Three sample preparation protocols for polymerase chain reaction based detection of Cryptosporidium parvum in environmental samples

Cryptosporidium parvum is a protozoan parasite responsible for an increasing number of outbreaks of gastrointestinal illness worldwide. In this report, we describe development of sample preparation protocols for polymerase chain reaction (PCR)-based detection of C. parvum in fecal material and environmental water samples. Two of these methods were found adequate for isolation of Cryptosporidium DNA from filtered water pellet suspensions. The first involved several filtration steps, immunomagnetic separation and freeze-thaw cycles. The second method involved filtration, addition of EnviroAmp lysis reagent, freeze-thaw cycles and precipitation of the DNA with isopropanol. Using nested PCR, we detected 100 oocysts/ml of filtered water pellet suspension, with either of the above sample preparation procedures. Nested PCR increased sensitivity of the assay by two to three orders of magnitude as compared to the primary PCR. The detection limit for seeded fecal samples was 10-fold higher than for filtered environmental water pellet suspension. Nested PCR results showed 62.4 and 91.1% correlation with immunofluorescence assay (IFA) for fecal samples and filtered environmental water pellet suspensions, respectively. This correlation decreased to 47.2% and 44.4%, respectively, when only IFA positive samples were analyzed. However, in fecal samples contaminated with a high number (> 10(5)/g) of C. parvum oocysts, this correlation was 100%.     

The use of NASBA for the detection of microbial pathogens in food and environmental samples

The isothermal amplification method nucleic acid sequence-based amplification (NASBA), which amplifies RNA, has been reported as useful for the detection of microbial pathogens in food and environmental samples. Methods have been published for Campylobacter spp., Listeria monocytogenes and Salmonella enterica ser. Enteritidis in various foods and for Cryptosporidium parvum in water. Both 16S rRNA and various mRNAs have been used as target molecules for detection; the latter may have advantages in allowing specific detection of viable cells. Most of the methods to detect pathogens in foods have employed enrichment in nutrient medium prior to NASBA, as this can ensure sensitivity of detection and encourage the detection of only viable target cells. Although a relatively recent method, NASBA has the potential for adoption as a diagnostic tool for environmental pathogens.     

Bacterial colonization of pellet softening reactors used during drinking water treatment

Pellet softening reactors are used in centralized and decentralized drinking water treatment plants for the removal of calcium (hardness) through chemically induced precipitation of calcite. This is accomplished in fluidized pellet reactors, where a strong base is added to the influent to increase the pH and facilitate the process of precipitation on an added seeding material. Here we describe for the first time the opportunistic bacterial colonization of the calcite pellets in a full-scale pellet softening reactor and the functional contribution of these colonizing bacteria to the overall drinking water treatment process. ATP analysis, advanced microscopy, and community fingerprinting with denaturing gradient gel electrophoretic (DGGE) analysis were used to characterize the biomass on the pellets, while assimilable organic carbon (AOC), dissolved organic carbon, and flow cytometric analysis were used to characterize the impact of the biological processes on drinking water quality. The data revealed pellet colonization at concentrations in excess of 500 ng of ATP/g of pellet and reactor biomass concentrations as high as 220 mg of ATP/m(3) of reactor, comprising a wide variety of different microorganisms. These organisms removed as much as 60% of AOC from the water during treatment, thus contributing toward the biological stabilization of the drinking water. Notably, only a small fraction (about 60,000 cells/ml) of the bacteria in the reactors was released into the effluent under normal conditions, while the majority of the bacteria colonizing the pellets were captured in the calcite structures of the pellets and were removed as a reusable product.     

PCR-based quantitation of Cryptosporidium parvum in municipal water samples.

