scholarly journals Protocol for Specific Isolation of Virulent Strains of Vibrio vulnificus Serovar E (Biotype 2) from Environmental Samples

2004 ◽  
Vol 70 (12) ◽  
pp. 7024-7032 ◽  
Author(s):  
Eva Sanjuán ◽  
Carmen Amaro
Keyword(s):  
Environmental Samples ◽  
Vibrio Vulnificus ◽  
Growth Yield ◽  
Field Studies ◽  
Epidemiological Studies ◽  
Alkaline Peptone Water ◽  
Virulent Strains ◽  
Peptone Water ◽  
Human Sera ◽  
Human Infections

ABSTRACT The eel pathogen Vibrio vulnificus biotype 2 comprises at least three serovars, with serovar E being the only one involved in both epizootics of eel vibriosis and sporadic cases of human infections. The virulent strains of this serovar (VSE) have only been recovered from clinical (mainly eel tissue) sources. The main objective of this work was to design and validate a new protocol for VSE-specific isolation from environmental samples. The key element of the new protocol is the broth used for the first step (saline eel serum broth [SEB]), which contains eel serum as a nutritive and selective component. This approach takes advantage of the ability of VSE cells to grow in eel serum and thus to separate themselves from the pool of competitors. The growth yield in SEB after 8 h of incubation was 1,000 times higher for VSE strains than for their putative competitors (including biotype 1 strains of the species). The selective and differential agar Vibrio vulnificus medium (VVM) was selected from five selective media for the second step because it gave the highest plating efficiency not only for the VSE group but also for other V. vulnificus groups, including biotype 3. The entire protocol was validated by field studies, with alkaline peptone water plus VVM as a control. V. vulnificus was isolated by both protocols, but serovar E was only recovered by the new method described here. All selected serovar E isolates were identified as VSE since they were virulent for both eels and iron-overloaded mice and resisted the bactericidal action of eel and iron-overloaded human sera. In conclusion, this new protocol is a suitable method for the isolation of VSE strains from environmental samples and is recommended for epidemiological studies of the pathogenic serovar E.

Comparison of Five Selective Enrichment Broths and Two Selective Agars For Recovery of Vibrio vulnificus From Oysters

1992 ◽  
Vol 55 (5) ◽  
pp. 356-359 ◽  
Author(s):  
EDNA M. SLOAN ◽  
CURTIS J. HAGEN ◽  
GAYLE A. LANCETTE ◽  
JAMES T. PEELER ◽  
JOHN N. SOFOS
Keyword(s):  
Vibrio Vulnificus ◽  
Sodium Dodecyl ◽  
Error Recovery ◽  
Polymyxin B ◽  
Rank Test ◽  
Selective Enrichment ◽  
Alkaline Peptone Water ◽  
Peptone Water ◽  
Average Standard Error ◽  
Selection Of

The Bacteriological Analytical Manual (6th edition) specifies use of glucose-salt-teepol (GST) broth for detection of Vibrio vulnificus and other halophilic vibrios in seafood. Since teepol is no longer commercially available, this study compared five enrichment broths for their ability to recover V. vulnificus. Ten samples of seeded oysters were analyzed using a three-tube MPN and enriched in each of five broths in duplicate. Broth cultures were then streaked onto cellobiose-polymyxin B-colistin (CPC) agar and sodium dodecyl sulfate-polymyxin B-sucrose (SDS) agar plates. Average (± standard error) recovery (log MPN/g) from each broth was as follows: Alkaline-peptone-water (APW), 4.16 ± 0.20; Marine (MRN) broth, 3.63 ± 0.16; Horie's broth, 2.88 ± 0.17; Monsur's broth, 2.43 ± 0.16; and GST broth, 1.28 ± 0.28. APW and MRN broths yielded significantly (P<0.05) higher recovery than other broths by the Kruskal-Wallis nonparametric rank test. Vibrio vulnificus was isolated with higher frequency from CPC (81%) as compared with SDS (61%) agar plates. Background growth was minimal on CPC agar, facilitating selection of V. vulnificus colonies. Based on these results, APW enrichment broth and CPC isolation agar were more efficient for recovery of V. vulnificus from oysters than other broth and agar combinations

Effect of Frozen Storage and Vacuum-Packaging on Survival of Vibrio Vulnificus in Gulf Coast Oysters (Crassostrea virginica)

