Role of surface proteins in the deposition kinetics of Cryptosporidium parvum oocysts

A radial stagnation point flow system was used to investigate the influence of Cryptosporidium parvum surface properties on oocyst deposition kinetics onto solid surfaces. To determine the role of oocyst surface proteins in adhesion, the deposition kinetics of viable oocysts were compared with the deposition kinetics of oocysts treated (inactivated) with either heat or formalin. Results showed a significantly higher deposition rate with formalin and heat-treated oocysts compared to viable oocysts under identical solution ionic strengths. Low deposition rates and corresponding attachment efficiencies were observed with viable oocysts over the entire range of solution conditions investigated, even at high ionic strengths where DLVO theory predicts the absence of an electrostatic energy barrier. An "electrosteric" repulsion between the viable Cryptosporidium oocyst and the quartz substrate, attributed to proteins on the oocyst surface, is surmised to cause this low deposition rate. Inactivation of the oocysts with either formalin or heat resulted in increased attachment efficiencies over the entire range of ionic strengths examined. It is hypothesized that formalin and heat treatments alter the structure of surface proteins and thus reduce steric repulsion. Formalin treatment was also found to impart an increased hydrophobicity to the oocyst surface and thus greater enhancement in oocyst deposition kinetics compared to heat treatment.     

Microelectrophoresis of Cryptosporidium parvum oocysts in aqueous solutions of inorganic and surfactant cations

Cryptosporidium parvum is a protozoan parasite associated with waterborne outbreaks of diarrhoeal disease. The life cycle of this parasite includes the production of a spheroidal oocyst that is of 4–6 microns in diameter. The thickness of the oocyst wall and its capacity to strongly adhere to both organic and inorganic surfaces are features of the oocysts which could be attributed to its survival in the environment for extended periods. Hence, the need to study their surface chemistry in the aqueous environment. The surface charging properties of the intact C. parvum oocysts were derived from microelectrophoresis measurements on these robust biological species. The ζ potentials of Cryptosporidium oocysts were measured in a range of inorganic electrolyte solutions and in solutions of a multivalent cationic surfactant. The surface potential of the oocyst was found to be pH dependent, with an isoelectric point in mM NaCl of ∼2, suggesting the presence of surface carboxylate groups associated with glycoproteins or phosphate groups. The area/charge for the fully ionised oocysts was found to be ∼80 nm², corresponding to a total maximum charge of 1.6×10⁻¹³ C per oocyst. The effect of a highly charged novel cage surfactant known as CS12 on the Cryptosporidium oocyst surface potential provided valuable insight into its uptake and possible surface activity. Uptake of CS12 was detected at concentrations as low as 2×10⁻⁸ M. At ∼2×10⁻⁵ M CS12 the oocyst surface was uncharged and became positively charged at higher concentrations. These findings suggest that there could be improvements to current concentration methods by manipulation of the surface charge.     

Disinfection of drinking water contaminated with Cryptosporidium parvum oocysts under natural sunlight and using the photocatalyst TiO2

The results of a batch-process solar disinfection (SODIS) and solar photocatalytic disinfection (SPCDIS) on drinking water contaminated with Cryptosporidium are reported. Cryptosporidium parvum oocyst suspensions were exposed to natural sunlight in Southern Spain and the oocyst viability was evaluated using two vital dyes [4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI)]. SODIS exposures (strong sunlight) of 8 and 12h reduced oocyst viability from 98% (+/-1.3%) to 11.7% (+/-0.9%) and 0.3% (+/-0.33%), respectively. SODIS reactors fitted with flexible plastic inserts coated with TiO2 powder (SPCDIS) were found to be more effective than those which were not. After 8 and 16 h of overcast and cloudy solar irradiance conditions, SPCDIS reduced oocyst viability from 98.3% (+/-0.3%) to 37.7% (+/-2.6%) and 11.7% (+/-0.7%), respectively, versus to that achieved using SODIS of 81.3% (+/-1.6%) and 36.0% (+/-1.0%), respectively. These results confirm that solar disinfection of drinking water can be an effective household intervention against Cryptosporidium contamination.     