A PCR method for the quantitation of Cryptosporidium parvum oocysts in municipal drinking water samples was investigated. Quantitative PCR uses an internal standard (IS) template with unknown target numbers to compare to standards of known concentrations in a standard curve. The IS template was amplified using the same primers used to amplify a portion of a 358 bp gene fragment that encodes a repetitive oocyst wall protein in C. parvum. Municipal water samples spiked with known numbers of C. parvum oocysts were tested by quantitative PCR using the IS and the Digene SHARP Signal System Assay for PCR product detection. The absorbance readings for target DNA and IS templates versus the number of molecules of the target DNA were plotted to generate standard curves for estimating oocyst numbers. The method allowed the quantitation of oocysts from log 3 to log 5 spiked into municipal water samples.     

Rapid extraction of DNA From Escherichia coli and Cryptosporidium parvum for use in PCR.

The Xtra Amp tube, Isocode paper, Instagene matrix, and PrepMan matrix methods were evaluated for their ability to rapidly extract PCR-quality DNAs from Escherichia coli O157:H7 and Cryptosporidium parvum. All methods provided satisfactory DNA from E. coli, and the Xtra Amp and Instagene reagents provided satisfactory DNA from C. parvum.     

Effects of divalent cations and EDTA on spectral properties of phytochrome in particulate fractions from etiolated pea epicotyls

The photoreversible absorbance change of phytochrome in suspensionsof a 20,000xg particulate fraction (20kP) prepared from a 1,000xgsupernatant (1kS) of etiolated pea epicotyl extracts decreasedremarkably in the presence of 5 mM Cu²⁺, Zn²⁺ and Co²⁺, butremained unchanged in 5 mM Ca²⁺, Mg²⁺, Fe²⁺ or Mn²⁺. This spectraldistortion of phytochrome was more evident in soluble preparationsand in suspensions of pellets prepared from red light (R)-irradiatedtissues than it was in suspensions of pellets prepared in thedark from etiolated tissues that received no actinic irradiation. When Cu²⁺ was added to the red-light-absorbing form of phytochrome(Pr) in resuspended pellets prepared from R-irradiated tissues,the distortion of its difference spectrum took place after irradiationwith the first actinic R. In contrast, when Cu²⁺ was added tothe far-red-light-absorbing form of phytochrome (Pfr) in thesame resuspended pellet, no distortion was seen, unless thePfr in the pellet was first photoconverted to Pr and then photoconvertedback to Pfr. Spectral distortion of Pr remained small during dark incubationat 25°C when suspensions of 20kPs were prepared and incubatedwith a buffer containing EDTA, whether the 20kP was preparedfrom nonirradiated tissue or from R-irradiated tissues. But,when EDTA was added to a suspension of 20kP prepared from 1kS,after the 1kS was irradiated with R in the presence of 10 mMCaCl₂, the spectral distortion of Pr in 20kP occurred instantaneously. (Received April 14, 1980; )     

Leader of the pack: faecal pellet deposition order impacts PCR amplification in wombats

DNA sourced from faeces is notoriously less reliable than that from tissue. Hence, understanding whether faecal pellet quality varies within faecal piles may be important for sample selection. We hypothesized that the order in which faecal pellets are deposited may influence microsatellite polymerase chain reaction (PCR) amplification success from sampled faeces, more specifically, that first pellets deposited will have signatures of greater success than later ones. In a first test of the hypothesis, first and later-deposited pellets, as determined from the direction of footprints, were collected from fresh (overnight) faecal piles of northern hairy-nosed wombats (Lasiorhinus krefftii). DNA extracts were typed for seven microsatellite loci. We found that faecal deposition order significantly affected optical density of bands on autoradiographs (a measure of PCR amplification success) when the first faecal pellet was compared with the last one, but not when the first pellet was only distinguishable from later ones. The absence of a difference in amplification rate between first and later pellets is likely a reflection of the overall high amplification success in this study. That first pellets deposited yield more product suggests they contain more intestinal cells. Although further comparisons are needed, these results may inform sample selection in species for which success of microsatellite PCR amplification of faecal DNA is low. Deposition order may have more of an impact on amplification success and genotyping errors as faecal age increases.     

