A simplified immunofluorescence technique for detection of viable cells of Vibrio cholerae O1 and O139

Chowdhury, M. A. R.; Xu, Bo; Montilla, Rafael; Hasan, Jafrul; Huq, Anwar; Colwell, Rita R.

doi:10.1016/0167-7012(95)00066-6

Cited by 28 publications

(17 citation statements)

References 16 publications

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“…Agar plates were incubated overnight at 37 C. Five or more presumptive V. cholerae colonies from each agar plate were placed into tryptic soy agar (TSA) transport tubes and incubated overnight at 37 C. For DFA-DVC testing, 10 μL each of 2.5% yeast extract and 0.2% nalidixic acid were added to 1-mL aliquots of homogenized PLK and PFW samples. 11 After overnight incubation in the dark at 37 C, samples were fixed with 112 μL of formaldehyde (37% by weight), and stored at 4 C. Alkaline peptone water enrichments in lysis buffer, culture isolates in TSA transport vials, and DFA samples were shipped to the Centers for Disease Control and Prevention (CDC) in Atlanta, GA for subsequent analysis. One replicate set of the APW enrichments in lysis buffer and the DFA-DVC samples were shipped to the University of Maryland (UMD).…”

Section: Methodsmentioning

confidence: 99%

“…26 The DFA-DVC was performed to obtain a direct viable count of V. cholerae O1 and O139 from PLK and PFW samples. 11 At UMD, formalin-fixed samples were analyzed by microscopy using DFA kits for V. cholerae O1 and O139 (New Horizons Diagnostics, Baltimore, MD).…”

Section: Methodsmentioning

confidence: 99%

“…9 When V. cholerae cannot be detected by traditional culture methods, viable cells can be detected in environmental samples using fluorescent antibody and direct viable count methods (DFA-DVC). 10,11 Because DFA-DVC allows for the detection of plankton-associated V. cholerae, data obtained from DFA-DVC can be used to evaluate whether V. cholerae may be present in an environmental niche. Additionally, gene sequences specific to toxigenic V. cholerae can be used to determine the presence of viable V. cholerae cells in enrichment cultures.…”

Section: Introductionmentioning

confidence: 99%

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Environmental Surveillance for Toxigenic Vibrio cholerae in Surface Waters of Haiti

Kahler¹,

Haley²,

Chen³

et al. 2015

The American Society of Tropical Medicine and Hygiene

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Abstract. Epidemic cholera was reported in Haiti in 2010, with no information available on the occurrence or geographic distribution of toxigenic Vibrio cholerae in Haitian waters. In a series of field visits conducted in Haiti between 2011 and 2013, water and plankton samples were collected at 19 sites. Vibrio cholerae was detected using culture, polymerase chain reaction, and direct viable count methods (DFA-DVC). Cholera toxin genes were detected by polymerase chain reaction in broth enrichments of samples collected in all visits except March 2012. Toxigenic V. cholerae was isolated from river water in 2011 and 2013. Whole genome sequencing revealed that these isolates were a match to the outbreak strain. The DFA-DVC tests were positive for V. cholerae O1 in plankton samples collected from multiple sites. Results of this survey show that toxigenic V. cholerae could be recovered from surface waters in Haiti more than 2 years after the onset of the epidemic.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Environmental Surveillance for Toxigenic Vibrio cholerae in Surface Waters of Haiti

Kahler¹,

Haley²,

Chen³

et al. 2015

The American Society of Tropical Medicine and Hygiene

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“…The copepods were rinsed and resuspended in 1 ml of filter-sterilized water. All samples were prepared for direct viable counts (DVC) (10,21) and fixed with 5% formalin.…”

Section: Methodsmentioning

confidence: 99%

“…To determine if there were quantitative differences in colonization by V. cholerae of adults and nauplii, V. cholerae O1 El Tor was added to microcosms containing either 10 to 20 E. affinis adults or 20 to 45 nauplii (all naupliar stages, N1 to N6). Microcosms were prepared, inoculated, and run under the same conditions as above and were sampled at 24 h. All samples were prepared for direct microscope counting, following methods previously described (10,21).…”

