Sensitive immunoassay of Listeria monocytogenes with highly fluorescent bioconjugated silica nanoparticles probe

In this paper, a sensitive immunoassay method was proposed for Listeria monocytogenes detection by using highly fluorescent bioconjugated nanoparticles probe. (FITC-IgG)-doped fluorescent silica nanoparticles (fsNPs) firstly were synthesized by a microemulsion method and characterized by TEM and fluorescent spectra. Then the prepared fsNPs were conjugated with polyclonal rabbit anti-L. monocytogenes antibody (pAb) and used as indicator probe. A sandwich-type immune affinity reaction between polyclonal rabbit anti-L. monocytogenes antibody coated onto microplate wells, target bacteria and the fsNPs-antibody conjugates subsequently was conducted to detect target L. monocytogenes and assemble the indicator probe onto the wells. The target L. monocytogenes was measured by the fluorescent signals of the assembled indicator probes. Under the optimal conditions, the calibration graph of fluorescent intensity is proportional to the amount of target bacteria over the range of 50-10,320 CFU/mL with a detection limit of 50 CFU/mL. The proposed method has been successfully applied to detect L. monocytogenes in food samples offering the advantages of sensitivity, simplicity, and stability.     

Toxigenic Vibrio cholerae in the Aquatic Environment of Mathbaria, Bangladesh

Toxigenic Vibrio cholerae, rarely isolated from the aquatic environment between cholera epidemics, can be detected in what is now understood to be a dormant stage, i.e., viable but nonculturable when standard bacteriological methods are used. In the research reported here, biofilms have proved to be a source of culturable V. cholerae, even in nonepidemic periods. Biweekly environmental surveillance for V. cholerae was carried out in Mathbaria, an area of cholera endemicity adjacent to the Bay of Bengal, with the focus on V. cholerae O1 and O139 Bengal. A total of 297 samples of water, phytoplankton, and zooplankton were collected between March and December 2004, yielding eight V. cholerae O1 and four O139 Bengal isolates. A combination of culture methods, multiplex-PCR, and direct fluorescent antibody (DFA) counting revealed the Mathbaria aquatic environment to be a reservoir for V. cholerae O1 and O139 Bengal. DFA results showed significant clumping of the bacteria during the interepidemic period for cholera, and the fluorescent micrographs revealed large numbers of V. cholerae O1 in thin films of exopolysaccharides (biofilm). A similar clumping of V. cholerae O1 was also observed in samples collected from Matlab, Bangladesh, where cholera also is endemic. Thus, the results of the study provided in situ evidence for V. cholerae O1 and O139 in the aquatic environment, predominantly as viable but nonculturable cells and culturable cells in biofilm consortia. The biofilm community is concluded to be an additional reservoir of cholera bacteria in the aquatic environment between seasonal epidemics of cholera in Bangladesh.     

Susceptibility to Vibrio cholerae infection in a cohort of household contacts of patients with cholera in Bangladesh

Background

Despite recent progress in understanding the molecular basis of Vibrio cholerae pathogenesis, there is relatively little knowledge of the factors that determine the variability in human susceptibility to V. cholerae infection.

Methods and Findings

We performed an observational study of a cohort of household contacts of cholera patients in Bangladesh, and compared the baseline characteristics of household members who went on to develop culture-positive V. cholerae infection with individuals who did not develop infection. Although the vibriocidal antibody is the only previously described immunologic marker associated with protection from V. cholerae infection, we found that levels of serum IgA specific to three V. cholerae antigens—the B subunit of cholera toxin, lipopolysaccharide, and TcpA, the major component of the toxin–co-regulated pilus—also predicted protection in household contacts of patients infected with V. cholerae O1, the current predominant cause of cholera. Circulating IgA antibodies to TcpA were also associated with protection from V. cholerae O139 infection. In contrast, there was no association between serum IgG antibodies specific to these three antigens and protection from infection with either serogroup. We also found evidence that host genetic characteristics and serum retinol levels modify susceptibility to V. cholerae infection.

