Now showing items 21-39 of 39
Abstract: | Hepcidin is a family of short cysteine-rich antimicrobial peptides (AMPs) participating in various physiological functions with inevitable role in host immune responses. Present study deals with identification and characterisation of a novel hepcidin isoform from coral fish Zanclus cornutus. The 81 amino acid (aa) preprohepcidin obtained from Z. cornutus consists of a hydrophobic aa rich 22 mer signal peptide, a highly variable proregion of 35 aa and a bioactive mature peptide with 8 conserved cysteine residues which contribute to the disulphide back bone. The mature hepcidin, Zc-hepc1 has a theoretical isoelectric point of 7.46, a predicted molecular weight of 2.43 kDa and a net positive charge of ?1. Phylogenetic analysis grouped Z. cornutus hepcidin with HAMP2 group hepcidins confirming the divergent evolution of hepcidin-like peptide in fishes. Zc-hepc1 can attain a b-hairpin-like structure with two antiparallel b-sheets. This is the first report of an AMP from the coral fish Z. cornutus. |
Description: | Probiotics & Antimicro. Prot. (2013) 5:187–194 DOI 10.1007/s12602-013-9139-x |
URI: | http://dyuthi.cusat.ac.in/purl/4078 |
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Molecular Chara ... rnutus (Linnaeus,1758).pdf | (574.2Kb) |
Abstract: | Antimicrobial peptides (AMPs) play a major role in innate immunity. Penaeidins are a family of AMPs that appear to be expressed in all penaeid shrimps. Penaeidins are composed of an N-terminal proline-rich domain, followed by a C-terminal domain containing six cysteine residues organized in two doublets. This study reports the first penaeidin AMP sequence, Fi-penaeidin (GenBank accession number HM243617) from the Indian white shrimp, Fenneropenaeus indicus. The full length cDNA consists of 186 base pairs encoding 61 amino acidswith an ORF of 42 amino acids and contains a putative signal peptide of 19 amino acids. Comparison of F. indicus penaeidin (Fi-penaeidin) with other known penaeidins showed that it shared maximum similarity with penaeidins of Farfantepenaeus paulensis and Farfantepenaeus subtilis (96% each). Fi-penaeidin has a predicted molecular weight (MW) of 4.478 kDa and theoretical isoelectric point (pI) of 5.3 |
Description: | Aquaculture 319 (2011) 298–301 |
URI: | http://dyuthi.cusat.ac.in/purl/4067 |
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Molecular chara ... Fenneropenaeus indicus.pdf | (1.453Mb) |
Abstract: | A crustinlike antimicrobial peptide from the haemocytes of giant tiger shrimp, Penaeus monodon was partially characterized at the molecular level and phylogenetic analysis was performed. The partial coding sequence of 299 bp and 91 deduced amino acid residues possessed conserved cysteine residues characteristic of the shrimp crustins. Phylogenetic tree and sequence comparison clearly confirmed divergence of this crustinlike AMP from other shrimp crustins. The differential expression of the crustinlike AMP in P. monodon in response to the administration of various immunostimulants viz., two marine yeasts (Candida haemulonii S27 and Candida sake S165) and two bglucan isolates (extracted from C. haemulonii S27 and C. sake S165) were noted during the study. Responses to the application of two grampositive probiotic bacteria (Bacillus MCCB101 and Micrococcus MCCB104) were also observed. The immune profile was recorded preand postchallenge white spot syndrome virus (WSSV) by semiquantitative RTPCR. Expressions of seven WSSV genes were also observed for studying the intensity of viral infection in the experimental animals. The crustinlike AMP was found to be constitutively expressed in the animal and a significant downregulation could be noted postchallenge WSSV. Remarkable downregulation of the gene was observed in the immunostimulant fed animals prechallenge followed by a significant upregulation postchallenge WSSV. Tissuewise expression of crustinlike AMP on administration of C. haemulonii and Bacillus showed maximum transcripts in gill and intestine. The marine yeast, C. haemulonii and the probiotic bacteria, Bacillus were found to enhance the production of crustinlike AMP and confer significant protection to P. monodon against WSSV infection |
Description: | Immunobiology 216 (2011) 184–194 |
URI: | http://dyuthi.cusat.ac.in/purl/4035 |
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Molecular chara ... ts and challenge withW.pdf | (840.4Kb) |
Description: | Fish & Shellfish Immunology 28 (2010) 216-220 |