1994 ◽  
Vol 57 (7) ◽  
pp. 604-606 ◽  
Author(s):  
ROGER W. PARKER ◽  
ELLEN M. MAURER ◽  
A. BILL CHILDERS ◽  
DONALD H. LEWISI
Keyword(s):  
Vibrio Vulnificus ◽  
Gulf Coast ◽  
Health Hazard ◽  
Frozen Storage ◽  
Polymyxin B ◽  
Aerobic Bacteria ◽  
Most Probable Number ◽  
Probable Number ◽  
Alkaline Peptone Water ◽  
Peptone Water

Vibrio vulnificus contamination of raw oysters is a serious public health hazard, therefore, it is necessary to investigate the persistence of V. vulnificus in harvested and stored oysters. For this study, triplicate oyster samples were split into four treatment groups: control, normal-packaged; control, vacuum-packaged; inoculated, normal-packaged; and inoculated, vacuum-packaged. Oysters in the inoculated groups were individually injected with V. vulnificus to a level of approximately 1 × 106 CFU/g. Control oysters were already naturally contaminated to a level of approximately 1 × 104 CFU/g. Oysters were then packaged, frozen and stored at −20°C. On day 0 and days 7, 14, 30 and 70 post-freezing, concentrations of total aerobic bacteria and V. vulnificus were determined using a 3-tube most probable number (MPN) estimation from enrichment Alkaline Peptone Water tubes with subsequent presumptive V. vulnificus growth on modified Cellobiose-Polymyxin B-Colistin agar. Length of frozen storage had a significant effect on decreasing total aerobic bacteria (from approximately 106 CFU/g to approximately 102.5 CFU/g) and V. vulnificus (from approximately 105 CFU/g to approximately 101 CFU/g). Also, vacuum-packaged samples showed significantly lower concentrations of V. vulnificus over the length of the study than did the normal-sealed samples.

Enumeration of Vibrio parahaemolyticus and Vibrio vulnificus in Various Seafoods with Two Enrichment Broths

1994 ◽  
Vol 57 (5) ◽  
pp. 403-409 ◽  
Author(s):  
CURTIS J. HAGEN' ◽  
EDNA M. SLOAN ◽  
GAYLE A. LANCETTE ◽  
JAMES T. PEELER ◽  
JOHN N. SOFOS
Keyword(s):  
Cold Stress ◽  
Vibrio Parahaemolyticus ◽  
Vibrio Vulnificus ◽  
Polymyxin B ◽  
Most Probable Number ◽  
Geometric Means ◽  
Probable Number ◽  
Alkaline Peptone Water ◽  
Peptone Water ◽  
Stressed Cells

This study compares recoveries of Vibrio parahaemolyticus and Vibrio vulnificus with salt-polymyxin B broth (SPB) and alkaline peptone water (APW) from samples of crab legs, oysters, shrimp, lobster and shark, which were inoculated at three levels (approximately 101 to 102, 102 to 103 and 104 to 105/g) with each of the pathogens. Six samples of each product were analyzed [3-tube most probable number (MPN)] with each broth. Inoculated samples of oysters and slurries of crab and lobster were also tested after cold stress (refrigerated at 2 to 4°C, 3 or 7 days, or frozen at −15°C for 21 or 28 days). For each seafood, geometric means of cells recovered with APW were significantly (P < 0.05) higher than the corresponding means of recovery with SPB. In addition, 12 of 15 calculated estimates of 50% relative detectable levels (RDL50) were lower (P < 0.05) for APW than for SPB. In these samples, the level of detection by APW was found to be 40 to 32,000 and 6- to 42-fold lower for V parahaemolyticus and V. vulnificus, respectively, than the level of detection by SPB. In cold-stored samples, overall detection of the pathogens was greatly reduced, but APW was also more efficient than SPB in recovering stressed cells.