Conclusions

Our observation that levels of serum IgA (but not serum IgG) directed at certain V. cholerae antigens are associated with protection from infection underscores the need to better understand anti–V. cholerae immunity at the mucosal surface. Furthermore, our data suggest that susceptibility to V. cholerae infection is determined by a combination of immunologic, nutritional, and genetic characteristics; additional factors that influence susceptibility to cholera remain unidentified.     

Fluorescent antibody staining method for enumeration of viable environmental Vibrio cholerae 01

A membrane filtration method has been developed which is useful for enumeration of viable Vibriocholerae 01 in environmental water samples by immunofluorescent staining. The samples are incubated with yeast extract and nalidixic acid. Substrate responsive cells, i.e., viable cells, elongate and after staining with specific antiserum and fluorescein conjugate, viable V. cholera cells appear as long, peripheral fluorescent green banded bacilli when viewed under the microscope. Using an ocular reticule, the number of viable cells per ml can be calculated. The procedure has been adapted for use with other bacterial species if specific antisera are employed.     

Detection of Vibrio parahaemolyticus in Penaeus chinensis using an indirect fluorescent antibody staining procedure

An indirect fluorescent antibody technique (iFAT) incorporating fluorescein-isothiocyanate conjugated anti-rabbit globulin goat serum, and rhodamine-isothiocyanate conjugated bovine serum albumin as background stain has been developed for the detection of Vibrio parahaemolyticus.     

Distribution ofVibrio cholerae in two Florida estuaries

The distribution ofVibrio cholerae was examined in 2 Florida estuaries, Apalachicola and Tampa Bay.Vibrio cholerae serotype non-01 was the most abundant serotype, being isolated from 45% of the oyster samples, 30% of the sediments, 50% of the waters, and 75% of the blue crabs.Vibrio cholerae serotype 01 was isolated from only one oyster sample. Strong linear correlations betweenV. cholerae and temperature, salinity, or the other physical/chemical parameters measured,Escherichia coli, or fecal coliforms were not observed, but a range of temperatures and salinities appeared relevant to the distribution of the organism. The organism was present in the highest concentrations when salinities were 10‰–25‰ and temperatures were 20?C–35?C.In vitro growth curves of 95V. cholerae environmental isolates further supported that 10‰–25‰ was an ideal salinity range for the organisms. The results suggest thatV. cholerae is a widely distributed organism in the nutrient-rich warm waters of the Gulf Coast estuaries.     

Genetic Differences in the Immunocyte Response of Mice to Separate Determinants on One Bacterial Antigen

WE wish to report studies on the relationship between the antibody responses in different strains of mice, using a newly developed immunoplaque assay. Our method permits direct enumeration of antibody plaque forming cells (p.f.c.) to distinct somatic antigens of Vibrio cholerae bacilli, rather than mere demonstration of serum antibody titres. We found that the immune response of mice to two different antigens present on cholera organisms does not seem to depend on the presence of any particular allele of the H ₂ locus.     

Identification of Vibrio cholerae serotypes in high-risk marine products with non-gel sieving capillary electrophoresis

Vibrio cholerae, a natural inhabitant of the marine environment, poses a threat to human health, and its new epidemic variants have been reported. A method of multiplex polymerase chain reaction–capillary electrophoresis–laser-induced fluorescence (PCR–CE–LIF) detection has been developed to detect and identify V. cholerae in marine products sensitively, rapidly, and reliably. Four sets of primers were selected to amplify genus-specific VCC gene, O139 serogroup-specific O139 gene, O1 serogroup-specific O1 gene, and ctxA gene associated with the CT toxin of enterotoxigenic V. cholerae. The PCR products were detected using CE–LIF with SYBR Gold serving as the DNA fluorescent dye. The parameters of PCR and the separation conditions of CE–LIF were optimized. Under the optimal conditions, V. cholerae was detected and four serotypes were identified simultaneously within 8 min. The alignment analysis showed that the PCR products had good agreement with the published sequences from GenBank, indicating that the primers selected in this study had high specificity and the PCR results were reliable. The proposed method could detect 5 to 20 cfu/ml V. cholerae. The intraday precisions of migration time and peak area of DNA marker and PCR products were in the ranges of 1.60–2.56% and 1.60–6.29%, respectively. The specificity results showed that only five standard bacteria used in this study showed the specific peaks when the target bacteria were mixed with seven other common intestinal pathogenic bacteria at the same concentration. The assay was applied to 71 high-risk marine products, and different serotypes of V. cholerae could be identified sensitively and reliably.     

