Dispersion and transport of Cryptosporidium Oocysts from fecal pats under simulated rainfall events

The dispersion and initial transport of Cryptosporidium oocysts from fecal pats were investigated during artificial rainfall events on intact soil blocks (1,500 by 900 by 300 mm). Rainfall events of 55 mm h(-1) for 30 min and 25 mm h(-1) for 180 min were applied to soil plots with artificial fecal pats seeded with approximately 10(7) oocysts. The soil plots were divided in two, with one side devoid of vegetation and the other left with natural vegetation cover. Each combination of event intensity and duration, vegetation status, and degree of slope (5 degrees and 10 degrees ) was evaluated twice. Generally, a fivefold increase (P < 0.05) in runoff volume was generated on bare soil compared to vegetated soil, and significantly more infiltration, although highly variable, occurred through the vegetated soil blocks (P < 0.05). Runoff volume, event conditions (intensity and duration), vegetation status, degree of slope, and their interactions significantly affected the load of oocysts in the runoff. Surface runoff transported from 10(0.2) oocysts from vegetated loam soil (25-mm h(-1), 180-min event on 10 degrees slope) to up to 10(4.5) oocysts from unvegetated soil (55-mm h(-1), 30-min event on 10 degrees slope) over a 1-m distance. Surface soil samples downhill of the fecal pat contained significantly higher concentrations of oocysts on devegetated blocks than on vegetated blocks. Based on these results, there is a need to account for surface soil vegetation coverage as well as slope and rainfall runoff in future assessments of Cryptosporidium transport and when managing pathogen loads from stock grazing near streams within drinking water watersheds.     

Detection of UV-Induced Thymine Dimers in Individual Cryptosporidium parvum and Cryptosporidium hominis Oocysts by Immunofluorescence Microscopy

To investigate the effect of UV light on Cryptosporidium parvum and Cryptosporidium hominis oocysts in vitro, we exposed intact oocysts to 4-, 10-, 20-, and 40-mJ·cm⁻² doses of UV irradiation. Thymine dimers were detected by immunofluorescence microscopy using a monoclonal antibody against cyclobutyl thymine dimers (anti-TDmAb). Dimer-specific fluorescence within sporozoite nuclei was confirmed by colocalization with the nuclear fluorogen 4′,6′-diamidino-2-phenylindole (DAPI). Oocyst walls were visualized using either commercial fluorescein isothiocyanate-labeled anti-Cryptosporidium oocyst antibodies (FITC-CmAb) or Texas Red-labeled anti-Cryptosporidium oocyst antibodies (TR-CmAb). The use of FITC-CmAb interfered with TD detection at doses below 40 mJ·cm⁻². With the combination of anti-TDmAb, TR-CmAb, and DAPI, dimer-specific fluorescence was detected in sporozoite nuclei within oocysts exposed to 10 to 40 mJ·cm⁻² of UV light. Similar results were obtained with C. hominis. C. parvum oocysts exposed to 10 to 40 mJ·cm⁻² of UV light failed to infect neonatal mice, confirming that results of our anti-TD immunofluorescence assay paralleled the outcomes of our neonatal mouse infectivity assay. These results suggest that our immunofluorescence assay is suitable for detecting DNA damage in C. parvum and C. hominis oocysts induced following exposure to UV light.     

Patterns of Cryptosporidium Oocyst Shedding by Eastern Grey Kangaroos Inhabiting an Australian Watershed

