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1.
Corrêa Ade A Rigotto C Moresco V Kleemann CR Teixeira AL Poli CR Simões CM Barardi CR 《Memórias do Instituto Oswaldo Cruz》2012,107(1):11-17
Within the country of Brazil, Santa Catarina is a major shellfish producer. Detection of viral contamination is an important step to ensure production quality and consumer safety during this process. In this study, we used a depuration system and ultraviolet (UV) disinfection to eliminate viral pathogens from artificially infected oysters and analysed the results. Specifically, the oysters were contaminated with hepatitis A virus (HAV) or human adenovirus type 5 (HAdV5). After viral infection, the oysters were placed into a depuration tank and harvested after 48, 72 and 96 h. After sampling, various oyster tissues were dissected and homogenised and the viruses were eluted with alkaline conditions and precipitated with polyethylene glycol. The oyster samples were evaluated by cell culture methods, as well as polymerase chain reaction (PCR) and quantitative-PCR. Moreover, at the end of the depuration period, the disinfected seawater was collected and analysed by PCR. The molecular assays showed that the HAdV5 genome was present in all of the depuration time samples, while the HAV genome was undetectable after 72 h of depuration. However, viral viability tests (integrated cell culture-PCR and immunofluorescence assay) indicated that both viruses were inactivated with 96 h of seawater recirculation. In conclusion, after 96 h of UV treatment, the depuration system studied in this work purified oysters that were artificially contaminated with HAdV5 and HAV. 相似文献
2.
Polymerase chain reaction identification of Vibrio vulnificus in artificially contaminated oysters 总被引:5,自引:0,他引:5
W E Hill S P Keasler M W Trucksess P Feng C A Kaysner K A Lampel 《Applied and environmental microbiology》1991,57(3):707-711
DNAs extracted from Vibrio vulnificus seeded into oyster homogenates were evaluated as templates for the polymerase chain reaction. Several extraction procedures were examined, and it was determined that DNA recovered from cells lysed by guanidine isothiocyanate, extracted with chloroform, and precipitated with ethanol was most suitable for use as a polymerase chain reaction template. The region targeted was a 519-bp portion of the cytotoxin-hemolysin gene of V. vulnificus. This region was amplified only when DNA from this species was present in the homogenate. V. vulnificus seeded into oyster homogenates at an initial level of 10(2) CFU/g of oyster meat was consistently observed after 24 h of incubation in alkaline peptone water. 相似文献
3.
Polymerase chain reaction identification of Vibrio vulnificus in artificially contaminated oysters. 下载免费PDF全文
W E Hill S P Keasler M W Trucksess P Feng C A Kaysner K A Lampel 《Applied microbiology》1991,57(3):707-711
DNAs extracted from Vibrio vulnificus seeded into oyster homogenates were evaluated as templates for the polymerase chain reaction. Several extraction procedures were examined, and it was determined that DNA recovered from cells lysed by guanidine isothiocyanate, extracted with chloroform, and precipitated with ethanol was most suitable for use as a polymerase chain reaction template. The region targeted was a 519-bp portion of the cytotoxin-hemolysin gene of V. vulnificus. This region was amplified only when DNA from this species was present in the homogenate. V. vulnificus seeded into oyster homogenates at an initial level of 10(2) CFU/g of oyster meat was consistently observed after 24 h of incubation in alkaline peptone water. 相似文献
4.
Use of an oligonucleotide probe to detect Vibrio parahaemolyticus in artificially contaminated oysters. 总被引:3,自引:0,他引:3
A 26-mer oligonucleotide specific to Vibrio parahaemolyticus was synthesized from a 1,275-bp thermostable direct hemolysin (tdh) gene. This oligonucleotide probe specifically reacted with DNA from 89 of 95 V. parahaemolyticus isolates but not with DNA from other vibrios or other enteric and nonenteric organisms (n = 48). The probe hybridized with Southern blots of 0.5-kb HindIII-restricted chromosomal DNA fragments from all but five V. parahaemolyticus test isolates. The probe could be used to directly identify V. parahaemolyticus in artificially contaminated food without an isolation step. 相似文献
5.
Use of an oligonucleotide probe to detect Vibrio parahaemolyticus in artificially contaminated oysters. 总被引:1,自引:0,他引:1 下载免费PDF全文
A 26-mer oligonucleotide specific to Vibrio parahaemolyticus was synthesized from a 1,275-bp thermostable direct hemolysin (tdh) gene. This oligonucleotide probe specifically reacted with DNA from 89 of 95 V. parahaemolyticus isolates but not with DNA from other vibrios or other enteric and nonenteric organisms (n = 48). The probe hybridized with Southern blots of 0.5-kb HindIII-restricted chromosomal DNA fragments from all but five V. parahaemolyticus test isolates. The probe could be used to directly identify V. parahaemolyticus in artificially contaminated food without an isolation step. 相似文献
6.