gbpA as a Novel qPCR Target for the Species-Specific Detection of Vibrio cholerae O1, O139, Non-O1/Non-O139 in Environmental,Stool, and Historical Continuous Plankton Recorder Samples

The Vibrio cholerae N-acetyl glucosamine-binding protein A (GbpA) is a chitin-binding protein involved in V. cholerae attachment to environmental chitin surfaces and human intestinal cells. We previously investigated the distribution and genetic variations of gbpA in a large collection of V. cholerae strains and found that the gene is consistently present and highly conserved in this species. Primers and probe were designed from the gbpA sequence of V. cholerae and a new Taq-based qPCR protocol was developed for diagnostic detection and quantification of the bacterium in environmental and stool samples. In addition, the positions of primers targeting the gbpA gene region were selected to obtain a short amplified fragment of 206 bp and the protocol was optimized for the analysis of formalin-fixed samples, such as historical Continuous Plankton Recorder (CPR) samples. Overall, the method is sensitive (50 gene copies), highly specific for V. cholerae and failed to amplify strains of the closely-related species Vibrio mimicus. The sensitivity of the assay applied to environmental and stool samples spiked with V. cholerae ATCC 39315 was comparable to that of pure cultures and was of 10² genomic units/l for drinking and seawater samples, 10¹ genomic units/g for sediment and 10² genomic units/g for bivalve and stool samples. The method also performs well when tested on artificially formalin-fixed and degraded genomic samples and was able to amplify V. cholerae DNA in historical CPR samples, the earliest of which date back to August 1966. The detection of V. cholerae in CPR samples collected in cholera endemic areas such as the Benguela Current Large Marine Ecosystem (BCLME) is of particular significance and represents a proof of concept for the possible use of the CPR technology and the developed qPCR assay in cholera studies.     

ERYTHROPOIESIS DURING AMPHIBIAN METAMORPHOSIS : III. Immunochemical Detection of Tadpole and Frog Hemoglobins (Rana catesbeiana) in Single Erythrocytes

Rabbit antibodies specific for the major tadpole and frog hemoglobin components of R. catesbeiana were used for the detection of the two hemoglobins inside single cells. The antisera, after fractionation by ammonium sulfate precipitation and diethylaminoethyl (DEAE)-cellulose chromatography, were conjugated with fluorescein isothiocyanate for the antifrog hemoglobin serum and tetramethylrhodamine isothiocyanate for the antitadpole hemoglobin serum. The conjugated fractions, refractionated by stepwise elution from a DEAE-cellulose column, were used for the fluorescent staining of blood smears, liver tissue imprints, and smears of liver cell suspensions. Both simultaneous and sequential staining with the two fluorescent preparations indicated that larval and adult hemoglobins were not present within the same erythrocyte during metamorphosis. In other experiments, erythroid cells from animals in metamorphosis were spread on agar containing specific antiserum. Precipitates were formed around the cells which contain the particular hemoglobin. The percentages of cells containing either tadpole or frog hemoglobin were estimated within the experimental error of the method. The data showed that the two hemoglobins are in different cells. It is concluded that the hemoglobin change observed during the metamorphosis of R. catesbeiana is due to the appearance of a new population of erythroid cells containing exclusively frog hemoglobin.     

Crystal structure of the Vibrio cholerae colonization factor TcpF and identification of a functional immunogenic site

Vibrio cholerae relies on two main virulence factors—toxin-coregulated pilus (TCP) and cholera toxin—to cause the gastrointestinal disease cholera. TCP is a type IV pilus that mediates bacterial autoagglutination and colonization of the intestine. TCP is encoded by the tcp operon, which also encodes TcpF, a protein of unknown function that is secreted by V. cholerae in a TCP-dependent manner. Although TcpF is not required for TCP biogenesis, a tcpF mutant has a colonization defect in the infant mouse cholera model that is as severe as a pilus mutant. Furthermore, TcpF antisera protect against V. cholerae infection. TcpF has no apparent sequence homology to any known protein. Here, we report the de novo X-ray crystal structure of TcpF and the identification of an epitope that is critical for its function as a colonization factor. A monoclonal antibody recognizing this epitope is protective against V. cholerae challenge and adds to the protection provided by an anti-TcpA antibody. These data suggest that TcpF has a novel function in V. cholerae colonization and define a region crucial for this function.     