Detection and discrimination of Cryptosporidium parvum and C. hominis in water samples by immunomagnetic separation-PCR

Cryptosporidium parvum and C. hominis have been the cause of large and serious outbreaks of waterborne cryptosporidiosis. A specific and sensitive recovery-detection method is required for control of this pathogen in drinking water. In the present study, nested PCR-restriction fragment length polymorphism (RFLP), which targets the divergent Cpgp40/15 gene, was developed. This nested PCR detected only the gene derived from C. parvum and C. hominis strains, and RFLP was able to discriminate between the PCR products from C. parvum and C. hominis. To evaluate the sensitivity of nested PCR, C. parvum oocysts inoculated in water samples of two different turbidities were recovered by immunomagnetic separation (IMS) and detected by nested PCR and fluorescent antibody assay (FA). Genetic detection by nested PCR and oocyst number confirmed by FA were compared, and the results suggested that detection by nested PCR depends on the confirmed oocyst number and that nested PCR in combination with IMS has the ability to detect a single oocyst in a water sample. We applied an agitation procedure with river water solids to which oocysts were added to evaluate the recovery and detection by the procedure in environmental samples and found some decrease in the rate of detection by IMS.     

An immunomagnetic separation-real-time PCR method for quantification of Cryptosporidium parvum in water samples

The protozoan parasite Cryptosporidium parvum is known to occur widely in both raw and drinking water and is the cause of waterborne outbreaks of gastroenteritis throughout the world. The routinely used method for the detection of Cryptosporidium oocysts in water is based on an immunofluorescence assay (IFA). It is both time-consuming and nonspecific for the human pathogenic species C. parvum. We have developed a TaqMan polymerase chain reaction (PCR) test that accurately quantifies C. parvum oocysts in treated and untreated water samples. The protocol consisted of the following successive steps: Envirochek capsule filtration, immunomagnetic separation (IMS), thermal lysis followed by DNA purification using Nanosep centrifugal devices and, finally, real-time PCR using fluorescent TaqMan technology. Quantification was accomplished by comparing the fluorescence signals obtained from test samples with those from standard dilutions of C. parvum oocysts. This IMS-real-time PCR assay permits rapid and reliable quantification over six orders of magnitude, with a detection limit of five oocysts for purified oocyst solutions and eight oocysts for spiked water samples. Replicate samples of spiked tap water and Seine River water samples (with approximately 78 and 775 oocysts) were tested. C. parvum oocyst recoveries, which ranged from 47.4% to 99% and from 39.1% to 68.3%, respectively, were significantly higher and less variable than those reported using the traditional US Environmental Protection Agency (USEPA) method 1622. This new molecular method offers a rapid, sensitive and specific alternative for C. parvum oocyst quantification in water.     

High quality biofuel pellet production from pre-compacted low density raw materials

In this study, pre-compaction was evaluated as a method to enhance stable reed canary grass pellet production. An experimental design of the factors raw material moisture content, steam addition, raw material bulk density, and die temperature was used to find production conditions for high quality pellets by multiple linear regression modelling of responses. Response variables being modelled were variability of pelletizer current (as a measurement of uneven production), pellet bulk density, and pellet durability. By pre-compacting the raw material from a bulk density of 150 kg/m3 to 270kg/m3, continuous production could be obtained at minimum raw material moisture content of 13.8%. Bulk density and durability were both highly correlated to raw material moisture content, but showed different optima. Multiple response optimization was used to target process settings for production of high quality reed canary grass pellets with bulk density >650kg/m3 and durability >97.5%.     

