A culture independent method for the detection of Aeromonas sp. from water samples

The genus Aeromonas is present in a wide variety of water environments and is recognised as potentially pathogenic to humans and animals. Members of this genus are often confused with Vibrio when using automated, commercial identification systems that are culture-dependent. This study describes a polymerase chain reaction (PCR) detection method for Aeromonas that is culture- independent and that targets the glycerophospholopid-cholesterol acyltransferase (gcat) gene, which is specific for this genus. The GCAT-PCR was 100% specific in artificially inoculated water samples, with a detection limit that ranged from 2.5 to 25 cfu/mL. The success at detecting this pathogen in 86 water samples using the GCAT-PCR method was identical to the conventional culturing method when a pre-enrichment step was carried out, yielding 83.7% positive samples. On the other hand, without a pre-enrichment step, only 77.9% of the samples were positive by culturing and only 15.1% with the GCATPCR. However, 83.7% positive samples were obtained for the GCAT-PCR when the water volume for the DNA extraction was increased from 400 μL to 4 mL. The proposed molecular method is much faster (5 or 29 h) than the culturing method (24 or 48 h) whether performed directly or after a pre-enrichment step and it will enable the fast detection of Aeromonas in water samples helping to prevent a possible transmission to humans

CERTIFICATION REPORT The certification of different mass fractions of DP-ØØ4114-3 in maize seed powder Certified Reference Materials ERM®-BF439a, ERM®-BF439b, ERM®-BF439c, ERM®-BF439d and ERM®-BF439e

This report describes the production of a set of Certified Reference Materials (CRMs) ERM BF439a, b, c, d and e, which are certified for their 4114 maize event mass fractions. These materials were produced following ISO Guide 34:2009 and are certified in accordance with ISO Guide 35:2006. The materials are intended for the calibration or quality control of real-time PCR measurements to identify 4114 maize and/or quantify its mass fraction. As with any reference material, they can also be used for establishing control charts or for carrying out validation studies. The CRMs were accepted as European Reference Material (ERM®) after peer evaluation by the partners of the European Reference Materials consortium.JRC.D.2-Standards for Innovation and sustainable Developmen

Development and evaluation of a multiplex PCR assay for simultaneous detection of Flavobacterium psychrophilum, Yersinia ruckeri and Aeromonas salmonicida subsp. salmonicida in culture fisheries

Bacterial cold water disease, enteric red mouth disease and frunculosis are the common bacterial diseases of fish worldwide. The etiologic agents of these diseases are Flavobacterium (F.) psychrophilum, Yersinia (Y.) ruckeri and Aeromonas (A.) salmonicida subsp. salmonicida, respectively. In this study, a multiplex polymerase chain reaction (m-PCR) method with YER8/10-Fer3/4-FP1/3 primer pairs which can identify these fish pathogens simultaneously was developed and optimized. In optimized conditions, neither false specific nor nonspecific amplification occurred. The detection limits of the m-PCR method using DNA extracts from dilutions of pure cultures of bacteria were 35 pg for Y. ruckeri and F. psychrophilum and 70 pg for A. salmonicida subsp. salmonicida. It was determined that 15 CFU Y. ruckeri and F. psychrophilum and 30 CFU A. salmonicida subsp. salmonicida could be detected by m-PCR developed using genomic DNA extracted from dilutions of the suspensions. The detection limits in the presence of tissue debris were 125 CFU for Y. ruckeri and F. psychrophilum and 250 CFU for A. salmonicida subsp. salmonicida. In conclusion, we submit that the m-PCR method developed and optimized in this study can be used for accurate and rapid identification of these bacteria

TagA is a secreted protease of Vibrio cholerae that specifically cleaves mucin glycoproteins

Vibrio cholerae is a human diarrhoeal pathogen that is a major cause of gastrointestinal disease and death worldwide. Pathogenic V. cholerae strains are characterized by the presence of a Vibrio pathogenicity island (VPI) that encodes virulence factors, including the toxin co-regulated pilus (TCP). TagA is encoded within the VPI and is positively co-regulated with cholera toxin and TCP. TagA is a sequelogue of the StcE mucinase of Escherichia coli O157 : H7. We investigated whether this sequence homology reflected a conserved enzymic substrate profile. TagA exhibited metalloprotease activity toward crude purified mucins, salivary mucin and LS174T goblet cell surface mucin. Like StcE, TagA did not cleave general protease substrates, but unlike StcE, TagA did not cleave the mucin-like serpin C1 esterase inhibitor. Both proteins cleaved the immune cell surface mucin CD43, but TagA demonstrated reduced enzymic efficiency relative to StcE. TagA was expressed and secreted by V. cholerae under ToxR-dependent conditions. A tagA-deficient V. cholerae strain showed no defect in a model of in vitro attachment to the HEp-2 cell line; however, overexpression of a proteolytically inactive mutant, TagA(E433D), caused a significant increase in attachment. The increased attachment was reduced by pretreatment of epithelial monolayers with active TagA. Our results indicate that TagA is a mucinase and suggest that TagA may directly modify host cell surface molecules during V. cholerae infection