I. A. Govorin 《Russian Journal of Marine Biology》2000,26(2):81-88
Bacterial contamination the inshore areas of seas, is currently one of the most common manifestations of increasing anthropogenic
pressure on aquatic ecosystems. Permanent sources of allochthonic microflora in the inshore areas of seas, are polluted with
untreated or insufficiently treated domestic and industrial sewage [5, 8, 12, 45, 66] and river run-off from areas of active
water use [22, 28], and suffer the effects of over-exploitation of beaches during the bathing season (in the summer). 相似文献
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Geffard A Amiard JC Amiard-Triquet C 《Comparative biochemistry and physiology. Toxicology & pharmacology : CBP》2002,131(3):281-293
In oysters Crassostrea gigas translocated from a metal-enriched estuary (Gironde, France) to a comparatively clean site, the Bay of Bourgneuf (France), Cd, Cu and Zn concentrations were determined monthly in the whole soft tissues, or in different fractions (cytosolic or insoluble) of gills and digestive glands. In all cases, the concentrations of all of the three metals decreased logarithmically and half-lives were always shortest for Cd (86-251 days). After 4 months, the Cd concentration had become not significantly different from the threshold considered safe for human consumption (1 mg kg(-1) wet wt.). In the digestive gland, half-lives were similar in cytosolic and insoluble fractions. In contrast, in the gills, elimination patterns differed markedly between these fractions. The long half-lives calculated for divalent metals in the insoluble fraction of the gills (1505 and 3010 days for Zn and Cu, respectively) is possibly due to a fossilization of metals in intracellular membrane-bound inclusions as shown previously in Ostrea edulis. It is interesting to underline that elimination is fastest for cytosolic metals compared to the insoluble fraction. 相似文献
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A biotin-labelled DNA probe was used in a dot-blot hybridization test to demonstrate the presence of Escherichia coli in a variety of artificially contaminated foodstuffs. Positive hybridization was detected by using a streptavidine/polyalkaline phosphatase conjugate to generate an insoluble coloured precipitate in the presence of an appropriate dye. The colour intensity was measured with a computer-controlled image analysis system which assessed objectively the hybridization signal produced by each sample. The method was capable of distinguishing positive hybridization at cell concentrations exceeding 10(4) cells/dot-blot, equivalent to 2 x 10(7) cells/g food, and had none of the drawbacks normally associated with the use of radioactively labelled DNA in hybridization techniques. The procedure is highly specific and takes less than 30 h. Many samples can be screened simultaneously and the procedure can be used to detect any species for which a suitable DNA probe is available. 相似文献
12.
A biotin-labelled DNA probe was used in a dot-blot hybridization test to demonstrate the presence of Escherichia coli in a variety of artificially contaminated foodstuffs. Positive hybridization was detected by using a streptavidine/polyalkaline phosphatase conjugate to generate an insoluble coloured precipitate in the presence of an appropriate dye. The colour intensity was measured with a computer-controlled image analysis system which assessed objectively the hybridization signal produced by each sample. The method was capable of distinguishing positive hybridization at cell concentrations exceeding 104 cells/dot-blot, equivalent to 2×107 cells/g food, and had none of the drawbacks normally associated with the use of radioactively labelled DNA in hybridization techniques. The procedure is highly specific and takes less than 30 h. Many samples can be screened simultaneously and the procedure can be used to detect any species for which a suitable DNA probe is available 相似文献
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Sunnotel O Snelling WJ McDonough N Browne L Moore JE Dooley JS Lowery CJ 《Applied and environmental microbiology》2007,73(16):5083-5087
When filter-feeding shellfish are consumed raw, because of their ability to concentrate and store waterborne pathogens, they are being increasingly associated with human gastroenteritis and have become recognized as important pathogen vectors. In the shellfish industry, UV depuration procedures are mandatory to reduce pathogen levels prior to human consumption. However, these guidelines are based around more susceptible fecal coliforms and Salmonella spp. and do not consider Cryptosporidium spp., which have significant resistance to environmental stresses. Thus, there is an urgent need to evaluate the efficiency of standard UV depuration against the survival of Cryptosporidium recovered from shellfish. Our study found that in industrial-scale shellfish depuration treatment tanks, standard UV treatment resulted in a 13-fold inactivation of recovered, viable C. parvum oocysts from spiked (1 x 10(6) oocysts liter (-1)) Pacific oysters. Depuration at half power also significantly reduced (P < 0.05; ninefold) the number of viable oocysts recovered from oysters. While UV treatment resulted in significant reductions of recovered viable oocysts, low numbers of viable oocysts were still recovered from oysters after depuration, making their consumption when raw a public health risk. Our study highlights the need for increased periodic monitoring programs for shellfish harvesting sites, improved depuration procedures, and revised microbial quality control parameters, including Cryptosporidium assessment, to minimize the risk of cryptosporidiosis. 相似文献
15.