Prevalence and Characterization of Vibrio vulnificus Isolated from Shrimp Products Imported into Denmark

1997 ◽  
Vol 60 (9) ◽  
pp. 1132-1134 ◽  
Author(s):  
ANDERS DALSGAARD ◽  
LISE HØI
Keyword(s):  
Vibrio Vulnificus ◽  
Polymyxin B ◽  
Potential Hazard ◽  
Alkaline Peptone Water ◽  
Heat Treated ◽  
Peptone Water ◽  
Frozen Shrimp ◽  
Low Prevalence ◽  
Raw Shrimp

Vibrio vulnificus is a naturally occurring bacterium in tropical aquatic environments. We investigated the prevalence of V. vulnificus in frozen shrimp products mainly originating from tropical countries by pre-enrichment of samples in alkaline peptone water supplemented with polymyxin B (APWP) followed by subculture onto modified cellobiose–polymyxin B–colistin (mCPC) agar. V. vulnificus was detected in 3 of 46 (7%) frozen raw shrimp samples analyzed. However, V. vulnificus was not recovered from any of the 61 frozen cooked products, suggesting that these products were adequately heat treated and that cross-contamination did not occur during processing. The pre-enrichment of shrimp samples in APWP followed by subculture on mCPC agar proved useful for the isolation of V. vulnificus. The absence of V. vulnificus in frozen cooked shrimp products and the low prevalence of V. vulnificus in frozen raw shrimp suggest that V. vulnificus in imported shrimp products does not constitute a potential hazard to public health.

scholarly journals Improved Isolation of Vibrio vulnificusfrom Seawater and Sediment with Cellobiose-Colistin Agar

1998 ◽  
Vol 64 (5) ◽  
pp. 1721-1724 ◽  
Author(s):  
Lise H�i ◽  
Inger Dalsgaard ◽  
Anders Dalsgaard
Keyword(s):  
Bile Salts ◽  
Vibrio Vulnificus ◽  
Polymyxin B ◽  
Selective Medium ◽  
Isolation Rate ◽  
Alkaline Peptone Water ◽  
Water And Sediment ◽  
Significant Difference ◽  
Peptone Water

ABSTRACT An improved selective medium, cellobiose-colistin (CC) agar, gave a significantly higher (P < 0.05) isolation rate ofVibrio vulnificus from water and sediment samples than did modified cellobiose-polymyxin B-colistin (mCPC) agar. In a total of 446 alkaline peptone water preenrichments amended with polymyxin B,V. vulnificus was isolated from 154 preenrichments (35%) with mCPC agar and from 179 preenrichments (40%) with CC agar. CC agar gave a higher plating efficiency of V. vulnificuscells than did cellobiose-polymyxin B-colistin (CPC) agar, mCPC agar, or thiosulfate-citrate-bile salts-sucrose (TCBS) agar; the only significant difference was observed with TCBS agar, which gave much lower plating efficiencies than the other selective media. Determination of MICs demonstrated that the concentrations of colistin and polymyxin B in CPC agar inhibit growth of a proportion ofV. vulnificus strains.

scholarly journals Detection and Enumeration of Vibrio vulnificus in Oysters from Two Estuaries along the Southwest Coast of India, Using Molecular Methods

2004 ◽  
Vol 70 (11) ◽  
pp. 6909-6913 ◽  
Author(s):  
Ammini Parvathi ◽  
H. Sanath Kumar ◽  
Indrani Karunasagar ◽  
Iddya Karunasagar
Keyword(s):  
Alkaline Phosphatase ◽  
Environmental Factors ◽  
Vibrio Vulnificus ◽  
Oligonucleotide Probe ◽  
Monsoon Season ◽  
Seasonal Distribution ◽  
Southwest Coast Of India ◽  
Colony Hybridization ◽  
Alkaline Peptone Water ◽  
Peptone Water

ABSTRACT This study was conducted to understand the seasonal distribution of Vibrio vulnificus in oysters from two estuaries and the effect of environmental factors on the abundance of V. vulnificus in tropical waters. V. vulnificus was detected in 56.6% of the samples tested by colony hybridization with an alkaline phosphatase-labeled oligonucleotide probe (VV-AP), and the counts ranged from <10/g during the summer months to 103/g in the monsoon season at both sites. The density of V. vulnificus appeared to be controlled more by salinity than by temperature. A nested PCR used in this study detected V. vulnificus in 85% of the samples following 18 h of enrichment in alkaline peptone water.

