A method for the detection of Norwalk virus and hepatitis A virus from shellfish tissues by PCR was developed. Virus was added to the stomach and hepatopancreatic tissues of oysters or hard-shell clams, and viral nucleic acids were purified by a modification of a previously described method (R. L. Atmar, T. G. Metcalf, F. H. Neill, and M. K. Estes, Appl. Environ. Microbiol. 59:631-635, 1993). The new method had the following advantages compared with the previously described method: (i) more rapid sample processing; (ii) increased test sensitivity; (iii) decreased sample-associated interference with reverse transcription-PCR; and (iv) use of chloroform-butanol in place of the chlorofluorocarbon trichlorotrifluoroethane. In addition, internal standards for both Norwalk virus and hepatitis A virus were made which demonstrated when inhibitors to reverse transcription-PCR were present and allowed quantitation of the viral nucleic acids present in samples. This assay can be used to investigate shellfish-associated gastroenteritis outbreaks and to study factors involved in virus persistence in shellfish.
Shellfish hatcheries are often affected by disease outbreaks. Three such episodes were investigated in different Galician hatcheries in order to establish the relationship between present microbiota and mortalities. Isolates were obtained from various parts of the hatcheries. Experimental tests for pathogenicity were carried out in microscale experiments using selected strains on Ostrea edulis larvae. The pathogenicity of 1 strain from each outbreak was demonstrated and shown to cause high mortalities (ranging from 98.5 to 100%) in 72 to 96 h after inoculation of larval cultures. All 3 strains belong to the genus Vibrio. One of the strains was identified as Vibrio neptunius and is the first description of this species as a molluscan pathogen. The other 2 strains showed low similarity with the Vibrio species analysed and may constitute new species within this genus. KEY WORDS: Flat oyster · Vibrio · Vibrio neptunius · Pathogenicity test · Shellfish hatchery Resale or republication not permitted without written consent of the publisherDis Aquat Org 67: [209][210][211][212][213][214][215] 2005 Tettelbach 1988, Lodeiros et al. 1992, Riquelme et al. 1995, Nicolas et al. 1996, Sáinz et al. 1998, Sugumar et al. 1998, Araya et al. 1999, Elston et al. 1999, Lacoste et al. 2001, Anguiano-Beltrán et al. 2004, Estes et al. 2004, Gay et al. 2004.In this paper, we present the results of bacteriological studies performed during 3 episodes of severe mortalities in 3 shellfish hatcheries in Galicia. The pathogenicity of isolated bacterial strains was tested in order to identify the aetiological agents. In addition, the first evidence of Vibrio neptunius as mollusc pathogen is presented. MATERIALS AND METHODS Description of facilities and bacterial isolation.The facilities where mortalities occurred are situated at different locations along the Galician coast in NW Spain (Fig. 1). Their management practices vary: one is a commercial hatchery beside a molluscan depuration facility (hatchery A); the second is a plant where molluscs and fishes are cultured (hatchery B); and the third is exclusively dedicated to molluscs (hatchery C). Two of the facilities (B and C) suffered mortalities in oyster larval stages and one (A) in post-larvae.Samples were taken in the different areas of the hatcheries: larvae, spat, broodstock, tank surfaces, phytoplankton and water. Larvae, spat and broodstock gonad (pieces excised aseptically) were washed, ground and homogenized in sterile seawater (SSW). Appropriate dilutions were made with these suspensions as well as with samples of water and phytoplankton used as larval feed. They were spread on Marine Agar (MA, Pronadisa) and Thiosulphate Citrate Bile Sucrose (TCBS, Oxoid). Samples taken directly from inner surfaces of the tanks containing the oysters were spread on the above mentioned media with sterile swabs.Plates were incubated at 22°C and selected colonies were isolated after 24 h (TCBS) or 7 d (MA) on MA, and further restreaked to purity. Pure cultures of strains were frozen at -8...
A multiplex-PCR approach, employing 2 primer pairs directed to internal regions of the 16s rRNA and ureC genes, was utilized to analyze a collection of Photobacterium damselae strains, including 25 isolates of subspecies piscicida and 15 isolates of subspecies darnselae. With this procedure, all the P. darnselae subsp. damselae strains yielded 2 amplification products, one of 267 bp and the other of 448 bp, corresponding to internal fragments of the 16s rRNA and ureCgenes, respectively. However, P. damselae subsp. piscicida isolates only showed the PCR product of 267 bp (16s rRNA fragment), indicating the absence of the urease gene in its genome. We have constructed a DNA probe directed to an internal region of the ureC gene, and corroborated by dot blot hybridization that the P. darnselae subsp. piscicida lacks this gene, whereas it is present in the subspecies darnselae. This constitutes the first successful discrimination between both subspecies using a PCR procedure, which could become a useful tool for diagnosis of pasteurellosis in the field. In addition, since these 2 subspecies have been shown to share nearly the same rrn operon sequence, our results provided evidence that one of the steps in the P, damselae speciation proccess included gainfloss events associated with the ure operon.
Wastewater treatment plants (WWTPs) are an effective barrier in the protection of human and environment health around the world, although WWTPs also are suggested to be selectors and-or reservoirs of antibiotic resistance genes (ARGs) before entering the environment. The dogma about WWTPs as “ARG selectors” presumes that biotreatment compartments (e.g., activated sludge; AS) are single densely populated ecosystems with elevated horizontal gene transfer. However, recent work has suggested WWTP biotreatment compartments may be different than previously believed relative to antibiotic resistance (AR) fate, and other process factors, such as bacterial separation and specific waste sources, may be key to ARGs released to the environment. Here we combined 16S rRNA metagenomic sequencing and high-throughput qPCR to characterise microbial communities and ARGs across a wastewater network in Spain that includes both community (i.e., non-clinical urban) and hospital sources. Contrary to expectations, ARGs found in downstream receiving waters were not dominated by AS biosolids (RAS), but more resembled raw wastewater sources. In fact, ARGs and microbial communities in liquid-phase WWTP effluents and RAS were significantly different (Bray–Curtis dissimilarity index = 0.66 ± 0.11), with a consequential fraction of influent ARGs and organisms passing directly through the WWTP with limited association with RAS. Instead, ARGs and organisms in the RAS may be more defined by biosolids separation and biophysical traits, such as flocculation, rather than ARG carriage. This explains why RAS has significantly lower ARG richness (47 ± 4 ARGs) than liquid-phase effluents (104 ± 5 ARGs), and downstream water column (135 ± 4 ARGs) and river sediments (120 ± 5 ARGs) (Tukey's test, p < 0.001). These data suggest RAS and liquid-phase WWTP effluents may reflect two parallel ecosystems with potentially limited ARG exchange. As such, ARG mitigation in WWTPs should more focus on removing bacterial hosts from the liquid phase, AR source reduction, and possibly disinfection to reduce ARG releases to the environment.
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