rates during outbreaks. The identification of pathogenic species using a fast and reliable method of diagnosis is essential for successful epidemiological studies and disease control. The present study evaluated the use of direct colony PCR in combination with 16S rRNA gene sequencing to diagnose fish bacterial diseases, with the goal of reducing the costs and time necessary for bacterial identification. The method was successful for all 178 isolates tested and produced bands with the same intensity as the standard PCR performed using pure DNA. In conclusion, the genetics methods allowed detecting the most common and important pathogens in Aquaculture, including 12 species of occurrence in Brazilian fish farms. The results of the present study constitute an advance in the available diagnostic methods for bacterial pathogens in fish farms.

Evaluation of antibacterial activity and immunostimulant of red seaweed Chondrococcus hornemanni(Kuetzing,1847)against marine ornamental fish pathogens

Objective:To evaluate the antibacterial activity and immunostimulant of red seaweedChondrococcus hornemanni pathogens.Methods:(C.hornemanni)(Kuetzing,1847)against marine ornamental fish antimicrobial activity against two fish pathogens and the partially purified potential compound was characterized by GC-MS.Purified seaweed extract was injected to the clownfish,Amphiprion sebae to study the innate immune response of these fishes.Results:In the present study,seaweed extract of C.hornemanni showed significant Aeromonas hydrophila(20 mm)and Vibrio parahaemolyticus(19 mm)at a concentration(25 mg/mL)shown higher antimicrobial activity.The white blood cell count and respirator burst activity was significantly increased in the experimental tanks(E1)and(E2)when compared with control.The extracts was found effective and had more than 80%inhibitory activity against Conclusions:Hence,it is concluded that the seaweed extracts of C.hornemanni had potential bioactive compounds and act an immunostimulant and improve the immune response to fish.

Effect of fucoidan from Turbinaria ornata against marine ornamental fish pathogens

Objective:To evaluate the antimicrobial capacity of fucoidan from the brown seaweed Turbinaria ornata against the marine ornamental fish bacterial pathogens.Methods:Fucoidan was isolated by the ethanol extraction method and the functional groups were identified by Fourier Transform Infrared Spectroscopy analysis.Subsequently,structural characterization was done by 1H NMR analysis.In vitro antibacterial activity of fucoidan was performed by the agar plate diffusion method and Minimum Inhibitory Concentration.Results:The characteristic C-0-S bending vibration of sulfate substituent the axial C-4 was observed at 839 cm^(-1).Characterisitic signal of the fucoidan was detected in a different ppm of 1H NMR analysis.The maximum antibacterial activity(16.23±0.11)mm was obtained for Vibrio parahaemolyticus and the minimum activity(5.1±0.24)mm was recorded for Yersinia enterocolitica.The Minimum Inhibitory Concentration value was recorded between 2.5 to 10 mg/mL to the respective pathogens.Conclusions:The present study proved that fucoidan possessed the significant antibacterial activity against the tested fish bacterial pathogens.It could be further used as a natural antibiotic in an aquaculture system to control the bacterial diseases.However,the present study suggested that the further in vivo study is required to get the better understanding of the mode of action of fucoidan.

Taxonomy of Ochroconis, genus including opportunistic pathogens on humans and animals

The genus Ochroconis(Sympoventuriaceae,Venturiales)is revised and currently contains 13 species for which the phylogenetic position has been determined using multilocus sequencing.The older generic name Scolecobasidium is considered to be of doubtful identity because the type specimen is ambiguous.Within the Ochroconis lineage,phylogenetic distances of all markers analyzed are exceptionally large,both between and within species.A new genus Verruconis is proposed for the neurotropic opportunist Ochroconis gallopava.Species accepted within the lineages are keyed out on the basis of phenotypic characters.Main ecological traits within each species are discussed.Verruconis species are thermophilic and one of them is an important agent of infection in the brain,while Ochroconis is mesophilic,several species causing infections in cold-blooded animals.

Evaluation of antibacterial activity of bioactive compounds obtained from the seaweed Chondrococcus hornemanni on ichthyopathogenic bacteria affecting marine ornamental fish

Objective:To investigate antibacterial effects of extracts from the seaweed Chondrococcushornemanni(C.hornemanni)on bacterial pathogens of marine ornamental fish.Method:Methanol extract obtained from C.hornemanni showed a broad and high antibacterial activity against four fish pathogens including Providencia rettgeri,Aeromonas hydrophila,Vibrioalginoticus and Vibrio parahaemolyticus.The crude extract obtained from the dried seaweeds was fractionated and purified using column chromatography.Purified extracts were analyzed with Fourier transform infrared spectroscopy(FTIR)for identifying the functional groups.Phytoconstituents of the active fraction were further identified by means of gas chromatography and mass spectrometric(GC-MS)analysis.Result:The first fraction of the extracts showed effective inhibitory activity against Aeromonashydrophila and Vibrio parahaemolyticus at a concentration of 100μL.However,Vibrio alginolyticus and Providencia rettgeri had shown a moderately lesser inhibitory response to the extract.Conclusion:Hence,it is concluded that extracts of seaweed C.hornemanni,contain potential bioactive compounds with a considerable antibiotic activity.

Screening of marine seaweeds for bioactive compound against fish pathogenic bacteria and active fraction analysed by gas chromatography-mass spectrometry

Objective:To isolate bioactive molecules from marine seaweeds and check the antimicrobial activity against the fish pathogenic bacteria.Methods:Fresh marine seaweeds Gracilaria edulis,Kappaphycus spicifera,Sargassum wightii(S.wightii)were collected.Each seaweed was extracted with different solvents.In the study,test pathogens were collected from microbial type culture collection.Antibacterial activity was carried out by using disc diffusion method and minimum inhibition concentration(MIC)was calculated.Best seaweed was analysed by fourier transform infrared spectroscopy.The cured extract was separated by thin layer chromatography(TLC).Fraction was collected from TLC to check the antimicrobial activity.Best fraction was analysed by gas chromatography mass spectrometer(GCMS).Results:Based on the disc diffusion method,S.wightii showed a better antimicrobial activity than other seaweed extracts.Based on the MIC,methanol extract of S.wightii showed lower MIC than other solvents.S.wightii were separated by TLC.In this TLC,plate showed a two fraction.These two fractions were separated in preparative TLC and checked for their antimicrobial activity.Fraction 2 showed best MIC value against the tested pathogen.Fraction 2 was analysed by GCMS.Based on the GCMS,fraction 2 contains n-hexadecanoic acid(59.44%).Conclusions:From this present study,it can be concluded that S.wightii was potential sources of bioactive compounds.