Occurrence of Vibrio vulnificus Biotypes in Danish Marine Environments

1998 ◽  
Vol 64 (1) ◽  
pp. 7-13 ◽  
Author(s):  
L. Høi ◽  
J. L. Larsen ◽  
I. Dalsgaard ◽  
A. Dalsgaard
Keyword(s):  
Vibrio Vulnificus ◽  
Low Frequency ◽  
Polymyxin B ◽  
Wild Fish ◽  
Strongly Correlated ◽  
Marine Environments ◽  
Alkaline Peptone Water ◽  
Warm Summer ◽  
Peptone Water ◽  
Lower Temperature

ABSTRACT During the unusually warm summer in Denmark in 1994, 11 clinical cases of Vibrio vulnificus infection were reported. These reports initiated an investigation of the occurrence of V. vulnificus biotypes in Danish marine environments. Samples of coastal water, sediment, shellfish, and wild fish were analyzed by preenrichment in alkaline peptone water amended with polymyxin B (2.0 × 104 U/liter) followed by streaking onto modified cellobiose-polymyxin B-colistin agar. V. vulnificus-like colonies were tested with a V. vulnificus-specific DNA probe. Low densities of V. vulnificus were detected in water (0.8 to 19 CFU/liter) from June until mid-September and in sediment (0.04 to >11 CFU/g) from July until mid-November. The presence of V. vulnificus was strongly correlated with water temperature. However, we isolatedV. vulnificus from water from a mussel farm at a lower temperature than previously reported (7°C). In 1 of the 13 locations studied, V. vulnificus was found in mussels in 7 of 17 samples analyzed; this is the first report of V. vulnificusin European shellfish. V. vulnificus was also isolated from gills, intestinal contents, and mucus from wild fish. Although biotyping of 706 V. vulnificus strains isolated during our investigations revealed that the majority of the strains (99.6%) belonged to biotype 1, biotype 2 was detected in seawater at a low frequency (0.4%). Our findings provide further evidence that seawater can serve as a reservoir and might facilitate spread of V. vulnificus biotype 2 to eels, with subsequent spread to persons handling eels. In conclusion, our data demonstrate that V. vulnificus is ubiquitous in a temperate marine environment and that V. vulnificus biotype 2 is not strictly confined to eels.

Evaluation of DNA Colony Hybridization and Real-Time PCR for Detection of Vibrio parahaemolyticus and Vibrio vulnificus in Postharvest-Processed Oysters

2009 ◽  
Vol 72 (10) ◽  
pp. 2106-2109 ◽  
Author(s):  
JESSICA L. JONES ◽  
KATHY E. NOE ◽  
ROBIN BYARS ◽  
ANGELO DePAOLA
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Parahaemolyticus ◽  
Vibrio Vulnificus ◽  
The United States ◽  
Most Probable Number ◽  
Probable Number ◽  
Colony Hybridization ◽  
Alkaline Peptone Water ◽  
Peptone Water

The applicability of real-time PCR was examined for detection of vibrios from postharvest-processed (PHP) oysters to allow for a more rapid assay and higher sample throughput than currently used. During June to October 2004, 68 PHP oyster samples were collected directly from PHP firms or from retail markets across the United States. PHP oysters were examined to determine the effectiveness of treatments in the reduction of vibrio levels and to compare the analytical methods utilized. The latter is the focus of the data presented here. Each sample was analyzed for Vibrio parahaemolyticus and V. vulnificus by using a 2-dilution, three-tube most-probable-number (MPN) and a 25-g presence/absence enrichment in alkaline peptone water. Following 6-h and overnight enrichment, aliquots from each MPN tube and the 25-g sample were streaked onto selective media and tested by real-time PCR. Colonies from the selective agar were confirmed as V. parahaemolyticus or V. vulnificus by DNA colony hybridization. DNA hybridization and real-time PCR results for each MPN tube and the 25-g enrichment at both time points were analyzed individually for each organism. The methods were in agreement for 857 (95%) of 901 and for 882 (98%) of 903 tubes for detection of V. parahaemolyticus and V. vulnificus, respectively. Overall, there was 96% agreement between real-time and DNA colony hybridization. The results obtained by real-time PCR were comparable to those from DNA colony hybridization, but analysis time was significantly reduced for the detection of vibrios in PHP-treated oysters.

scholarly journals Ergonomic Evaluation of Current Advancements in Blueberry Harvesting

Agronomy ◽  
2018 ◽  
Vol 8 (11) ◽  
pp. 266 ◽  
Author(s):  
Eunsik Kim ◽  
Andris Freivalds ◽  
Fumiomi Takeda ◽  
Changying Li
Keyword(s):  
Occupational Safety ◽  
Health And Safety ◽  
Field Studies ◽  
The United States ◽  
Epidemiological Studies ◽  
Component Ratio ◽  
Social Scientists ◽  
Private Industry ◽  
Work Related ◽  
Trauma Disorders

