scholarly journals Evaluation of a Tissue Culture–Based Approach for Differentiating between Virulent and Avirulent Vibrio parahaemolyticus Strains Based on Cytotoxicity

2007 ◽  
Vol 70 (2) ◽  
pp. 348-354 ◽  
Author(s):  
P. S. MARIE YEUNG ◽  
MARTIN WIEDMANN ◽  
KATHRYN J. BOOR
Keyword(s):  
Tissue Culture ◽  
Cell Lines ◽  
Vibrio Parahaemolyticus ◽  
Cell Damage ◽  
Human Infection ◽  
Clinical Isolates ◽  
Food Sources ◽  
Oyster Tissue ◽  
Assay Sensitivity ◽  
Mammalian Cell Lines

The ability of only a subset of Vibrio parahaemolyticus strains to cause human infection underscores the need for an analytical method that can effectively differentiate between pathogenic strains and those that do not cause disease. We tested the feasibility of a tissue culture–based assay to determine whether clinical isolates could be differentiated from nonclinical isolates based on relative isolate cytopathogenicity. To screen for cytotoxic capability, we measured relative extracellular lactate dehydrogenase as an indicator of host cell damage in five different mammalian cell lines in the presence of V. parahaemolyticus. Isolates originating from clinical sources exhibited 15.5 to 59.3% relative cytotoxicity, whereas those originating from food sources exhibited 4.4 to 54.9% relative cytotoxicity. In the presence of ∼1.2 × 106 cells, cytotoxicity was 1.6- to 3.5-fold higher (P < 0.05) for clinical isolates than for nonclinical isolates in L2, Henle 407, and Caco-2 cell lines. V. parahaemolyticus serotype O3:K6 clinical isolates had 1.6- to 2.1-fold higher cytotoxicity than did the non-O3:K6 clinical isolates, with significantly higher cytotoxicity in HeLa, J774A.1, and Henle 407 cells than in L2 and Caco-2 cells. Because V. parahaemolyticus often is found in oysters, the effect of the presence of an oyster matrix on assay efficacy was also tested with L2 cells. The cytotoxicity elicited by a highly cytotoxic V. parahaemolyticus isolate was not affected by the presence of oyster tissue, suggesting that an oyster matrix will not interfere with assay sensitivity. In the present format, this assay can detect the presence of >105 cells of a virulent V. parahaemolyticus strain in an oyster matrix.

scholarly journals Vibrio parahaemolyticus Growth under Low-Iron Conditions and Survival under High-Magnesium Conditions

2006 ◽  
Vol 69 (5) ◽  
pp. 1040-1045 ◽  
Author(s):  
CHIA-HSIN JU ◽  
P. S. MARIE YEUNG ◽  
JESSICA OESTERLING ◽  
DANIEL A. SEIGERMAN ◽  
KATHRYN J. BOOR
Keyword(s):  
Environmental Conditions ◽  
Vibrio Parahaemolyticus ◽  
Human Exposure ◽  
Growth Characteristics ◽  
Clinical Isolates ◽  
Food Sources ◽  
Entire Group ◽  
Phenotypic Characteristic ◽  
Environmental Survival ◽  
Recent Emergence

Since 1996, Vibrio parahaemolyticus serotype O3:K6 and closely related strains have been associated with an increased incidence of V. parahaemolyticus gastroenteritis worldwide, suggesting the emergence of strains with enhanced abilities to cause disease. One hypothesis for the recent emergence of V. parahaemolyticus O3:K6 and related strains is an enhanced capacity for environmental survival relative to other strains, which might result in increased human exposure to these organisms. Therefore, the objective of this study was to test the hypothesis that survival or growth characteristics of clinical V. parahaemolyticus isolates differ from those of nonclinical isolates under different environmental conditions. Twenty-six V. parahaemolyticus isolates selected to represent either clinical or food sources were monitored for either survival following exposure to high magnesium (300 mM) or growth under iron-limited conditions. Isolates in each category (clinical or food) differed widely in survival capabilities following 24 h of exposure to 300 mM Mg2+. Although 4 of 15 clinical isolates grew better at approximately 0.96 μM Fe2+ (iron-limited conditions) than at 50 μM Fe2+ (iron-rich conditions), as an entire group clinical isolates in this study were not more effective at growing under iron-limited conditions than were strains not associated with disease. Within the diverse collection of strains examined in these experiments, neither growth characteristics in low-iron environments nor survival capabilities following exposure to high magnesium concentrations were uniformly different between clinical and nonclinical V. parahaemolyticus isolates. Therefore, neither phenotypic characteristic can be used to reliably differentiate potentially pathogenic V. parahaemolyticus strains.

scholarly journals Genetic Diversity and Population Structure of Vibrio parahaemolyticus Isolated From Clinical and Food Sources