URI: | http://dyuthi.cusat.ac.in/purl/4039 |
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Molecular chara ... Fenneropenaeus indicus.pdf | (687.6Kb) |
Abstract: | The addition of commercial nitrifying bacterial products has resulted in significant improvement of nitrification efficiency in recirculating aquaculture systems (RAS). We developed two nitrifying bacterial consortia (NBC) from marine and brackish water as start up cultures for immobilizing commercialized nitrifying bioreactors for RAS. In the present study, the community compositions of the NBC were analyzed by universal 16S rRNA gene and bacterial amoA gene sequencing and fluorescence in situ hybridization (FISH). This study demonstrated that both the consortia involved autotrophic nitrifiers, denitrifiers as well as heterotrophs. Abundant taxa of the brackish water heterotrophic bacterial isolates were Paenibacillus and Beijerinckia spp. whereas in the marine consortia they were Flavobacterium, Cytophaga and Gramella species. The bacterial amoA clones were clustered together with high similarity to Nitrosomonas sp. and uncultured beta Proteobacteria. FISH analysis detected ammonia oxidizers belonging to b subclass of proteobacteria and Nitrosospira sp. in both the consortia, and Nitrosococcus mobilis lineage only in the brackish water consortium and the halophilic Nitrosomonas sp. only in the marine consortium. However, nitrite oxidizers, Nitrobacter sp. and phylum Nitrospira were detected in both the consortia. The metabolites from nitrifiers might have been used by heterotrophs as carbon and energy sources making the consortia a stable biofilm. |
Description: | International Biodeterioration & Biodegradation 78 (2013) 74e81 |
URI: | http://dyuthi.cusat.ac.in/purl/4070 |
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Molecular chara ... ne aquaculture systems.pdf | (856.4Kb) |
Abstract: | Development of continuous cell lines from shrimp is essential to investigate viral pathogens. Unfortunately, there is no valid cell line developed from crustaceans in general and shrimps in particular to address this issue. Lack of information on the requirements of cells in vitro limits the success of developing a cell line, where the microenvironment of a cell culture, provided by the growthmedium, is of prime importance. Screening and optimization of growth medium components based on statistical experimental designs have been widely used for improving the efficacy of cell culture media. Accordingly, we applied Plackett–Burman design and response surface methodology to study multifactorial interactions between the growth factors in shrimp cell culture medium and to identify the most important ones for growth of lymphoid cell culture from Penaeus monodon. The statistical screening and optimization indicated that insulin like growth factor-I (IGF-I) and insulin like growth factor-II (IGF-II) at concentrations of 100 and 150 ng ml-1, respectively, could significantly influence the metabolic activity and DNA synthesis of the lymphoid cells. An increase of 53 % metabolic activity and 24.8 % DNA synthesis could be obtained, which suggested that IGF-I and IGFII had critical roles in metabolic activity and DNA synthesis of shrimp lymphoid cells |
Description: | Cytotechnology DOI 10.1007/s10616-014-9697-0 |
URI: | http://dyuthi.cusat.ac.in/purl/4076 |
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Multifactorial ... olic activity in vitro.pdf | (2.346Mb) |
Abstract: | Anti-lipopolysaccharide factors are small proteins that bind and neutralize lipopolysaccharide and exhibit potent antimicrobial activities. This study presents the molecular characterization and phylogenetic analysis of the first ALF isoform (Pp-ALF1; JQ745295) identified from the hemocytes of Portunus pelagicus. The full length cDNA of Pp-ALF1 consisted of 880 base pairs encoding 293 amino acids with an ORF of 123 amino acids and contains a putative signal peptide of 24 amino acids. Pp-ALF1 possessed a predicted molecular weight (MW) of 13.86 kDa and theoretical isoelectric point (pI) of 8.49. Two highly conserved cysteine residues and putative LPS binding domain were observed in Pp-ALF1. Peptide model of Pp-ALF1 consisted of two α-helices crowded against a four-strand β-sheet. Comparison of amino acid sequences and neighbor joining tree showed that Pp-ALF1 has a maximum similarity (46%) to ALF present in Portunus trituberculatus followed by 39% similarity to ALF of Eriocheir sinensis and 38% similarity to ALFs of Scylla paramamosain and Scylla serrata. Pp-ALF1 is found to be a new isoform of ALF family and its characteristic similarity with other known ALFs signifies its role in protection against invading pathogens. |