Human pathogenic Cryptosporidium species bioanalytical detection method with single oocyst detection capability

A bioanalytical detection method for specific detection of viable human pathogenic Cryptosporidium species, C. parvum, C. hominis, and C. meleagridis is described. Oocysts were isolated from water samples via immunomagnetic separation, and mRNA was extracted with oligo-dT magnetic beads, amplified using nucleic acid sequence-based amplification (NASBA), and then detected in a nucleic acid hybridization lateral flow assay. The amplified target sequence employed was hsp70 mRNA, production of which is stimulated via a brief heat shock. The described method was capable of detecting one oocyst in 10 μL using flow-cytometer-counted samples. Only viable oocysts were detected, as confirmed using 4′,6-diamidino-2-phenylindole and propidium iodide (DAPI/PI) staining. The detection system was challenged by detecting oocysts in the presence of large numbers of common waterborne microorganisms and packed pellet material filtered from environmental water samples. When the method was compared with EPA Method 1622 for C. parvum detection, highly comparable results were obtained. Since the described detection system yields unambiguous results within 4.5 h, it is an ideal method for monitoring the safety of drinking water.     

Generation of a polyclonal Fab phage display library to the protozoan parasite Cryptosporidium parvum

We had developed a technology for creation of recombinant polyclonal antibody libraries, standardized perpetual mixtures of polyclonal whole antibodies for which the genes are available and can be altered as desired. We report here the first phase of generating a polyclonal antibody library to Cryptosporidium parvum, a protozoan parasite that causes severe disease in AIDS patients, for which there is no effective treatment. BALB/c mice, immunized by neonatal oral infection with oocysts followed by intraperitoneal immunization with a sporozoite/oocyst preparation of C. parvum, were used for construction of a Fab phage display library in a specially-designed bidirectional vector. This library was selected for reactivity to an oocyst/sporozoite preparation, and was shown to be antigen-specific and diverse. Following mass transfer of the selected variable region gene pairs to appropriate mammalian expression vectors, such anti-C. parvum Fab phage display libraries could be used to develop chimeric polyclonal antibody libraries, with mouse variable regions and human constant regions, for passive immunotherapy of C. parvum infection.     

Improved method for plasma ADMA, SDMA, and arginine quantification by field-amplified sample injection capillary electrophoresis UV detection

Here, we describe an easy field-amplified sample injection capillary electrophoresis method with UV detection for the separation and detection of free plasma arginine and dimethylated arginines. The analytes were baseline-separated within 22?min by using 50?mmol/L Tris phosphate pH 2.3 as running buffer. The plasma samples were treated with acetonitrile/ammonia for protein elimination, the supernatants were dried, re-swollen in water and directly injected in the capillary without complex cleanup by solid phase extraction and/or tedious sample derivatization procedures. Due to the stacking effects of the electrokinetic injection, it was possible to operate a consistent on-line pre-concentration of the analytes before running the electrophoresis. This procedure allowed to reach a detection limit in the real sample of 10?nmol/L for dimethylated arginines and 20?nmol/L for arginine, thus improving about threefold our previous method, that required a more complicated pre-analytical procedure to concentrate samples. The recovery of plasma ADMA was 99-104% and inter-day CV was less than 3%. The assay performance was evaluated measuring the levels of arginine and its dimethyl derivatives in 50 subjects. The statistical tests for the methods comparison suggest that the data obtained by our new method and by our previous CE assay are similar.     

Validation of a high-performance thin-layer chromatographic method for trace analysis for some generic drugs affecting gastrointestinal function

To prevent cross-contamination between pharmaceutical products manufactured with the same equipment, cleanup procedures must be introduced before the manufacture of a new product begins. From an analytical point of view, it is crucial to select and validate a suitable analytical method to determine contaminants in the rinse water, swabs, and the placebo of the next product. High performance thin-layer chromatography (HPTLC) was chosen in our laboratory for this purpose and was optimized to meet the requirements of trace determination. The method was validated in terms of the limit of detection, limit of quantitation (LOQ), linearity close to the LOQ, sample preparation from the swab media and from the placebo of the next product made with the same equipment (recovery), precision, selectivity (interference from the swab and placebo matrixes), resolution (from related compounds), and robustness. The HPTLC method was applied to 2 different generic drugs affecting gastrointestinal function--the water-soluble H2-receptor antagonist ranitidine hydrochloride (RHCl) and the water-insoluble choleretic drug ursodeoxycholic acid (UDCA). Chromatography was performed on silica plates by using toluene-methanol-diethylamine (9 + 1 + 1, v/v/v) and n-heptane-ethyl acetate-glacial acetic acid (5 + 5 + 1, v/v/v) as the mobile phases for RHCl and UDCA, respectively. RHCl was measured in situ at 320 nm, whereas the detection of UDCA was performed at 502 nm after postchromatographic derivatization. The method was used for the determination of RHCl and UDCA in the swabs, the final rinse water, and the placebo batch after the cleanup process.     