2021 ◽  
Vol 12 ◽  
Author(s):  
Min He ◽  
Tao Lei ◽  
Fufeng Jiang ◽  
Jumei Zhang ◽  
Haiyan Zeng ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Vibrio Parahaemolyticus ◽  
Large Scale ◽  
Foodborne Pathogen ◽  
Guangdong Province ◽  
Housekeeping Genes ◽  
Clinical Isolates ◽  
Food Sources ◽  
Clinical Samples ◽  
High Genetic Diversity

Vibrio parahaemolyticus is a common foodborne pathogen that causes gastroenteritis worldwide. Determining its prevalence and genetic diversity will minimize the risk of infection and the associated economic burden. Multilocus sequence typing (MLST) is an important tool for molecular epidemiology and population genetic studies of bacteria. Here, we analyzed the genetic and evolutionary relationships of 162 V. parahaemolyticus strains isolated in the Guangdong Province, China, using MLST. In the study, 120 strains were isolated from food samples, and 42 strains were isolated from clinical samples. All strains were categorized into 100 sequence types (STs), of which 58 were novel (48 from the food isolates and 10 from the clinical isolates). ST415 was the most prevalent ST among the food isolates, while ST3 was the most prevalent ST among the clinical isolates. Further, 12 clonal complexes, 14 doublets, and 73 singletons were identified in all ST clusters, indicating high genetic diversity of the analyzed strains. At the concatenated sequence level, non-synonymous sites in both, food and clinical isolates, were associated with purifying selection. Of note, the dN/dS ration was greater than 1 for some housekeeping genes in all isolates. This is the first time that some loci under positive selection were identified. These observations confirm frequent recombination events in V. parahaemolyticus. Recombination was much more important than mutation for genetic heterogeneity of the food isolates, but the probabilities of recombination and mutations were almost equal for the clinical isolates. Based on the phylogenetic analysis, the clinical isolates were concentrated in the maximum-likelihood tree, while the food isolates were heterogeneously distributed. In conclusion, the food and clinical isolates of V. parahaemolyticus from the Guangdong Province are similar, but show different evolutionary trends. This may help prevent large-scale spread of highly virulent strains and provides a genetic basis for the discovery of microevolutionary relationships in V. parahaemolyticus populations.

scholarly journals Molecular, Serological, and Virulence Characteristics of Vibrio parahaemolyticus Isolated from Environmental, Food, and Clinical Sources in North America and Asia

2003 ◽  
Vol 69 (7) ◽  
pp. 3999-4005 ◽  
Author(s):  
Angelo DePaola ◽  
Jodie Ulaszek ◽  
Charles A. Kaysner ◽  
Bradley J. Tenge ◽  
Jessica L. Nordstrom ◽  
...  
Keyword(s):  
United States ◽  
New York ◽  
Vibrio Parahaemolyticus ◽  
Gulf Coast ◽  
Atlantic Coast ◽  
The United States ◽  
Clinical Isolates ◽  
Food Sources ◽  
Environmental Isolates ◽  
The Pacific

ABSTRACT Potential virulence attributes, serotypes, and ribotypes were determined for 178 pathogenic Vibrio parahaemolyticus isolates from clinical, environmental, and food sources on the Pacific, Atlantic, and Gulf Coasts of the United States and from clinical sources in Asia. The food and environmental isolates were generally from oysters, and they were defined as being pathogenic by using DNA probes to detect the presence of the thermostable direct hemolysin (tdh) gene. The clinical isolates from the United States were generally associated with oyster consumption, and most were obtained from outbreaks in Washington, Texas, and New York. Multiplex PCR was used to confirm the species identification and the presence of tdh and to test for the tdh-related hemolysin trh. Most of the environmental, food, and clinical isolates from the United States were positive for tdh, trh, and urease production. Outbreak-associated isolates from Texas, New York, and Asia were predominantly serotype O3:K6 and possessed only tdh. A total of 27 serotypes and 28 ribogroups were identified among the isolates, but the patterns of strain distribution differed between the serotypes and ribogroups. All but one of the O3:K6 isolates from Texas were in a different ribogroup from the O3:K6 isolates from New York or Asia. The O3:K6 serotype was not detected in any of the environmental and food isolates from the United States, and none of the food or environmental isolates belonged to any of the three ribogroups that contained all of the O3:K6 and related clinical isolates. The combination of serotyping and ribotyping showed that the Pacific Coast V. parahaemolyticus population appeared to be distinct from that of either the Atlantic Coast or Gulf Coast. The fact that certain serotypes and ribotypes contained both clinical and environmental isolates while many others contained only environmental isolates implies that certain serotypes or ribotypes are more relevant for human disease.

scholarly journals Virological and molecular investigation on specimens from buffaloes suspected to be infected by buffalopox virus in Egypt 2019