Description: | Aquaculture 356-357 (2012) 119–122 |
URI: | http://dyuthi.cusat.ac.in/purl/4058 |
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A new isoform o ... eristics and phylogeny.pdf | (788.2Kb) |
Abstract: | BACKGROUND: A packed bed bioreactor (PBBR) activated with an indigenous nitrifying bacterial consortia was developed and commercialized for rapid establishment of nitrification in brackish water and marine hatchery systems in the tropics. The present study evaluated nitrification in PBBR integrated into a Penaeus monodon recirculating maturation system under different substrate concentrations and flow rates. RESULTS:Instantnitrificationwasobservedafter integration ofPBBRinto thematuration system.TANandNO2-Nconcentrations were always maintained below0.5 mg L−1 during operation. The TANandNO2-N removalwas significant (P < 0.001) in all the six reactor compartments of the PBBR having the substrates at initial concentrations of 2, 5 and 10 mg L−1. The average volumetric TAN removal rates increased with flow rates from 43.51 (250 L h−1) to 130.44 (2500 L h−1) gTAN m−3 day−1 (P < 0.05). FISH analysis of the biofilms after 70 days of operation gave positive results with probes NSO 190 ((β ammonia oxidizers), NsV 443 (Nitrosospira spp.) NEU (halophilic Nitrosomonas), Ntspa 712 (Phylum Nitrospira) indicating stability of the consortia. CONCLUSION: The PBBR integrated into the P. monodon maturation system exhibited significant nitrification upon operation for 70 days as well as at different substrate concentrations and flow rates. This system can easily be integrated into marine and brackish water aquaculture systems, to establish instantaneous nitrification |
Description: | J Chem Technol Biotechnol 2011; 86: 790–797 |
URI: | http://dyuthi.cusat.ac.in/purl/4050 |
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Nitrification i ... rations and flow rates.pdf | (243.5Kb) |
Abstract: | Hepcidin is cysteine-rich short peptide of innate immune system of fishes, equipped to perform prevention and proliferation of invading pathogens like bacteria and viruses by limiting iron availability and activating intracellular cascades. Hepcidins are diverse in teleost fishes, due to the varied aquatic environments including exposure to pathogens, oxygenation and iron concentration. In the present study, we report a 87-amino acid (aa) preprohepcidin (Hepc-CB1) with a signal peptide of 24 aa, a prodomain of 39 aa and a bioactive mature peptide of 24 aa from the gill mRNA transcripts of the deep-sea fish spinyjaw greeneye, Chlorophthalmus bicornis. Molecular characterisation and phylogenetic analysis categorised the peptide to HAMP2-like group with a mature peptide of 2.53 kDa; a net positive charge (?3) and capacity to form b-hairpin-like structure configured by 8 conserved cysteines. The present work provides new insight into the mass gene duplication events and adaptive evolution of hepcidin isoforms with respect to environmental influences and positive Darwinian selection. This work reports a novel hepcidin isoform under the group HAMP2 from a nonacanthopterygian deep-sea fish, C. bicornis |
Description: | Probiotics & Antimicro. Prot. (2013) 5:1–7 DOI 10.1007/s12602-012-9120-0 |
URI: | http://dyuthi.cusat.ac.in/purl/4071 |
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A Novel Isoform ... acterisation and Phylo.pdf | (609.9Kb) |
Abstract: | Lack of a valid shrimp cell line has been hampering the progress of research on shrimp viruses. One of the reasons identified was the absence of an appropriate medium which would satisfy the requirements of the cells in vitro. We report the first attempt to formulate an exclusive shrimp cell culture medium (SCCM) based on the haemolymph components of Penaeus monodon prepared in isosmotic seawater having 27 % salinity. The SCCM is composed of 22 amino acids, 4 sugars, 6 vitamins, cholesterol, FBS, phenol red, three antibiotics, potassium dihydrogen phosphate and di-sodium hydrogen phosphate at pH 6.8–7.2. Osmolality was adjusted to 720 ± 10 mOsm kg-1 and temperature of incubation was 25 8C. The most appropriate composition was finally selected based on the extent of attachment of cells and their proliferation by visual observation. Metabolic activity of cultured cells was measured by MTT assay and compared with that in L-15 (29), modified L-15 and Grace’s insect medium, and found better performance in SCCM especially for lymphoid cells with 107 % increase in activity and 85 ± 9 days of longevity. The cells from ovary and lymphoid organs were passaged twice using the newly designed shrimp cell dissociation ‘‘cocktail’’. |