Detection of Vibrio cholerae by Real-Time Nucleic Acid Sequence-Based Amplification

A multitarget molecular beacon-based real-time nucleic acid sequence-based amplification (NASBA) assay for the specific detection of Vibrio cholerae has been developed. The genes encoding the cholera toxin (ctxA), the toxin-coregulated pilus (tcpA; colonization factor), the ctxA toxin regulator (toxR), hemolysin (hlyA), and the 60-kDa chaperonin product (groEL) were selected as target sequences for detection. The beacons for the five different genetic targets were evaluated by serial dilution of RNA from V. cholerae cells. RNase treatment of the nucleic acids eliminated all NASBA, whereas DNase treatment had no effect, showing that RNA and not DNA was amplified. The specificity of the assay was investigated by testing several isolates of V. cholerae, other Vibrio species, and Bacillus cereus, Salmonella enterica, and Escherichia coli strains. The toxR, groEL, and hlyA beacons identified all V. cholerae isolates, whereas the ctxA and tcpA beacons identified the O1 toxigenic clinical isolates. The NASBA assay detected V. cholerae at 50 CFU/ml by using the general marker groEL and tcpA that specifically indicates toxigenic strains. A correlation between cell viability and NASBA was demonstrated for the ctxA, toxR, and hlyA targets. RNA isolated from different environmental water samples spiked with V. cholerae was specifically detected by NASBA. These results indicate that NASBA can be used in the rapid detection of V. cholerae from various environmental water samples. This method has a strong potential for detecting toxigenic strains by using the tcpA and ctxA markers. The entire assay including RNA extraction and NASBA was completed within 3 h.     

Antagonistic Interactions among Marine Bacteria Impede the Proliferation of Vibrio cholerae

Changes in global climate have raised concerns about the emergence and resurgence of infectious diseases. Vibrio cholerae is a reemerging pathogen that proliferates and is transported on marine particles. Patterns of cholera outbreaks correlate with sea surface temperature increases, but the underlying mechanisms for rapid proliferation of V. cholerae during ocean warming events have yet to be fully elucidated. In this study, we tested the hypothesis that autochthonous marine bacteria impede the spread of V. cholerae in the marine environment. It was found that some marine bacteria are capable of inhibiting the growth of V. cholerae on surfaces and that bacterial isolates derived from pelagic particles show a greater frequency of V. cholerae inhibition than free-living bacteria. Vibrio cholerae was less susceptible to antagonism at higher temperatures, such as those measured during El Niño-Southern Oscilliation and monsoonal events. Using a model system employing green fluorescent protein-labeled bacteria, we found that marine bacteria can directly inhibit V. cholerae colonization of particles. The mechanism of inhibition in our model system was linked to the biosynthesis of andrimid, an antibacterial agent. Antibiotic production by the model antagonistic strain decreased at higher temperatures, thereby explaining the increased competitiveness of V. cholerae under warmer conditions. These findings suggest that bacterium-bacterium antagonism is a contributing mechanism in regulating the proliferation of V. cholerae on marine particles.     