The occurrence of Cryptosporidium oocysts in feces from a population of wild eastern grey kangaroos inhabiting a protected watershed in Sydney, Australia, was investigated. Over a 2-year period, Cryptosporidium oocysts were detected in 239 of the 3,557 (6.7%) eastern grey kangaroo fecal samples tested by using a combined immunomagnetic separation and flow cytometric technique. The prevalence of Cryptosporidium in this host population was estimated to range from 0.32% to 28.5%, with peaks occurring during the autumn months. Oocyst shedding intensity ranged from below 20 oocysts/g feces to 2.0 × 10⁶ oocysts/g feces, and shedding did not appear to be associated with diarrhea. Although morphologically similar to the human-infective Cryptosporidium hominis and the Cryptosporidium parvum “bovine” genotype oocysts, the oocysts isolated from kangaroo feces were identified as the Cryptosporidium “marsupial” genotype I or “marsupial” genotype II. Kangaroos are the predominant large mammal inhabiting Australian watersheds and are potentially a significant source of Cryptosporidium contamination of drinking water reservoirs. However, this host population was predominantly shedding the marsupial-derived genotypes, which to date have been identified only in marsupial host species.     

Modeling the Impact of Soil and Water Conservation on Surface and Ground Water Based on the SCS and Visual Modflow

Soil and water conservation measures can impact hydrological cycle, but quantitative analysis of this impact is still difficult in a watershed scale. To assess the effect quantitatively, a three-dimensional finite-difference groundwater flow model (MODFLOW) with a surface runoff model–the Soil Conservation Service (SCS) were calibrated and applied based on the artificial rainfall experiments. Then, three soil and water conservation scenarios were simulated on the sand-box model to assess the effect of bare slope changing to grass land and straw mulching on water volume, hydraulic head, runoff process of groundwater and surface water. Under the 120 mm rainfall, 60 mm/h rainfall intensity, 5 m² area, 3° slope conditions, the comparative results indicated that the trend was decrease in surface runoff and increase in subsurface runoff coincided with the land-use converted from bare slope to grass land and straw mulching. The simulated mean surface runoff modulus was 3.64×10⁻² m³/m²/h in the bare slope scenario, while the observed values were 1.54×10⁻² m³/m²/h and 0.12×10⁻² m³/m²/h in the lawn and straw mulching scenarios respectively. Compared to the bare slope, the benefits of surface water reduction were 57.8% and 92.4% correspondingly. At the end of simulation period (T = 396 min), the simulated mean groundwater runoff modulus was 2.82×10⁻² m³/m²/h in the bare slope scenario, while the observed volumes were 3.46×10⁻² m³/m²/h and 4.91×10⁻² m³/m²/h in the lawn and straw mulching scenarios respectively. So the benefits of groundwater increase were 22.7% and 60.4% correspondingly. It was concluded that the soil and water conservation played an important role in weakening the surface runoff and strengthening the underground runoff. Meanwhile the quantitative analysis using a modeling approach could provide a thought for the study in a watershed scale to help decision-makers manage water resources.     

Effectiveness of Standard UV Depuration at Inactivating Cryptosporidium parvum Recovered from Spiked Pacific Oysters (Crassostrea gigas)

When filter-feeding shellfish are consumed raw, because of their ability to concentrate and store waterborne pathogens, they are being increasingly associated with human gastroenteritis and have become recognized as important pathogen vectors. In the shellfish industry, UV depuration procedures are mandatory to reduce pathogen levels prior to human consumption. However, these guidelines are based around more susceptible fecal coliforms and Salmonella spp. and do not consider Cryptosporidium spp., which have significant resistance to environmental stresses. Thus, there is an urgent need to evaluate the efficiency of standard UV depuration against the survival of Cryptosporidium recovered from shellfish. Our study found that in industrial-scale shellfish depuration treatment tanks, standard UV treatment resulted in a 13-fold inactivation of recovered, viable C. parvum oocysts from spiked (1 × 10⁶ oocysts liter ⁻¹) Pacific oysters. Depuration at half power also significantly reduced (P < 0.05; ninefold) the number of viable oocysts recovered from oysters. While UV treatment resulted in significant reductions of recovered viable oocysts, low numbers of viable oocysts were still recovered from oysters after depuration, making their consumption when raw a public health risk. Our study highlights the need for increased periodic monitoring programs for shellfish harvesting sites, improved depuration procedures, and revised microbial quality control parameters, including Cryptosporidium assessment, to minimize the risk of cryptosporidiosis.     