Description: | Cytotechnology (2013) 65:307–322 DOI 10.1007/s10616-012-9491-9 |
URI: | http://dyuthi.cusat.ac.in/purl/4063 |
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A novel medium ... m from Penaeus monodon.pdf | (681.8Kb) |
Abstract: | A marine isolate of jáÅêçÅçÅÅìë MCCB 104 has been identified as an aquaculture probiotic antagonistic to sáÄêáç. In the present study different carbon and nitrogen sources and growth factors in a mineral base medium were optimized for enhanced biomass production and antagonistic activity against the target pathogen, sáÄêáç=Ü~êîÉóá, following response surface methodology (RSM). Accordingly the minimum and maximum limits of the selected variables were determined and a set of fifty experiments programmed employing central composite design (CCD) of RSM for the final optimization. The response surface plots of biomass showed similar pattern with that of antagonistic activity, which indicated a strong correlation between the biomass and antagonism. The optimum concentration of the carbon sources, nitrogen sources, and growth factors for both biomass and antagonistic activity were glucose (17.4 g/L), lactose (17 g/L), sodium chloride (16.9 g/L), ammonium chloride (3.3 g/L), and mineral salts solution (18.3 mL/L). © KSBB |
Description: | Biotechnology and Bioprocess Engineering 2007, 12: 548-555 |
URI: | http://dyuthi.cusat.ac.in/purl/4044 |
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Optimization of ... ntral composite design.pdf | (479.9Kb) |
Abstract: | To demonstrate pathological changes due to white spot virus infection in Fenneropenaeus indicus, a batch of hatchery bred quarantined animals was experimentally infected with the virus. Organs such as gills, foregut, mid-gut, hindgut, nerve, eye, heart, ovary and integument were examined by light and electron microscopy. Histopathological analyses revealed changes hitherto not reported in F. indicus such as lesions to the internal folding of gut resulted in syncytial mass sloughed off into lumen, thickening of hepatopancreatic connective tissue with vacuolization of tubules and necrosis of rectal pads in hindgut. Virus replication was seen in the crystalline tract region of the compound eye and eosinophilic granules infiltrated from its base. In the gill arch, dilation and disintegration of median blood vessel was observed. In the nervous tissues, encapsulation and subsequent atrophy of hypertrophied nuclei of the neurosecretory cells were found. Transmission electron microscopy showed viral replication and morphogenesis in cells of infected tissue. De novo formed vesicles covered the capsid forming a bilayered envelop opened at one end inside the virogenic stroma. Circular vesicles containing nuclear material was found fused with the envelop. Subsequent thickening of the envelop resulted in the fully formed virus. In this study, a correlation was observed between the stages of viral multiplication and the corresponding pathological changes in the cells during the WSV infection. Accordingly, gill and foregut tissues were found highly infected during the onset of clinical signs itself, and are proposed to be used as the tissues for routine disease diagnosis. |
Description: | Journal of Invertebrate Pathology 102 (2009) 225–232 |
URI: | http://dyuthi.cusat.ac.in/purl/4060 |
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Pathological ch ... nd virus morphogenesis.pdf | (666.4Kb) |
Abstract: | This study shows that the disease resistance and survival rate of Penaeus monodon in a larval rearing systems can be enhanced by supplementing with antagonistic or non-antagonistic probiotics. The antagonistic mode of action of Pseudomonas MCCB 102 and MCCB 103 against vibrios was demonstrated in larval mesocosm with cultures having su⁄cient concentration of antagonistic compounds in their culture supernatant. Investigations on the antagonistic properties of Bacillus MCCB 101, Pseudomonas MCCB 102 and MCCB 103 and Arthrobacter MCCB 104 against Vibrio harveyi MCCB111under in vitro conditions revealed that Pseudomonas MCCB 102 and MCCB 103 were inhibitory to the pathogen.These inhibitory propertieswere further con¢rmed in the larval rearing systems of P. monodon. All these four probionts signi¢cantly improved larval survival in long-term treatments as well as when challengedwith a pathogenic strain ofV. harveyiMCCB111. We could demonstrate that Pseudomonas MCCB 102 andMCCB103 accorded disease resistance and a higher survival rate in P. monodon larval rearing systems throughactive antagonism of vibrios,whereas Bacillus MCCB 101 and Arthrobacter MCCB 104 functioned as probiotics through immunostimulatory and digestive enzyme-supporting modes of action. |