Collaborative study on the isolation of salmonella from artificially contaminated milk powder 总被引:1,自引:0,他引:1
H J Beckers F M van Leusden D Roberts O Pietzsch T H Price M van Schothorst P D Tips P Vassiliadis E H Kampelmacher 《The Journal of applied bacteriology》1985,59(1):35-40
The suitability of artificially contaminated milk powder as a substrate for salmonella reference samples and its stability under different storage conditions were studied. The need for a reconstitution step in the standard isolation method for salmonellas from milk powders was also investigated. When milk powder was examined in this way with a reconstitution step, differences in laboratory methods and/or storage times had no significant effect on the results after storage at 4 degrees C. With powder stored at room temperature there was a systematic decrease in the number of samples positive as the storage time increased. It is concluded therefore that milk powder contaminated with salmonellas should be stored at 4 degrees C. Examination of such milk powder with a reconstitution step yielded better results than without it and this step is therefore necessary for improving the reproducibility of the method. No significant differences were encountered between the standard isolation method and that used in the authors' laboratories. The results of this study indicate that milk powder is suitable as basic material for reference samples and that a reconstitution step should be included in the standard salmonella isolation procedure. 相似文献
16.
H.J. Beckers F.M. Van Leusden D. Roberts † O. Pietzsch ‡ T.H. Price § M. Van Schothorst† P.O. Tips P. Vassiliadis E.H. Kampelmacher ¶ 《Journal of applied microbiology》1985,59(1):35-40
B eckers , H.J., van L eusden , F.M., R oberts , D., P ietzsch , O., P rice , T.H., van S chothorst , M., T ips , P.D., V assiliadis , P. & K ampelmacher , E.M. 1985. Collaborative study on the isolation of salmonella from artificially contaminated milk powder. Journal of Applied Bacteriology 59 , 35–40.
The suitability of artificially contaminated milk powder as a substrate for salmonella reference samples and its stability under different storage conditions were studied. The need for a reconstitution step in the standard isolation method for salmonellas from milk powders was also investigated. When milk powder was examined in this way with a reconstitution step, differences in laboratory methods and/or storage times had no significant effect on the results after storage at 4C. With powder stored at room temperature there was a systematic decrease in the number of samples positive as the storage time increased. It is concluded therefore that milk powder contaminated with salmonellas should be stored at 4C. Examination of such milk powder with a reconstitution step yielded better results than without it and this step is therefore necessary for improving the reproducibility of the method. No significant differences were encountered between the standard isolation method and that used in the authors' laboratories. The results of this study indicate that milk powder is suitable as basic material for reference samples and that a reconstitution step should be included in the standard salmonella isolation procedure. 相似文献
The suitability of artificially contaminated milk powder as a substrate for salmonella reference samples and its stability under different storage conditions were studied. The need for a reconstitution step in the standard isolation method for salmonellas from milk powders was also investigated. When milk powder was examined in this way with a reconstitution step, differences in laboratory methods and/or storage times had no significant effect on the results after storage at 4C. With powder stored at room temperature there was a systematic decrease in the number of samples positive as the storage time increased. It is concluded therefore that milk powder contaminated with salmonellas should be stored at 4C. Examination of such milk powder with a reconstitution step yielded better results than without it and this step is therefore necessary for improving the reproducibility of the method. No significant differences were encountered between the standard isolation method and that used in the authors' laboratories. The results of this study indicate that milk powder is suitable as basic material for reference samples and that a reconstitution step should be included in the standard salmonella isolation procedure. 相似文献
17.