Transport of Cryptosporidium parvum Oocysts through Vegetated Buffer Strips and Estimated Filtration Efficiency

Vegetated buffer strips were evaluated for their ability to remove waterborne Cryptosporidium parvum from surface and shallow subsurface flow during simulated rainfall rates of 15 or 40 mm/h for 4 h. Log₁₀ reductions for spiked C. parvum oocysts ranged from 1.0 to 3.1 per m of vegetated buffer, with buffers set at 5 to 20% slope, 85 to 99% fescue cover, soil textures of either silty clay (19:47:34 sand-silt-clay), loam (45:37:18), or sandy loam (70:25:5), and bulk densities of between 0.6 to 1.7 g/cm³. Vegetated buffers constructed with sandy loam or higher soil bulk densities were less effective at removing waterborne C. parvum (1- to 2-log₁₀ reduction/m) compared to buffers constructed with silty clay or loam or at lower bulk densities (2- to 3-log₁₀ reduction/m). The effect of slope on filtration efficiency was conditional on soil texture and soil bulk density. Based on these results, a vegetated buffer strip comprised of similar soils at a slope of ≤20% and a length of ≥3 m should function to remove ≥99.9% of C. parvum oocysts from agricultural runoff generated during events involving mild to moderate precipitation.     

Steady-State Effects of 2,5,2′,5′-Tetrachlorobiphenyl on Growth, Photosynthesis, and P Uptake in Selenastrum capricornutum

The steady-state effect of 2,5,2′,5′-tetrachlorobiphenyl (TCBP) on the green alga Selenastrum capricornutum was investigated in a P-limited two-stage chemostat system. The partition coefficient of this polychlorinated biphenyl congener was 5.9 × 10⁴ in steady-state cultures. At a cellular TCBP concentration of 12.2 × 10⁻⁸ ng · cell⁻¹, growth rate was not affected. However, photosynthetic capacity (P_max) was significantly enhanced by TCBP (56 × 10⁻⁹ μmol of C · cell⁻¹ · h⁻¹ versus 34 × 10⁻⁹ μmol of C · cell⁻¹ · h⁻¹ in the control). Photosynthetic efficiency, or the slope of the photosynthesis-irradiance curve, was also significantly higher. There was little difference in the cell chlorophyll a content, and therefore the difference in these photosynthetic characteristics was the same even when they were expressed on a per-chlorophyll a basis. Cell C content was higher in TCBP-containing cells than in TCBP-free cells, but approximately 36% of the C fixed by cells with TCBP was not incorporated as cell C. The maximum P uptake rate was also enhanced by TCBP, but the half-saturation concentration appeared to be unaffected.     

Improved Quantitative Estimates of Low Environmental Loading and Sporadic Periparturient Shedding of Cryptosporidium parvum in Adult Beef Cattle

Our primary goal was to generate an accurate estimate of the daily environmental loading rate of Cryptosporidium parvum oocysts for adult beef cattle, using immunomagnetic separation coupled with direct immunofluorescence microscopy for a highly sensitive diagnostic assay. An additional goal was to measure the prevalence and intensity of fecal shedding of C. parvum oocysts in pre- and postparturient cows as an indicator of their potential to infect young calves. This diagnostic method could detect with a ≥90% probability oocyst concentrations as low as 3.2 oocysts g of feces⁻¹, with a 54% probability of detecting just one oocyst g of feces⁻¹. Using this diagnostic method, the overall apparent prevalence of adult beef cattle testing positive for C. parvum was 7.1% (17 of 240), with 8.3 and 5.8% of cattle shedding oocysts during the pre- and postcalving periods, respectively. The mean intensity of oocyst shedding for test-positive cattle was 3.38 oocysts g of feces⁻¹. The estimated environmental loading rate of C. parvum ranged from 3,900 to 9,200 oocysts cow⁻¹ day⁻¹, which is substantially less than a previous estimate of 1.7 × 10⁵ oocysts cow⁻¹ day⁻¹ (range of 7.7 × 10⁴ to 2.3 × 10⁵ oocysts cow⁻¹ day⁻¹) (B. Hoar, E. R. Atwill, and T. B. Farver, Quant. Microbiol. 2:21-36, 2000). Use of this highly sensitive assay functioned to detect a greater proportion of low-intensity shedders in our population of cattle, which reduced the estimated mean intensity of shedding and thereby reduced the associated environmental loading rate compared to those of previous studies.     