Effect of Transport at Ambient Temperature on Detection and Isolation of Vibrio cholerae from Environmental Samples

It has long been assumed that prolonged holding of environmental samples at the ambient air temperature prior to bacteriological analysis is detrimental to isolation and detection of Vibrio cholerae, the causative agent of pandemic cholera. The present study was aimed at understanding the effect of transporting environmental samples at the ambient air temperature on isolation and enumeration of V. cholerae. For water and plankton samples held at ambient temperatures ranging from 31°C to 35°C for 20 h, the total counts did not increase significantly but the number of culturable V. cholerae increased significantly compared to samples processed within 1 h of collection, as measured by culture, acridine orange direct count, direct fluorescent-antibody-direct viable count (DFA-DVC), and multiplex PCR analyses. For total coliform counts, total bacterial counts, and DFA-DVC counts, the numbers did not increase significantly, but the culturable plate counts for V. cholerae increased significantly after samples were held at the ambient temperature during transport to the laboratory for analysis. An increase in the recovery of V. cholerae O1 and improved detection of V. cholerae O1 rfb and ctxA also occurred when samples were enriched after they were kept for 20 h at the ambient temperature during transport. Improved detection and isolation of toxigenic V. cholerae from freshwater ecosystems can be achieved by holding samples at the ambient temperature, an observation that has significant implications for tracking this pathogen in diverse aquatic environments.     

Rapid detection by multiplex PCR of Genomic Islands, prophages and Integrative Conjugative Elements in V. cholerae 7th pandemic variants

Vibrio cholerae poses a threat to human health, and new epidemic variants have been reported so far. Seventh pandemic V. cholerae strains are characterized by highly related genomic sequences but can be discriminated by a large set of Genomic Islands, phages and Integrative Conjugative Elements. Classical serotyping and biotyping methods do not easily discriminate among new variants arising worldwide, therefore the establishment of new methods for their identification is required. We developed a multiplex PCR assay for the rapid detection of the major 7th pandemic variants of V. cholerae O1 and O139. Three specific genomic islands (GI-12, GI-14 and GI-15), two phages (Kappa and TLC), Vibrio Seventh Pandemic Island 2 (VSP-II), and the ICEs of the SXT/R391 family were selected as targets of our multiplex PCR based on a comparative genomic approach. The optimization and specificity of the multiplex PCR was assessed on 5 V. cholerae 7th pandemic reference strains, and other 34 V. cholerae strains from various epidemic events were analyzed to validate the reliability of our method. This assay had sufficient specificity to identify twelve different V. cholerae genetic profiles, and therefore has the potential to be used as a rapid screening method.     

Immunological Biosensor for Detection of Vibrio cholerae O1in Environmental Water Samples

Environmental surveillance for the presence of Vibrio cholerae O1 is of utmost importance for the effective public health protection of cholera. In the present study, an amperometric immunosensor was developed for detection of Vibrio cholerae in environmental samples by using disposable screen-printed electrodes (SPEs). For this purpose, the experiments done include fabrication of SPEs by using carbon ink, electrochemical characterization of electrodes, optimization of dilutions of antibodies and immobilization of antibody. V. cholerae O1 bacteria were spiked in various environmental water samples in known number. The seeded samples were filtered through a 0.22 μm membrane, and the filters enriched in alkaline peptone water for 6 h and then used directly for detection of V. cholerae using the immunosensor. The immunosensor could detect as few as 8 c.f.u./ml in hand-pump water (ground water) and seawater, and 80 c.f.u./ml in sewer water and tap water. The total time taken in this detection assay was 55 min. Thus, the proposed method is simple and can be used for environmental monitoring of V.␣cholerae.     

Influence of climate factors on Vibrio cholerae dynamics in the Pearl River estuary,South China

Current research has seldom focused on the quantitative relationships between Vibrio cholerae (V. cholerae) and climate factors owing to the complexities and high cost of field observation in the aquatic environment. This study has focused on the relationships between V. cholerae and climate factors based on linear regression method and data partition method. Data gathered from 2008 to 2009 in the Pearl River estuary, South China, were adopted. Positive rate of V. cholerae was correlated closely with monthly climate factors of water temperature and air temperature, respectively in 2009. Quarterly data analysis from 2008 to 2009 showed that there existed seasonal characteristic for V. cholerae. Positive rate of V. cholerae was correlated positively with quarterly climate factors of land surface temperature, pH, water temperature, air temperature and rainfall, respectively and negatively with quarterly air pressure. Partition data analysis in 2009 showed that there existed geography region characteristic for V. cholerae. V. cholerae dynamics was closely correlated to climate factors in the downstream area. However, it was more greatly affected by human geography factors in the urban area. Positive annual rate of V. cholerae was higher in the downstream area than in the urban area both in 2008 and 2009. At last, a cellular automaton model was used to simulate V. cholerae diffusion downstream, and the distribution of V. cholerae obtained from this model was similar to that obtained from the field observations.     