Amphiphile disruption of pathogen attachment at the hematite (α-Fe2O3)-water interface

Prior studies have indicated that the subsurface transport of Cryptosporidium parvum oocysts is diminished in sediments containing iron oxides and that inner-sphere complexation of oocyst surficial carboxylate plays a role in the retardation. However, the impacts of natural organic matter (NOM) remain poorly understood. In this study, we used a model anionic surfactant, sodium dodecyl sulfate (SDS), as a surrogate for amphiphilic NOM components to examine the impacts of amphiphilic components on oocyst adhesion mechanisms. We employed in situ attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy to determine the effects of SDS on the molecular bonds that mediate interactions between oocyst surficial biomolecules and hematite (α-Fe(2)O(3)) surface functional groups over a wide range of solution pH. The results show that the presence of SDS significantly diminishes Fe-carboxylate complexation, as indicated by progressive decreases in intensity of asymmetric and symmetric stretching vibrations of carboxylate [ν(as)(COO(-)) and ν(s)(COO(-))] with reaction time. In addition, one of the ν(s)(COO(-)) bands shifted from 1370 to 1418 cm(-1) upon SDS introduction, suggesting that SDS also changed the complexation mode. The data indicate that competition from the sulfonate groups (OSO(3)(-)) of SDS at α-Fe(2)O(3) surface sites is a primary mechanism resulting in decreased Fe-carboxylate complexation. Sorptive competition from amphiphilic NOM components may therefore increase the mobility of C. parvum oocysts in the environment through disruption of interfacial pathogen-mineral surface bonds.     

A cleanup method of serum extracts with molecular sieves as SPE sorbents for the analysis of polybrominated diphenyl ethers

Based on the size- and shape-selective sorption, 13X molecular sieves were developed as solid-phase extraction adsorbents to cleanup serum extract for the determination of polybrominated diphenyl ethers. The important parameters affecting the cleanup efficiency were investigated including the amount of sorbents, the type, and volume of solvents. Under the optimized conditions, the capacity for removing impurities was evaluated via gel permeation chromatography and gas chromatography with mass spectrometry. The results demonstrated that up to 99% of lipids in corn oil (13 mg) can be removed after cleanup, and endogenous compounds in serum can also be effectively eliminated. The cleanup efficiency is not only superior to hydrophile-lipophile balance column, but also close to acid silica gel and multifunction impurity sorbents. Generally, the developed cleanup method exhibited higher recovery for polybrominated diphenyl ethers with more than four bromines, especially for nona- and deca-brominated diphenyl ethers (99.1˗117.8%). The cleanup method can be coupled with gas chromatography and tandem mass spectrometry for polybrominated diphenyl ethers analysis in human serum. The method detection limits were 0.01˗0.27 ng/mL and average recovery was 50.9˗113.3%, except 2,3',4',6-tetrabrominated, 2,3',4,4',6-pentabrominated, and 2,3,3',4,4',5',6-heptabrominated diphenyl ethers. 2,2',4,5'-Tetrabrominated diphenyl ethers had the highest detection frequency (95%) in human serum, whereas decabrominated diphenyl ethers had the maximum mean concentration (0.50 ng/mL).     