The fate of faecal pellets in the North Sea

During an intensive survey in May/June 1986, 129 stations in the North Sea were examined in different water depths with respect to several hydrographical parameters. The relations of planktonic faecal pellets to food (chl-a) and producers (zooplankton-wet weight) as well as to temperature, salinity and water depth were investigated. The amount of faecal pellets was positively correlated with chl-a and zooplankton. The length of the faecal pellets was negatively correlated with water depth indicating a degradation during sinking. Hardly any formed faecal material was detectable under 100 m water depth. The ratio of pellets to zooplankton was positively correlated to chlorophyll and influenced by daytime, indicating diel variations in pellet production. It is assumed that in summer most of the planktonic faecal material is disintegrated in the upper water layer and the role of faeces in sedimentation is minor.     

Concentration and detection of cryptosporidium oocysts in surface water samples by method 1622 using ultrafiltration and capsule filtration

The protozoan parasite Cryptosporidium parvum is known to occur widely in both source and drinking water and has caused waterborne outbreaks of gastroenteritis. To improve monitoring, the U.S. Environmental Protection Agency developed method 1622 for isolation and detection of Cryptosporidium oocysts in water. Method 1622 is performance based and involves filtration, concentration, immunomagnetic separation, fluorescent-antibody staining and 4',6-diamidino-2-phenylindole (DAPI) counterstaining, and microscopic evaluation. The capsule filter system currently recommended for method 1622 was compared to a hollow-fiber ultrafilter system for primary concentration of C. parvum oocysts in seeded reagent water and untreated surface waters. Samples were otherwise processed according to method 1622. Rates of C. parvum oocyst recovery from seeded 10-liter volumes of reagent water in precision and recovery experiments with filter pairs were 42% (standard deviation [SD], 24%) and 46% (SD, 18%) for hollow-fiber ultrafilters and capsule filters, respectively. Mean oocyst recovery rates in experiments testing both filters on seeded surface water samples were 42% (SD, 27%) and 15% (SD, 12%) for hollow-fiber ultrafilters and capsule filters, respectively. Although C. parvum oocysts were recovered from surface waters by using the approved filter of method 1622, the recovery rates were significantly lower and more variable than those from reagent grade water. In contrast, the disposable hollow-fiber ultrafilter system was compatible with subsequent method 1622 processing steps, and it recovered C. parvum oocysts from seeded surface waters with significantly greater efficiency and reliability than the filter suggested for use in the version of method 1622 tested.     

Effect of pellet size and pellet quality on pig performance

An experiment involving 1360 growing finishing pigs was undertaken to examine the effect of pellet size and pellet quality, as measured by the Holmen pellet durability test, on pig performance. A barley and soya bean meal diet was used and pellets were of two sizes, 5 and 10 mm diameter. Pellet quality was varied to give two types of pellet by steam conditioning and screening procedure during the pelleting process. The mean difference obtained in pellet durability was 11%.

Pig performance between 30 and 80 kg liveweight was not affected by either pellet size or pellet quality. There were small non-significant trends in favour of both the smaller pellets and the lower quality pellets. These trends, of the order of 1% or less, followed the same pattern as the dry matter content of the diets. The smaller diameter pellets were dried more efficiently in the cooling process and the low durability pellets had less steam added during the manufacturing process, which was reflected in the dry matter content of the finished diets.     

Phytochrome Pelletability Induced by Irradiation in Vivo: TEST FOR IN VITRO BINDING OF ADDED [S]PHYTOCHROME

Undegraded, highly purified [³⁵S]phytochrome was immunoaffinity-purified either from dark control oat (cv. Garry) shoots or from etiolated oat shoots that were previously irradiated first with red and then with far-red light so that, if proper extraction conditions had been utilized, about 60% of the total phytochrome would have been pelletable. When [³⁵S]phytochrome was added to extraction buffer immediately prior to homogenization of etiolated oat shoots, pelletability assays indicated that there was no preferential binding of [³⁵S]phytochrome regardless of (a) whether it was purified from dark control or irradiated shoots, (b) whether it was added as phytochrome-red-absorbing form or phytochrome-far-red-absorbing form, or (c) whether it was added to dark control or red-irradiated shoots. Similarly, binding of [³⁵S]phytochrome to resuspended pellets obtained from crude oat extracts was not specific for the source of [³⁵S]phytochrome, for its form, or for the irradiation treatment given to intact shoots used to prepare the resuspended pellets. No evidence was obtained to support the hypothesis that phytochrome binds with specificity to particulate material in vitro under conditions used to assay for light-enhanced, in vivo-induced phytochrome pelletability.     