Differentiation among the Vibrio cholerae serotypes O1, O139, O141 and non-O1, non-O139, non-O141 using specific monoclonal antibodies with dot blotting

Seven different monoclonal antibodies (MAbs) specific to only Vibrio cholerae were produced using a combination of five representative serotypes of V. cholerae for immunization. The first three MAbs (VC-93, VC-82 and VC-223) were specific to the V. cholerae serogroup O1 with different avidity for the serotypes O1 Inaba and O1 Ogawa. The fourth and the fifth MAbs were specific to V. cholerae O139 (VC-812) or O141 (VC-191) serogroups, respectively. The sixth MAb (VC-26) bound to all three serogroups of V. cholerae. The seventh MAb (VC-63) bound to all twenty five isolates of V. cholerae used in this study. None of the seven MAbs showed cross-reactivity with other Vibrio spp. or closely-related V. cholerae species, V. mimicus or other gram-negative bacteria. The eighth MAbs (VC-201) specific to almost all Vibrio spp. was also obtained. In dot blotting, these MAbs can be used to detect a diluted pure culture of V. cholerae in solution with a sensitivity range of from 10⁵ to 10⁷ CFU ml^− 1. However, the detection capability could be improved equivalent to that of PCR technique after preincubation of samples in alkaline peptone water (APW). Thus, these MAbs constitute convenient immunological tools that can be used for simple, rapid and simultaneous direct detection and differentiation of the individual serotypes of V. cholerae in complex samples, such as food and infected animals, without the requirement for bacterial isolation or biochemical characterization.     

Gold-conjugated Reagents for the Labeling of Carbohydrate-Recognition Domains and Glycoconjugates on Nematode Surfaces

Various fluorescent conjugated lectins have been used for the detection of glycoconjugates on nematode surfaces under light microscopy. Several problems have been experienced with these reagents including penetration of the cuticle by fluorescent lectins, non-glycoconjugate specificity, strong nematode autofluorescence at the emission wavelength of the fluorescent dye, and prevention of persistent visualization due to rapid quenching of the fluorescent components. Gold-conjugated reagents combined with silver enhancement alleviated these difficulties when working with three phytonematode species (Heterodera avenae, H. latipons, and Meloidogyne javanica) and two entomopathogenic species (Steinernema carpocapsae and S. glaseri) under light-microscopy visualization of binding by fluorescent lectins and neoglycoproteins. Moreover, gold-conjugated reagents resulted in stable bindings that enabled long-term observations.     

Characterization of non-membrane-damaging cytotoxin of non-toxigenic Vibrio cholerae O1 and its relevance to disease

The non-membrane-damaging cytotoxin which causes dramatic cell rounding of cultured HeLa cells was purified to homogeneity from a clinical strain (WO5) of non-toxigenic Vibrio cholerae O1 Inaba belonging to the El Tor biotype. The purified protein has a denatured molecular weight of 35 kDa and a native molecular weight of approximately 37 kDa indicating the monomeric nature of the protein. The 15 N-terminal amino acid sequence of non-membrane-damaging cytotoxin showed complete homology to the hemagglutinin protease previously purified and characterized from V. cholerae O1. Purified non-membrane-damaging cytotoxin from V. cholerae O1 was immunologically and biochemically identical to that previously purified from V. cholerae O26. Non-membrane-damaging cytotoxin was found to be enterotoxic in rabbit ileal loop assay inducing accumulation of non-hemorrhagic fluid at 100 μg and elicited a concentration dependent increase in short circuit current and tissue conductance of rabbit ileal mucosa mounted on Ussing chambers. A significant serum immunoglobulin G response against non-membrane-damaging cytotoxin was elicited by patients infected with V. cholerae O139 but not with V. cholerae O1. These properties make non-membrane-damaging cytotoxin a potential virulence factor of V. cholerae which should be taken into consideration while making live, attenuated recombinant vaccine strains against cholera.