Analysis of selected pharmaceuticals in fish and the fresh water bodies directly affected by reclaimed water using liquid chromatography-tandem mass spectrometry

A comprehensive method for the analysis of 11 target pharmaceuticals representing multiple commonly used therapeutic classes was developed for biological tissues (fish), reclaimed water, and the surface water directly affected by irrigation with reclaimed water. One gram of fish tissue homogenate was extracted by accelerated solvent extraction with methylene chloride followed by mixed-mode cation exchange solid phase extraction (SPE) cleanup and analyzed by liquid chromatography-tandem mass spectrometry. Compared to previously reported methods, the protocol produces cleaner extracts resulting in lower method detection limits. Similarly, an SPE method based on Oasis HLB cartridges was used to concentrate and cleanup reclaimed and surface water samples. Among the 11 target compounds analyzed, trimethoprim, caffeine, sulfamethoxazole, diphenhydramine, diltiazem, carbamazepine, erythromycin, and fluoxetine were consistently detected in reclaimed water. Caffeine, diphenhydramine, and carbamazepine were consistently detected in fish and surface water samples. Bioaccumulation factors for caffeine, diphenhydramine, and carbamazepine in mosquito fish (Gambusia holbrooki) were calculated at 29?±?26, 821?±?422, and 108?±?144, respectively. This is the first report of potential accumulation of caffeine in fish from a water body directly influenced by reclaimed water.

Determination of pesticide residues in lettuce by gas chromatography with electron-capture detection

A sensitive, rapid, and simple multiresidue method for the simultaneous determination of 19 pesticides in different varieties of lettuce (Lactuca sativum) was developed. Lettuce samples were extracted by homogenization with acetone and partitioned into ethyl acetate-cyclohexane. Subsequent sample cleanup was not needed. Final determination was made by capillary gas chromatography (GC) with electron-capture detection (ECD). Confirmation analysis of pesticides was performed by GC coupled with mass spectrometry in the selected ion monitoring mode. The average recovery by the GC-ECD method obtained for these compounds varied from 66.4 to 119.2% with relative standard deviations < 7.7%. The GC-ECD method has good linearity, and the detection limit for the pesticides studied varied from 0.1 to 3.8 microg/kg. The proposed method was used to determine pesticide levels in different types of lettuce grown in soils from experimental fields.     

Investigation of the interaction force between Cryptosporidium parvum oocysts and solid surfaces

Interaction force profiles between single Cryptosporidium parvum oocysts and positively charged, silane-coated silica particles were measured in aqueous solutions using an atomic force microscope. The oocysts were immobilized for the measurements by entrapment in Millipore polycarbonate membranes with 3 microm pore size. Experiments were performed in both NaCl and CaCl2 solutions at ionic strengths ranging from 1 to 100 mM. For both electrolytes, the decay length of the repulsive force profile was found to be nearly independent of the ionic strength and always much larger than the theoretical Debye length of the system. In addition, the magnitude of the force was found to be essentially the same for both electrolytes, suggesting that the long-range repulsive forces are primarily steric in nature. These results support the theory that the interaction force between oocysts and surfaces is controlled by an outer, weakly charged or uncharged carbohydrate layer. Measurements were also performed with oocysts that had been deactivated using either chemical (formalin) or heat treatment. The force profiles obtained with formalin-treated oocysts appear to be essentially the same as for the untreated oocysts, whereas the profiles measured with the heat-treated oocysts show a much stronger dependence on solution ionic strength. With either the heat-treated or formalin-treated oocysts, adhesion was observed much more frequently than with untreated oocysts, which is consistent with the increased deposition rate observed with treated oocysts by Kuznar and Elimelech (Kuznar, Z. A.; Elimelech, M. Langmuir 2005, 21, 710-716). These results also suggest that treated oocysts, especially ones that have been inactivated by heating, may not be good surrogates for viable oocysts in laboratory studies.     