Work-related musculoskeletal disorders (MSDs) accounted for 32% of days-away-from-work cases in private industry in 2016. Several factors have been associated with MSDs, such as repetitive motion, excessive force, awkward and/or sustained postures, and prolonged sitting and standing, all of which are required in farm workers’ labor. While numerous epidemiological studies on the prevention of MSDs in agriculture have been conducted, an ergonomics evaluation of blueberry harvesting has not yet been systematically performed. The purpose of this study was to investigate the risk factors of MSDs for several types of blueberry harvesting (hand harvesting, semi-mechanical harvesting with hand-held shakers, and over-the-row machines) in terms of workers’ postural loads and self-reported discomfort using ergonomics intervention techniques. Five field studies in the western region of the United States between 2017 and 2018 were conducted using the Borg CR10 scale, electromyography (EMG), Rapid Upper Limb Assessment (RULA), the Cumulative Trauma Disorders (CTD) index, and the NIOSH (National Institute for Occupational Safety and Health) lifting equation. In evaluating the workloads of picking and moving blueberries by hand, semi-mechanical harvesting with hand-held shakers, and completely mechanized harvesting, only EMG and the NIOSH lifting equation were used, as labor for this system is limited to loading empty lugs and unloading full lugs. Based on the results, we conclude that working on the fully mechanized harvester would be the best approach to minimizing worker loading and fatigue. This is because the total component ratio of postures in hand harvesting with a RULA score equal to or greater than 5 was 69%, indicating that more than half of the postures were high risk for shoulder pain. For the semi-mechanical harvesting, the biggest problem with the shakers is the vibration, which can cause fatigue and various risks to workers, especially in the upper limbs. However, it would be challenging for small- and medium-sized blueberry farms to purchase automated harvesters due to their high cost. Thus, collaborative efforts among health and safety professionals, engineers, social scientists, and ergonomists are needed to provide effective ergonomic interventions.

scholarly journals The rK39 Antigen from an Iranian Strain of Leishmania infantum: Detection of Anti-Leishmania Antibodies in Humans and Dogs

2020 ◽  
Author(s):  
Bibi Razieh HOSSEINI FARASH ◽  
Mehdi MOHEBALI ◽  
Bahram KAZEMI ◽  
Abdolmajid FATA ◽  
Reza RAOOFIAN ◽  
...  
Keyword(s):  
Leishmania Infantum ◽  
Field Studies ◽  
Severe Form ◽  
Recombinant Antigen ◽  
Agglutination Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Healthy Controls ◽  
Direct Agglutination Test ◽  
Human Sera ◽  
Extended Field

Background: Visceral leishmaniasis (VL) is the most severe form of leishmaniasis in Iran with high mortality rates in the case of inaccurate diagnosis and treatment. This study aimed to prepare and evaluate a new rk39 recombinant antigen from an Iranian strain of Leishmania infantum for diagnosis of VL in humans and dogs. Methods: rK39-based enzyme-linked immunosorbent assay (ELISA) was compared with the direct agglutination test (DAT) for the detection of anti L. infantum antibodies. We screened 84 human sera and 87 dog sera from clinical cases in the endemic area of Meshkin-Shahr, Iran along with 176 sera from healthy controls (collected from 86 humans and 90 dogs) during 2013 -2016. Results: Using the rK39 ELISA, a sensitivity of 85.7% (95% CI, 95-99%) and a specificity of 86.0% (95% CI, 95%-99%) were detected in human sera at a 1:800 (cut-off) titer when DAT-confirmed cases were compared with healthy controls; a sensitivity of 96.6% (95% CI, 95%-99%) and specificity of 94.4% (95% CI, 95%-99%) were found at a 1:80 (cut-off) titer compared with DAT. Kappa analysis indicated agreement between the rK39 ELISA and DAT (0.718) when using human sera at a 1:800 (cut-off) titer as well as (0.910) at a 1:80 (cut-off) titer when using dog sera (P<0.05). Conclusion: New rk39 recombinant antigen from an Iranian strain of Leishmania infantum seems to be used for diagnosis of VL in humans and dogs. Further extended field studies are recommended

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