A novel technique for the enumeration of bacteriophage from water

Abstract A novel method has been developed for concentrating and enumerating bacteriophages in water. Host bacterial cells are added to water samples to adsorb phage and then the infected bacteria are collected by centrifugation at 4000 rev./min for 30 min. The pellets are resuspended in small volumes and assayed for phages using the agar-overlay method. This procedure was more successful than three established techniques in recovering Bacillus phages from seeded samples of tap water. It also gave efficient recovery of phages from samples of river, lake and sea-water.     

Characterization and potential use of a Cryptosporidium parvum virus (CPV) antigen for detecting C. parvum oocysts

The purpose of this study was to characterize the viral symbiont (CPV) of Cryptosporidium parvum sporozoites and evaluate the CPV capsid protein (CPV40) as a target for sensitive detection of the parasite. Recombinant CPV40 was produced in Escherichia coli, purified by affinity chromatography, and used to prepare polyclonal rabbit sera specific for the viral capsid protein. Anti-rCPV40 recognized a 40 kDa and a 30 kDa protein in C. parvum oocysts and appeared to localize to the apical end of the parasite. Anti-rCPV40 serum was capable of detecting as few as 1 C. parvum oocyst in a dot blot assay, the sensitivity being at least 1000-fold greater than sera reactive with total native C. parvum oocyst protein or specific for the 41 kDa oocyst surface antigen. Water samples were seeded with C. parvum oocysts and incubated at 4, 20, or 25 degrees C for greater than 3 months to determine if CPV levels were correlated with oocyst infectivity. Samples were removed monthly and subjected to mouse and cell culture infectivity, as well as PCR analysis for infectivity and viral particle presence. While sporozoite infectivity declined by more than 75% after 1 month at 25 degrees C, the CPV signal was similar to that of control samples at 4 degrees C. By 3 months at 20 degrees C, the C. parvum oocysts were found to be non-infectious, but retained a high CPV signal. This study indicates that CPV is an excellent target for sensitive detection of C. parvum oocysts in water, but may persist for an indefinite time after oocysts become non-infectious.     

甘草次酸结肠靶向微丸的研制

目的:确定甘草次酸结肠靶向微丸的制剂处方,评价其释药特性。方法:采用挤出-滚圆法制备甘草次酸素丸,利用流化床包衣技术对甘草次酸素丸进行包衣,用浆法评价微丸的体外释药性能。结果:采用微晶纤维素和甘草次酸,同时加入黏合剂羧甲基纤维素钠,经过充分搅拌混合,以30%的乙醇作为润湿剂,通过挤出-滚圆制得甘草次酸素丸。以尤特奇S100为膜控材料,加入适量柠檬酸三乙酯与滑石粉配制包衣液,对甘草次酸素丸进行包衣,制得甘草次酸包衣微丸。释放度实验表明甘草次酸素丸在其增重20%时,在0.1 mo L/L的盐酸溶液中不释放,在p H6.8的磷酸缓冲液条件下6 h内其释放率不到20%。而在p H7.4的磷酸缓冲液条件下2 h内释放率达到80%以上。结论:所制的甘草次酸素丸处方合理,制剂工艺简便,通过流化床包衣技术所制的甘草次酸包衣微丸在模拟的胃液中不释放,在小肠液中释放缓慢,在结肠液中释药良好,具有良好的结肠靶向作用。     

Comparison of primers and optimization of PCR conditions for detection of Cryptosporidium parvum and Giardia lamblia in water.