Section: Vol 73 2007 Colonization Of Copepods By V Cholerae O1 Andmentioning

confidence: 99%

Association of Vibrio cholerae O1 El Tor and O139 Bengal with the Copepods Acartia tonsa and Eurytemora affinis

Rawlings

Ruiz

Colwell

2007

Appl Environ Microbiol

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The association of Vibrio cholerae with zooplankton has been suggested as an important factor in transmission of human epidemic cholera, and the ability to colonize zooplankton surfaces may play a role in the temporal variation and predominance of the two different serogroups (V. cholerae O1 El Tor and O139) in the aquatic environment. To date, interactions between specific serogroups and species of plankton remain poorly understood. Laboratory microcosm experiments were carried out to compare quantitatively the colonization of two copepod species, Acartia tonsa and Eurytemora affinis, by each of the epidemic serogroups. V. cholerae O1 consistently achieved higher abundances than V. cholerae O139 in colonizing adults of each copepod species as well as the multiple life stages of E. affinis. This difference in colonization may be significant in the general predominance of V. cholerae O1 in cholera epidemics in rural Bangladesh where water supplies are taken directly from the environment.Vibrio cholerae O1 El Tor and O139 Bengal are recognized as causative agents of cholera and are responsible for cholera epidemics in India and Bangladesh. More than 10 years ago, V. cholerae O139 Bengal was recognized as a newly emerged epidemic variant when it replaced V. cholerae O1 El Tor for two successive cholera seasons (9). It is not clear what factors contributed to the emergence of serogroup O139 as an epidemic variant or to its present coexistence with serogroup O1, but recent research provides strong evidence that horizontal transfer of genes among environmental strains of V. cholerae was a mechanism of its origin (4,12,15).Since the emergence of V. cholerae O139 in the Gangetic Delta region, both V. cholerae O1 and O139 have continued to cause cholera outbreaks with regular seasonality but with temporal variation in the prevalence of the two serogroups (3, 41). Yearly differences in reported cases of cholera caused by V. cholerae O1 and O139 suggest that differences may exist in resource utilization and responses to changing environmental conditions in the aquatic habitat as well as in the immunity of susceptible human populations. Publications that compare V. cholerae O1 and O139 emphasize molecular distinctions (15,16,34,36) and environmental distribution (1,30,43). No studies have explored the mechanisms by which the genotypes of V. cholerae may differ in their resource use. However, Simidu et al. (44) showed that different phenotypes of vibrios aggregated in different microhabitats, suggesting that temporal and spatial variation in the environment play important roles in the relative abundance of phenotypes.

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Detection, Isolation, and Identification of Vibrio cholerae from the Environment

et al. 2006

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Microbiological techniques for sampling the aquatic realm have become increasingly sophisticated, especially with advances in molecular biology. These techniques have been used to detect microorganisms that cannot be cultured by conventional bacteriological methods. This has resulted in a deeper and a clearer understanding of the ecology and epidemiology of microorganisms. Important advances have been made in isolation, detection, and identification of Vibrio cholerae over the past decade. The understanding that V. cholerae, like several other pathogenic bacteria, can enter into a state known as “viable but nonculturable” (VBNC) provided important clues on the epidemiology of the pathogen and its ability to cause sudden explosive epidemics at multiple places almost simultaneously. The advances in techniques have also allowed investigators to discern the intricate aspects of the ecology of this pathogen in the aquatic world. In this unit, we present the most accepted methods for the isolation and detection of V. cholerae.

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A simplified immunofluorescence technique for detection of viable cells of Vibrio cholerae O1 and O139

Cited by 28 publications

References 16 publications

Environmental Surveillance for Toxigenic Vibrio cholerae in Surface Waters of Haiti

Environmental Surveillance for Toxigenic Vibrio cholerae in Surface Waters of Haiti

Association of Vibrio cholerae O1 El Tor and O139 Bengal with the Copepods Acartia tonsa and Eurytemora affinis

Detection, Isolation, and Identification of Vibrio cholerae from the Environment

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