Behavior of Escherichia coli and male-specific bacteriophage in environmentally contaminated bivalve molluscs before and after depuration. 总被引:1,自引:0,他引:1 下载免费PDF全文
We monitored the differential reduction rates and elimination patterns of Escherichia coli and male-specific (F+) bacteriophage during UV depuration for 48 h in oysters (Crassostrea gigas) and mussels (Mytilus edulis) contaminated by short-term (1 to 3 weeks) and long-term (more than 6 months) exposure to sewage in the marine environment. The time taken to reduce levels of E. coli by 90% was 6.5 h or less in all cases. In contrast, the amounts of time needed to reduce levels of F+ bacteriophage by 90% were considerably longer: 47.3 and 41.3 h (after short- and long-term exposures, respectively) in mussels and 54.6 and 60.8 h (after short- and long-term exposures, respectively) in oysters. No differences in the rates of reduction of indicators of viral pollution following exposure of the shellfish to either short- or long-term sewage contamination were observed. Further experiments were conducted with mussels to determine the relative distributions of E. coli and F+ bacteriophage in tissue before and during depuration. Prior to depuration the majority of E. coli organisms (90.1%) and F+ bacteriophage (87.3%) were detected in the digestive tract (i.e., the digestive gland and intestine). E. coli and F+ bacteriophage were reduced in all tissues except the digestive gland to undetectable levels following depuration for 48 h. Within the digestive gland, levels of F+ bacteriophage were reduced to 30% of initial levels, whereas E. coli was reduced to undetectable levels.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
18.
A genetic probe was used to identify and enumerate virulent Yersinia enterocolitica colonies in 11 artificially contaminated foods. Efficiency of enumeration, determined by autoradiography after DNA colony hybridization, ranged from 66 to 100% (average, 86%) and was influenced by the number of indigenous bacteria. The use of nitrocellulose filters and agar medium had little effect on efficiency of enumeration. 相似文献
19.
Inactivation of caliciviruses 总被引:2,自引:0,他引:2
Duizer E Bijkerk P Rockx B De Groot A Twisk F Koopmans M 《Applied and environmental microbiology》2004,70(8):4538-4543
The viruses most commonly associated with food- and waterborne outbreaks of gastroenteritis are the noroviruses. The lack of a culture method for noroviruses warrants the use of cultivable model viruses to gain more insight on their transmission routes and inactivation methods. We studied the inactivation of the reported enteric canine calicivirus no. 48 (CaCV) and the respiratory feline calicivirus F9 (FeCV) and correlated inactivation to reduction in PCR units of FeCV, CaCV, and a norovirus. Inactivation of suspended viruses was temperature and time dependent in the range from 0 to 100 degrees C. UV-B radiation from 0 to 150 mJ/cm(2) caused dose-dependent inactivation, with a 3 D (D = 1 log(10)) reduction in infectivity at 34 mJ/cm(2) for both viruses. Inactivation by 70% ethanol was inefficient, with only 3 D reduction after 30 min. Sodium hypochlorite solutions were only effective at >300 ppm. FeCV showed a higher stability at pH <3 and pH >7 than CaCV. For all treatments, detection of viral RNA underestimated the reduction in viral infectivity. Norovirus was never more sensitive than the animal caliciviruses and profoundly more resistant to low and high pH. Overall, both animal viruses showed similar inactivation profiles when exposed to heat or UV-B radiation or when incubated in ethanol or hypochlorite. The low stability of CaCV at low pH suggests that this is not a typical enteric (calici-) virus. The incomplete inactivation by ethanol and the high hypochlorite concentration needed for sufficient virus inactivation point to a concern for decontamination of fomites and surfaces contaminated with noroviruses and virus-safe water. 相似文献
20.
AIMS: The aim of the present study was to investigate the behaviour of two pathogenic vibrios (Vibrio cholerae O1 and Vibrio parahaemolyticus) during depuration and to compare it with that of Escherichia coli, used as an indicator of suitability for consumption. METHODS AND RESULTS: Samples of Mytilus galloprovincialis were experimentally contaminated with E. coli, V. cholerae O1 and V. parahaemolyticus, depurated in a pilot plant using ozone and analysed at selected intervals. Numbers of E. coli and vibrios were estimated using an MPN method. The presence of vibrios was confirmed by the use of PCR. The target genes used were ctx for V. cholerae O1 and the restriction fragment pR72H for V. parahaemolyticus. There was a substantially smaller reduction in the numbers of both vibrios (approximately 1 log) during the depuration process than of E. coli (approximately 3 log). CONCLUSIONS: The results confirm the inadequacy of E. coli as an indicator that molluscs have been cleansed of other microbiological agents. SIGNIFICANCE AND IMPACT OF THE STUDY: The study confirms the risk associated with the consumption of mussels and the need to correctly conserve and cook them prior to consumption. 相似文献