Molecular Characterization of Clinical Isolates of Aeromonas Species from Malaysia

Background: Aeromonas species are common inhabitants of aquatic environments giving rise to infections in both fish and humans. Identification of aeromonads to the species level is problematic and complex due to their phenotypic and genotypic heterogeneity. Methodology/Principal Findings: Aeromonas hydrophila or Aeromonas sp were genetically re-identified using a combination of previously published methods targeting GCAT, 16S rDNA and rpoD genes. Characterization based on the genus specific GCAT-PCR showed that 94 (96%) of the 98 strains belonged to the genus Aeromonas. Considering the patterns obtained for the 94 isolates with the 16S rDNA-RFLP identification method, 3 clusters were recognised, i.e. A. caviae (61%), A. hydrophila (17%) and an unknown group (22%) with atypical RFLP restriction patterns. However, the phylogenetic tree constructed with the obtained rpoD sequences showed that 47 strains (50%) clustered with the sequence of the type strain of A. aquariorum, 18 (19%) with A. caviae, 16 (17%) with A. hydrophila, 12 (13%) with A. veronii and one strain (1%) with the type strain of A. trota. PCR investigation revealed the presence of 10 virulence genes in the 94 isolates as: lip (91%), exu (87%), ela (86%), alt (79%), ser (77%), fla (74%), aer (72%), act (43%), aexT (24%) and ast (23%). Conclusions/Significance: This study emphasizes the importance of using more than one method for the correct identification of Aeromonas strains. The sequences of the rpoD gene enabled the unambiguous identication of the 9

Adult spawning and early larval development of the endangered bivalve Pinna nobilis

[EN] The development of aquaculture activities has posed an alternative solution for the preservation of some overexploited shell¿sh ¿sheries worldwide. In the same way, endemic Mediterranean bivalves such as Pinna nobilis, highly threatened by habitat loss and coastal pollution, could found in aquaculture a solution for preserving the continuity of the species. Given the endangered status of the species, the biological and ecological processes regulating natural populations have been well studied, but there are still important knowledge gaps preventing the development of viable arti¿cial cultures. This study describes for the ¿rst time the larval development of P. nobilis (from fertilization until pediveliger larval stages) in captivity conditions. Moreover, di¿erent rearing tanks of 5, 16 and 80 L, larvae density from 1 to 600 larvae mL¿1, light conditions, food doses, were tested in order to establish the bases for the optimal rearing of the species and provide a source of individuals for restoring ¿eld populations. Results showed that 16 L tanks with a concentration of 2 larvae mL¿1, constant temperature of 21 °C, 12/12 h photoperiod and fed with an ¿optimal¿ mixture of 25 cells per ¿L of Chaetoceros calcitrans + 33.3 cells per¿L ofPavlova lutheri + 100 cells per¿L ofIsochrysis galbana¿ appear to be the best conditions to rearlarvae ofP. nobilis.Di¿erentcaptivity conditions such as loweror highertank volume, larvae density, or food doses; light privation did not report better results for larval development.The present study was financed by the Caisse d'Epargne Cote d'Azur. We are also grateful to the research crew of the Institut Oceanographique Paul Ricard and the Catholic University of Valencia for their technical support and help collecting and maintaining fan mussels. Special thanks to the reviewers for their constructive and necessary suggestions.Trigos, S.; Vicente, N.; Prado, P.; Espinos Gutierrez, FJ. (2018). Adult spawning and early larval development of the endangered bivalve Pinna nobilis. Aquaculture. 483:102-110. doi:10.1016/j.aquaculture.2017.10.015S10211048

Transcriptomics of In Vitro Immune-Stimulated Hemocytes from the Manila Clam Ruditapes philippinarum Using High-Throughput Sequencing

The Manila clam (Ruditapes philippinarum) is a worldwide cultured bivalve species with important commercial value. Diseases affecting this species can result in large economic losses. Because knowledge of the molecular mechanisms of the immune response in bivalves, especially clams, is scarce and fragmentary, we sequenced RNA from immune-stimulated R. philippinarum hemocytes by 454-pyrosequencing to identify genes involved in their immune defense against infectious diseases

Aeromonas spp. whole genomes and virulence factors implicated in fish disease

doi:10.1111/jfd.1202