Development and validation of a rapid method for microcystins in fish and comparing LC-MS/MS results with ELISA

Microcystins (MCs) are the most common cyanotoxins found worldwide in freshwater, brackish, and marine environments. The rapid and accurate analysis of MCs and nodularin (Nod-R) in fish tissue is important for determining occurrence, following trends, and monitoring exposure for risk assessment and other purposes. The aim of this study was to develop a streamlined and reliable sample preparation method for eight MCs (MC-RR, MC-YR, MC-LR, MC-WR, MC-LA, MC-LY, MC-LW, and MC-LF) and Nod-R in fish, and conduct a validation of the new method using liquid chromatography–tandem mass spectrometry (LC-MS/MS) for analysis and compare the results with a commercial enzyme-linked immunosorbent assay (ELISA) kit. Different sample preparation methods were compared, and a simple extraction protocol with acidified acetonitrile/water (3:1) followed by hexane partitioning cleanup was found to be most effective. Thorough validation of the final method was conducted, and 90–115% recoveries were achieved for all analytes except for MC-RR, which gave 130% average recovery (isotopically labeled internal standards were unavailable to correct for possible biases). The use of electrospray ionization in the negative mode gave few interferences and minimal matrix effects in the LC-MS/MS analysis overall. Precision was typically 10–20% RSD among multiple days in experiments, detection limits were <10 ng/g in the fish tissue (catfish, basa, and swai filets), and no false-positives or false-negatives occurred in blind analyses of many spiked samples. The ELISA was unable to distinguish between MCs but was found to correctly assess the presence or absence of MCs and Nod-R in the blind-fortified fish tissues. The capability of these approaches to measure covalently bound MCs was not assessed.     

超高效液相色谱串联质谱快速测定饮用水中9种N-亚硝胺的新方法

N-亚硝胺是潜在的人类致癌物,是近年来关注的一类饮用水消毒副产物,同时也是环境分析研究的热点.本文建立了超高效液相色谱(UPLC)串联质谱快速测定饮用水中9种N-亚硝胺的新方法,讨论了色谱柱和流动相对分离9种N-亚硝胺的影响,优化了多级反应质谱(MRM,MS/MS)条件.二甲基亚硝胺-d6为贮存和回收率内标,亚硝基二丙...     

Universal liposomes: preparation and usage for the detection of mRNA

Dye-encapsulating liposomes can serve as signaling reagents in biosensors and biochemical assays in place of enzymes or fluorophores. Detailed here is the use and preparation of streptavidin-coupled liposomes which offer a universal approach to biotinylated target detection. The universal approach provides two advantages, i.e. only one type of liposome is necessary despite varying target and probe sequences and the hybridization event can take place in the absence of potential steric hindrance occurring from liposomes directly conjugated to probes. One objective of this work was to optimize the one-step conjugation of SRB-encapsulating liposomes to streptavidin using EDC. Liposome, EDC, streptavidin concentrations, and reaction times were varied. The optimal coupling conditions were found to be an EDC:carboxylated lipid:streptavidin molar ratio of 600:120:1 and a reaction time of 15 min. The second goal was to utilize these liposomes in sandwich hybridization microtiter plate-based assays using biotinylated reported probes as biorecognition elements. The assay was optimized in terms of probe spacer length, probe concentration, liposome concentration, and streptavidin coverage. Subsequently, the optimized protocol was applied to the detection of DNA and RNA sequences. A detection limit of 1.7 pmol L⁻¹ and an assay range spanning four orders of magnitude (5 pmol L⁻¹−50 nmol L⁻¹) with a coefficient of variation ≤5.8% was found for synthetic DNA. For synthetic RNA the LOQ was half that of synthetic DNA. A comparison was made to alkaline phosphatase-conjugated streptavidin for detection which yielded a limit of quantitation approximately 80 times higher than that for liposomes in the same system. Thus, liposomes and the optimized sandwich hybridization method are well suited for detecting single-stranded nucleic acid sequences and compares favorably to other sandwich hybridization schemes recently described in the literature. The assay was then used successfully for the clear detection of mRNA amplified by nucleic acid sequence-based amplification (NASBA) isolated from as little as one Cryptosporidium parvum oocyst. The detection of mRNA from oocysts isolated from various water sample types using immunomagnetic separation was also assessed. Finally, to prove the wider applicability and sensitivity of this universal method, RNA amplified from the atxA gene of Bacillus anthracis was detected when the input to the preceding NASBA reaction was as low as 1.2 pg. This highly sensitive liposome-based microtiter plate assay is therefore a platform technology allowing for high throughput and wide availability for routine clinical and environmental laboratory applications.     