Effect of Specific Growth Rate on Fermentative Capacity of Baker’s Yeast

The specific growth rate is a key control parameter in the industrial production of baker’s yeast. Nevertheless, quantitative data describing its effect on fermentative capacity are not available from the literature. In this study, the effect of the specific growth rate on the physiology and fermentative capacity of an industrial Saccharomyces cerevisiae strain in aerobic, glucose-limited chemostat cultures was investigated. At specific growth rates (dilution rates, D) below 0.28 h⁻¹, glucose metabolism was fully respiratory. Above this dilution rate, respirofermentative metabolism set in, with ethanol production rates of up to 14 mmol of ethanol · g of biomass⁻¹ · h⁻¹ at D = 0.40 h⁻¹. A substantial fermentative capacity (assayed offline as ethanol production rate under anaerobic conditions) was found in cultures in which no ethanol was detectable (D < 0.28 h⁻¹). This fermentative capacity increased with increasing dilution rates, from 10.0 mmol of ethanol · g of dry yeast biomass⁻¹ · h⁻¹ at D = 0.025 h⁻¹ to 20.5 mmol of ethanol · g of dry yeast biomass⁻¹ · h⁻¹ at D = 0.28 h⁻¹. At even higher dilution rates, the fermentative capacity showed only a small further increase, up to 22.0 mmol of ethanol · g of dry yeast biomass⁻¹ · h⁻¹ at D = 0.40 h⁻¹. The activities of all glycolytic enzymes, pyruvate decarboxylase, and alcohol dehydrogenase were determined in cell extracts. Only the in vitro activities of pyruvate decarboxylase and phosphofructokinase showed a clear positive correlation with fermentative capacity. These enzymes are interesting targets for overexpression in attempts to improve the fermentative capacity of aerobic cultures grown at low specific growth rates.     

Sensitivity of an Immunomagnetic-Separation-Based Test for Detecting Escherichia coli O26 in Bovine Feces

The sensitivity of a test for cattle shedding Escherichia coli serogroup O26 was estimated using several fecal pats artificially inoculated at a range of concentrations with different E. coli O26 strains. The test involves the enrichment of fecal microflora in buffered peptone water, the selective concentration of E. coli O26 using antibody-coated immunomagnetic-separation beads, the identification of E. coli colonies on Chromocult tryptone bile X-glucuronide agar, and confirmation of the serogroup with E. coli serogroup O26-specific antisera using slide agglutination. The effective dose of E. coli O26 for an 80% test sensitivity (ED₈₀) was 1.0 × 10⁴ CFU g⁻¹ feces (95% confidence interval, 4.7 × 10³ to 2.4 × 10⁴). Differences in test sensitivity between different E. coli O26 strains and fecal pats were also observed. Individual estimates of ED₈₀ for each strain and fecal pat combination ranged from 4.2 × 10² to 4.8 × 10⁵ CFU g⁻¹. These results suggest that the test is useful for identifying individuals shedding a large number of E. coli O26 organisms or, if an appropriate number of individuals in a herd are sampled, for identifying affected herds. The study also provides a benchmark estimate of sensitivity that can be used to compare alternative tests for E. coli O26 and a methodological approach that can be applied to tests for other pathogenic members of the Enterobacteriaceae and other sample types.     