2021 ◽  
Vol 53 (4) ◽  
Author(s):  
Christine A. Mikhael ◽  
Farid F. Zaki ◽  
Fayez A. Salib
Keyword(s):  
Tissue Culture ◽  
Cell Lines ◽  
Cluster Formation ◽  
Clinical Signs ◽  
Chorioallantoic Membrane ◽  
Skin Lesions ◽  
Culture Cell ◽  
Human Infection ◽  
Tissue Culture Cell ◽  
Polymerase Chain

In this study, skin lesions from buffaloes showing clinical signs of buffalopox infection were tested to isolate and identify the buffalopox virus (BPXV). Clinical examination of infected buffaloes was performed and visible clinical signs recorded. Skin scabs from infected buffaloes were collected and used for virus isolation on embryonated chicken egg (ECE) and tissue culture cell lines. The isolated BPXV was identified and characterized using polymerase chain reaction (PCR). The infected buffaloes displayed fever, skin eruptions, enlargement of superficial lymph nodes, emaciation and drop in milk yield. The ECE inoculated with the prepared skin scab samples showed clear raised white pock lesions on the chorioallantoic membrane (CAM). The inoculated tissue cultures (VERO and BHK cell lines) revealed a cytopathic effect (CPE) including rounding, clumping with cytoplasmic granulation and cluster formation. PCR for the C18L specific BPXV gene was carried out on the virus infected tissue culture produced 368 bp bands. Human infection with BPXV was also recorded. It was concluded that BPXV is circulating in Egyptian buffaloes, causing economical losses and infection in contact humans.

scholarly journals Clinical Isolates of Measles Virus Use CD46 as a Cellular Receptor

2000 ◽  
Vol 74 (9) ◽  
pp. 3967-3974 ◽  
Author(s):  
Marianne Manchester ◽  
Danelle S. Eto ◽  
Alexandra Valsamakis ◽  
Paloma B. Liton ◽  
Rafael Fernandez-Muñoz ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Cell Lines ◽  
Measles Virus ◽  
Culture Cell ◽  
Clinical Isolates ◽  
Cell Surface Receptor ◽  
Tissue Culture Cell ◽  
Dependent Manner ◽  
Cell Type ◽  
Receptor Usage

ABSTRACT Laboratory strains of measles viruses (MV), such as Edmonston and Halle, use the complement regulatory protein CD46 as a cell surface receptor. The receptor usage of clinical isolates of MV, however, remains unclear. Receptor usage by primary patient isolates of MV was compared to isolates that had been passaged on a variety of tissue culture cell lines. All of the isolates could infect cells in a CD46-dependent manner, but their tropism was restricted according to cell type (e.g., lymphocytes versus fibroblasts). The results indicate that patient isolates that have not been adapted to tissue culture cell lines use CD46 as a receptor. In addition, passaging primary MV patient isolates in B95-8 cells selected variants that had alternate receptor usage compared to the original isolate. Thus, changes in receptor usage by MV are dependent upon the cell type used for isolation. Furthermore, our results confirm the relevance of the CD46 receptor to natural measles infection.

High Level Expression of Active Human Prothrombin in a Vaccina Virus Expression System

1992 ◽  
Vol 68 (02) ◽  
pp. 119-124 ◽  
Author(s):  
F G Falkner ◽  
P L Turecek ◽  
R T A MacGillivray ◽  
W Bodemer ◽  
F Scheiflinger ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Cell Lines ◽  
Expression System ◽  
Human Cell Line ◽  
Cell System ◽  
Time Saving ◽  
Expression Levels ◽  
Mammalian Cell Lines ◽  
Cell Line Hela ◽  
Virus Expression

SummaryWe have worked out an efficient and time saving procedure for the expression of recombinant human prothrombin. The glycoprotein was expressed in the vaccinia virus expression system in several mammalian cell lines. The kidney cell lines Vero and BHK and the human cell line Hela were found to efficiently secrete prothrombin. Expression levels of 3–4 µg of factor II per 106 cells per day corresponding to 18–23 mU per 106 cells per day were achieved. Since the expression levels obtained with the vaccinia virus/Vero cell system were comparable to those obtained in amplified transformed CHO cells it provides an alternative system for the efficient expression of human prothrombin and may allow to further elucidate structure-function relationships of (pro)thrombin and its various effectors.

scholarly journals Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alessandro T. Caputo ◽  
Oliver M. Eder ◽  
Hana Bereznakova ◽  
Heleen Pothuis ◽  
Albert Ardevol ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Recombinant Proteins ◽  
Cultured Cells ◽  
Hek293 Cells ◽  
Reaction Products ◽  
Enzyme Form ◽  
X Ray Crystallography ◽  
Mammalian Cell Lines ◽  
Apparent Loss

AbstractPuromycin and the Streptomyces alboniger-derived puromycin N-acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N-acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins.

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