Bufferized solvent extraction and HPLC fluorometric detection method for sarafloxacin in pig and chicken muscles

In this study, a method for the detection of sarafloxacin in pig and chicken muscles was developed using HPLC‐FLD as a regulatory residue technique. Good extraction efficiency was achieved using a mixture of 1% orthophosphoric acid–0.2 m MgCl₂ in water and acetonitrile as an extraction solvent, and n‐hexane partitioning and centrifugation for cleanup was used in the absence of dehydration. Specificity, linearity, detection and quantification limits, recovery, accuracy and precision were all validated, and all results were sufficient for the SARA regulatory residue method in pig and chicken muscles. The method developed and described herein was not only simple but also reliable, and was applied to market samples to determine their residue contents. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of herbicide terbuthylazine and its major hydroxy and dealkylated metabolites in constructed wetland sediments using solid phase extraction and high performance liquid chromatography-diode array detection

This paper describes the development of a new analytical method for the analysis of the herbicide TER and its degradation products in sediment samples. This method, based on high-performance liquid chromatography with diode-array detection, was validated for the simultaneous determination of TER and its major metabolites, desethylterbuthylazine, desisopropyatrazine, hydroxyterbuthylazine, desethylhydroxyatrazine and desethylhydroxyterbuthylazine. This method includes a cleanup and a solid-phase extraction step, using ultra-pure water and MCX cartridges respectively, with an overall recovery efficiency ranging from 89.3 to 97.9%. The statistical evaluation demonstrates good linearity with correlation coefficients >0.999 and adequate accuracy and precision for all analytes, with% Er and RSD values up to 10.5% and 8.3% respectively. The limit of detection for all substances was found to be 3.3?ng?g^?1. This method can be employed in remediation studies of TER and its major metabolites in sediments of constructed wetlands leading to useful results for the degradation and dispersion of TER in the vertical profile of wetland sediment substrates.     

Rapid multiplug filtration cleanup method for the determination of 124 pesticide residues in rice,wheat, and corn

A simple and rapid multiplug filtration cleanup method based on multiwalled carbon nanotubes was developed to determine 124 pesticide residues in rice, wheat, and corn, which could be done in a few seconds without conditioning and elution steps. Various combinations of sorbents were optimized for each matrix with a dispersive solid‐phase extraction procedure to get a satisfactory recovery and clean‐up performance. Good linearity was obtained for all pesticides with calibration curve coefficients larger than 0.9958. Most recoveries for the majority pesticides were between 70 and 120% (n = 5) with relative standard deviations below 20%. The limit of detection was 0.1–1.3 μg/kg, and the limit of quantification was 0.2–4.3 μg/kg for the pesticides in all matrices. The work suggests that the multiplug filtration cleanup method is better than the dispersive solid‐phase extraction method and it could be applied to routinely monitor pesticide residues in market samples.     

Quantification of deoxynivalenol in wheat using an immunoaffinity column and liquid chromatography

A simple and accurate method to quantify the mycotoxin deoxynivalenol (DON) in wheat is described. The method uses immunoaffinity chromatography for DON isolation and liquid chromatography (LC) for toxin detection and quantification. Wheat samples are extracted in water, filtered twice and applied to an immunoaffinity column. Following a water wash, DON is eluted from the column with methanol and injected onto an LC system with a UV detector for quantification. Test performance was evaluated in terms of antibody specificity, limit of detection, percentage recovery, precision, column capacity, assay linearity and comparison with the GC-electron-capture detection (ECD) method of Tacke and Casper. Specificity of the immunoaffinity column cleanup procedure was confirmed with only DON (>80%) and its 15-C derivatives (40-50%) being recognized by the antibody while 3-C DON derivatives, nivalenol, T-2 and fusarenon-X did not bind. The limit of detection is at least 0.10 microg/g. Percentage recovery for the entire assay range averages 90% with an average relative standard deviation of 8.3%. Naturally contaminated samples showed comparable precision. Column capacity was determined to be 3.3 microg. The assay showed a high degree of linearity (r2=0.999) and an optimum assay range of 0.10 to 10.0 microg/g. Comparative analysis of 28 naturally or artificially contaminated wheat samples using DONtest-HPLC and the GC-ECD method of Tacke and Casper showed that DONtest-HPLC is a statistically significant predictor of the GC-ECD method (r2=0.982).