Structure-Activity Relationships in Microbial Transformation of Phenols

The second-order rate constants for the microbial transformation of a series of phenols were correlated with the physicochemical properties of the phenols. The compounds studied were phenol, p-methylphenol, p-chlorophenol, p-bromophenol, p-cyanophenol, p-nitrophenol, p-acetylphenol, and p-methoxyphenol. Phenol-grown cells of Pseudomonas putida U transformed these compounds. Microbial transformation rate constants ranged from (1.5 ± 0.99) × 10⁻¹⁴ liter · organism⁻¹ · h⁻¹ for p-cyanophenol to (7.0 ± 1.3) × 10⁻¹² liter · organism⁻¹ · h⁻¹ for phenol. Linear regression analyses of rate constants and electronic, steric, and hydrophobic parameters showed that van der Waal's radii gave the best coefficient of determination (r² = 0.956). Products identified by thin-layer chromatography and liquid chromatography indicated that the phenols were microbially oxidized to the corresponding catechols.     

Accumulation,Allocation, and Risk Assessment of Polycyclic Aromatic Hydrocarbons (PAHs) in Soil-Brassica chinensis System

Farmland soil and leafy vegetables accumulate more polycyclic aromatic hydrocarbons (PAHs) in suburban sites. In this study, 13 sampling areas were selected from vegetable fields in the outskirts of Xi’an, the largest city in northwestern China. The similarity of PAH composition in soil and vegetation was investigated through principal components analysis and redundancy analysis (RDA), rather than discrimination of PAH congeners from various sources. The toxic equivalent quantity of PAHs in soil ranged from 7 to 202 μg/kg d.w., with an average of 41 μg/kg d.w., which exceeded the agricultural/horticultural soil acceptance criteria for New Zealand. However, the cancer risk level posed by combined direct ingestion, dermal contact, inhalation of soil particles, and inhalation of surface soil vapor met the rigorous international criteria (1×10⁻⁶). The concentration of total PAHs was (1052±73) μg/kg d.w. in vegetation (mean±standard error). The cancer risks posed by ingestion of vegetation ranged from 2×10⁻⁵ to 2×10⁻⁴ with an average of 1.66×10⁻⁴, which was higher than international excess lifetime risk limits for carcinogens (1×10⁻⁴). The geochemical indices indicated that the PAHs in soil and vegetables were mainly from vehicle and crude oil combustion. Both the total PAHs in vegetation and bioconcentration factor for total PAHs (the ratio of total PAHs in vegetation to total PAHs in soil) increased with increasing pH as well as decreasing sand in soil. The total variation in distribution of PAHs in vegetation explained by those in soil reached 98% in RDA, which was statistically significant based on Monte Carlo permutation. Common pollution source and notable effects of soil contamination on vegetation would result in highly similar distribution of PAHs in soil and vegetation.     

Perfusion Method for Assaying Microbial Activities in Sediments: Applicability to Studies of N2 Fixation by C2H2 Reduction

A perfusion method for assaying nitrogenase activity (acetylene reduction) in marine sediments was developed. The method was used to assay sediment cores from Spartina alterniflora (salt marsh), Zostera marina (sea grass), and Thalassia testudinum (sea grass) communities, and the results were compared with those of conventional sealed-flask assays. Rates of ethylene production increased progressively with time in the perfusion assays, reaching plateau values of 2 to 3 nmol · g of dry sediment⁻¹ · h⁻¹ by 10 to 20 h. Depletion of interstitial NH₄⁺ was implicated in this stimulation of nitrogenase activity. Initial acetylene reduction rates determined by the perfusion assay of cores from the Spartina community ranged from 0.15 to 0.60 nmol of C₂H₄ · g of dry sediment⁻¹ · h⁻¹. These rates were similar to those for sediments assayed in sealed flasks without seawater when determined over linear periods of C₂H₄ production. Initial values obtained by using the perfusion method were 0.66 nmol of C₂H₄ · g of dry sediment⁻¹ · h⁻¹ for sediments from Zostera communities and 0.70 nmol of C₂H₄ · g of dry sediment⁻¹ · h⁻¹ for sediments from Thalassia communities. In all cases, rates determined by simultaneous slurry assays were lower than those determined by the perfusion method.     