Eight pairs of published PCR primers were evaluated for the specific detection of Cryptosporidium parvum and Giardia lamblia in water. Detection sensitivities ranged from 1 to 10 oocysts or cysts for purified preparations and 5 to 50 oocysts or cysts for seeded environmental water samples. Maximum sensitivity was achieved with two successive rounds of amplification and hybridization, with oligonucleotide probes detected by chemiluminescence. Primer annealing temperatures and MgCl2 concentrations were optimized, and the specificities of the primer pairs were determined with closely related species. Some of the primers were species specific, while others were only genus specific. Multiplex PCR for the simultaneous detection of Cryptosporidium and Giardia was demonstrated with primers amplifying 256- and 163-bp products from the 18S rRNA gene of Cryptosporidium and the heat shock protein gene of Giardia, respectively. The results demonstrate the potential utility of PCR for the detection of pathogenic protozoa in water but emphasize the necessity of continued development.     

Combination of ARAD microfibre filtration and LAMP methodology for simple, rapid and cost-effective detection of human pathogenic Giardia duodenalis and Cryptosporidium spp. in drinking water

Aims:  In this study, we report a new, simple methodology for the monitoring of Cryptosporidium oocysts and Giardia cysts in drinking water samples, ranging from 10- to 1000-l, which combines a new ARAD microfibre filtration of the (oo)cysts from drinking water and loop-mediated isothermal amplification (LAMP) of a human pathogenic Cryptosporidium parvum , Cryptosporidium hominis , Cryptosporidium meleagridis and Giardia duodenalis Assemblage A and B specific DNA sequence.
Methods and Results:  During the evaluation of the new concentration and detection technique, spiked reagent and matrix water samples plus blank samples were filtered and tested. In total, 27 samples have been investigated. The results clearly demonstrate that the methodology of using a new ARAD filter, which passed through 1000 l of drinking water with high turbidity (2 NTU), and followed by the LAMP assay was able to detect at least one (oo)cyst in 10 l of drinking water based on a 1000-l sample, taken over a 24-h period.
Conclusions:  The described protozoa detection methodology is sensitive, rapid and cost-effective.
Significance and Impact of the Study:  This effective procedure will be useful for small waterworks to achieve continuous monitoring and is also of value for screening catchments to identify those that require further treatment and more detailed microscopic counts.     

Immunomagnetic separation (IMS)-fluorescent antibody detection and IMS-PCR detection of seeded Cryptosporidium parvum oocysts in natural waters and their limitations

Detection and enumeration of Cryptosporidium parvum in both treated and untreated waters are important to facilitate prevention of future cryptosporidiosis incidents. Immunomagnetic separation (IMS)-fluorescent antibody (FA) detection and IMS-PCR detection efficiencies were evaluated in two natural waters seeded with nominal seed doses of 5, 10, and 15 oocysts. IMS-FA detected oocysts at concentrations at or below the three nominal oocyst seed doses, illustrating that IMS-FA is sensitive enough to detect low oocyst numbers. However, the species of the oocysts could not be determined with this technique. IMS-PCR, targeting the 18S rRNA gene in this study, yielded positive amplification for 17 of the 18 seeded water samples, and the amplicons were subjected to restriction fragment length polymorphism digestion and DNA sequencing for species identification. Interestingly, the two unseeded, natural water samples were also PCR positive; one amplicon was the same base pair size as the C. parvum amplicon, and the other amplicon was larger. These two amplified products were determined to be derived from DNA of Cryptosporidium muris and a dinoflagellate. These IMS-PCR results illustrate that (i) IMS-PCR is able to detect low oocyst numbers in natural waters, (ii) PCR amplification alone is not confirmatory for detection of target DNA when environmental samples are used, (ii) PCR primers, especially those designed against the rRNA gene region, need to be evaluated for specificity with organisms closely related to the target organism, and (iv) environmental amplicons should be subjected to appropriate species-specific confirmatory techniques.