Description: | Aquaculture Research, 2010, 41, 847-860 |
URI: | http://dyuthi.cusat.ac.in/purl/4041 |
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Penaeus monodon ... c bacterial probiotics.pdf | (1.413Mb) |
Abstract: | Immortal cell lines have not yet been reported from Penaeus monodon, which delimits the prospects of investigating the associated viral pathogens especially white spot syndrome virus (WSSV). In this context, a method of developing primary hemocyte culture from this crustacean has been standardized by employing modified double strength Leibovitz-15 (L-15) growth medium supplemented with 2% glucose, MEM vitamins (1 ), tryptose phosphate broth (2.95 g l 1), 20% FBS, N-phenylthiourea (0.2 mM), 0.06 lgml 1 chloramphenicol, 100 lgml 1 streptomycin and 100 IU ml 1 penicillin and hemolymph drawn from shrimp grown under a bio-secured recirculating aquaculture system (RAS). In this medium the hemocytes remained viable up to 8 days. 5-Bromo-20-deoxyuridine (BrdU) labeling assay revealed its incorporation in 22 ± 7% of cells at 24 h. Susceptibility of the cells to WSSV was confirmed by immunofluoresence assay using a monoclonal antibody against 28 kDa envelope protein of WSSV. A convenient method for determining virus titer as MTT50/ml was standardized employing the primary hemocyte culture. Expression of viral genes and cellular immune genes were also investigated. The cell culture could be demonstrated for determining toxicity of a management chemical (benzalkonium chloride) by determining its IC50. The primary hemocyte culture could serve as a model for WSSV titration and viral and cellular immune related gene expression and also for investigations on cytotoxicity of aquaculture drugs and chemicals |
Description: | Journal of Invertebrate Pathology 105 (2010) 312–321 |
URI: | http://dyuthi.cusat.ac.in/purl/4031 |
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Primary hemocyt ... andcytotoxicity assays.pdf | (1.345Mb) |
Abstract: | For establishing nitrification in prawn (non-penaeid, salinity 10–15 ppt) and shrimp (penaeid, salinity 30–35 ppt) larval production systems, a stringed bed suspended bioreactor (SBSBR) was designed, fabricated, and validated. It was fabricated with 5 mm polystyrene and low density polyethylene beads as the substrata for ammonia and nitrite oxidizing bacterial consortia, respectively, with an overall surface area of 684 cm2. The reactors were activated in a prototype activator and were transported in polythene bags to the site of testing. Performance of the reactors activated with the nitrifying bacterial consortia AMONPCU-1 (ammonia oxidizers for non-penaeid culture) and NIONPCU-1 (nitrite oxidizers for non-penaeid culture) was evaluated in a Macrobrachium rosenbergii larval rearing system and those activated with AMOPCU-1 (ammonia oxidizers for penaeid culture) and NIOPCU-1 (nitrite oxidizers for penaeid culture) in a Penaeus monodon seed production system. Rapid setting up of nitrification could be observed in both the static systems which resulted in a higher relative per cent survival of larvae |
Description: | Aquacult Int (2009) 17:479–489 DOI 10.1007/s10499-008-9218-2 |
URI: | http://dyuthi.cusat.ac.in/purl/4068 |
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Stringed bed su ... naeid hatchery systems.pdf | (347.8Kb) |
Abstract: | Anti-lipopolysaccharide factors (ALFs), a type of cationic antimicrobial peptides (AMPs), and their derivatives are becoming predominant candidates for potential drugs in viral and bacterial diseases. This study reports the first ALF from the mud crab Scylla tranquebarica (StALF, JQ899453) and the second ALF isoform from the blue swimmer crab Portunus pelagicus (PpALF2, JQ899452). Both sequences encoded for precursor molecules, starting with a signal peptide containing 26 amino acid residues, followed by a highly cationic mature peptide, containing two conserved cysteine residues flanking a putative lipopolysaccharide (LPS)-binding domain. BLAST analysis revealed that both PpALF2 and StALF exhibited significant similarity with crustacean ALF sequences. The predicted molecular mass of the mature ALFs was 11.2 kDa with an estimated pI of 10.0. PpALF2 and StALF also showed the typical pattern of alternating hydrophobic and hydrophilic residues in their putative disulphide loop, suggesting that they comprise the same functional domain. Phylogenetic analysis showed that PpALF2 and StALF have similar evolutionary status and they were phylogenetically ancient immune effector molecules which may play an essential role in the host defense mechanism. The spatial structures of PpALF2 and StALF possessed four beta-strands and two alpha-helices. The results indicated that there were more than one ALF involved in crab immunity against various pathogens. ALFs would provide candidate promising therapeutic or prophylactic agents in health management and diseases control in crustacean aquaculture |
Description: | Molecular Immunology 52 (2012) 258– 263 |
URI: | http://dyuthi.cusat.ac.in/purl/4065 |
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Two isoforms of ... d Scylla tranquebarica.pdf | (1.310Mb) |
Description: | Winter School on 'RECENT ADVANCES IN DIAGNOSIS AND MANAGEMENT OF DISEASES IN MARICULTURE' 7-27 November, 2002 Course Manual Indian Council of Agricultural Research Central ^Aarine Fisheries Research Institute |
URI: | http://dyuthi.cusat.ac.in/purl/4086 |
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Use Of Immunost ... Aquaculture Management.pdf | (248.9Kb) |
Abstract: | Surveys for bacteriological analysis of larval samples to isolate the associated vibrios were carried out during 1985^1992, 2001 and 2002 in three di¡erent hatcheries located on the southwest coast of India. Vibrio isolates were examined for their species diversity, virulence based on haemolysis in prawn blood agar, lipolysis, proteolysis and chitinolysis and antibiotic sensitivity.Vibrio cholerae was the predominant species in the apparently healthy larval samples, whereas V. alginolyticus and V. vulni¢cus dominated during disease and morbidity. No correlation was found between the hydrolytic properties and haemolytic activity of the vibrios associated with the larvae. All isolates were resistant to erythromycin and resistance to oxytetracycline, ampicillin and streptomycin sulphate was prevalent among the larger section of the Vibrio population. This suggested that antibiotic application may not be of much use to protect the larvae fromvibriosis. This is the ¢rst report on the diversity of Vibrio species associated with Macrobrachium rosenbergii larvae and their virulence characteristics based on haemolysis in prawn blood agar |
Description: | Aquaculture Research, 2006, 37, 351^358 doi:10.1111/j.1365-2109.2005.01432.x |
URI: | http://dyuthi.cusat.ac.in/purl/4045 |
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Vibrios associa ... Indian southwest coast.pdf | (132.8Kb) |
Abstract: | Aeromonas spp. are ubiquitous aquatic organisms, associated with multitude of diseases in several species of animals, including fishes and humans. In the present study, water samples from two ornamental fish culture systems were analyzed for the presence of Aeromonas. Nutrient agar was used for Aeromonas isolation, and colonies (60 No) were identified through biochemical characterization. Seven clusters could be generated based on phenotypic characters, analyzed by the programme NTSYSpc, Version 2.02i, and identified as: Aeromonas caviae (33.3%), A. jandaei (38.3%) and A. veronii biovar sobria (28.3%). The strains isolated produced highly active hydrolytic enzymes, haemolytic activity and slime formation in varying proportions. The isolates were also tested for the enterotoxin genes (act, alt and ast), haemolytic toxins (hlyA and aerA), involved in type 3 secretion system (TTSS: ascV, aexT, aopP, aopO, ascF–ascG, and aopH), and glycerophospholipid-cholesterol acyltransferase (gcat). All isolates were found to be associated with at least one virulent gene. Moreover, they were resistant to frequently used antibiotics for human infections. The study demonstrates the pathogenic potential of Aeromonas, associated with ornamental fish culture systems suggesting the emerging threat to public health |
Description: | Brazilian Journal of Microbiology (2012): 754-765 |
URI: | http://dyuthi.cusat.ac.in/purl/4059 |
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Virulence Poten ... hreat To public Health.pdf | (192.5Kb) |
Now showing items 21-39 of 39
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