Inactivation of Cryptosporidium parvum Oocysts by Ammonia

The survival of Cryptosporidium parvum oocysts in soil and water microhabitats may be affected by the environmental production and release of free ammonia. The objective of this study was to determine the effects of increasing free ammonia concentrations and times of exposure on oocyst viability. Wild-type oocysts were obtained from naturally infected calf feces by chemical (continuous-flow) centrifugation and sucrose gradients. Ammonia (NH₃) from a commercial solution was applied in concentrations ranging from 0.007 to 0.148 M. Exposure times ranged from 10 min to 24 h at a constant temperature of 24 ± 1°C. Viability of oocysts was determined with a dye permeability assay and an in vitro excystation assay (M. B. Jenkins, L. J. Anguish, D. D. Bowman, M. J. Walker, and W. C. Ghiorse, Appl. Environ. Microbiol. 63:3844–3850, 1997). Even the lowest concentration of ammonia decreased significantly the viability of oocysts after 24 h of exposure. Increasing concentrations of ammonia increased inactivation rates, which ranged from 0.014 to 0.066 h⁻¹. At the highest concentration of ammonia, a small fraction of viable oocysts still remained. Exposure to pH levels corresponding to those associated with the ammonia concentrations showed minimal effects of alkaline pH alone on oocyst viability. This study shows that environmentally relevant concentrations of free ammonia may significantly increase the inactivation of oocysts in ammonia-containing environments.     

Occurrence of Cryptosporidium and Giardia in Wild Ducks along the Rio Grande River Valley in Southern New Mexico

Fecal samples were taken from wild ducks on the lower Rio Grande River around Las Cruces, N. Mex., from September 2000 to January 2001. Giardia cysts and Cryptosporidium oocysts were purified from 69 samples by sucrose enrichment followed by cesium chloride (CsCl) gradient centrifugation and were viewed via fluorescent-antibody (FA) staining. For some samples, recovered cysts and oocysts were further screened via PCR to determine the presence of Giardia lamblia and Crytosporidium parvum. The results of this study indicate that 49% of the ducks were carriers of Cryptosporidium, and the Cryptosporidium oocyst concentrations ranged from 0 to 2,182 oocysts per g of feces (mean ± standard deviation, 47.53 ± 270.3 oocysts per g); also, 28% of the ducks were positive for Giardia, and the Giardia cyst concentrations ranged from 0 to 29,293 cysts per g of feces (mean ± standard deviation, 436 ± 3,525.4 cysts per g). Of the 69 samples, only 14 had (oo)cyst concentrations that were above the PCR detection limit. Samples did test positive for Cryptosporidium sp. However, C. parvum and G. lamblia were not detected in any of the 14 samples tested by PCR. Ducks on their southern migration through southern New Mexico were positive for Cryptosporidium and Giardia as determined by FA staining, but C. parvum and G. lamblia were not detected.     

High rates of denitrification and nitrous oxide emission in arid biological soil crusts from the Sultanate of Oman

Using a combination of process rate determination, microsensor profiling and molecular techniques, we demonstrated that denitrification, and not anaerobic ammonium oxidation (anammox), is the major nitrogen loss process in biological soil crusts from Oman. Potential denitrification rates were 584±101 and 58±20 μmol N m⁻² h⁻¹ for cyanobacterial and lichen crust, respectively. Complete denitrification to N₂ was further confirmed by an ¹⁵NO₃⁻ tracer experiment with intact crust pieces that proceeded at rates of 103±19 and 27±8 μmol N m⁻² h⁻¹ for cyanobacterial and lichen crust, respectively. Strikingly, N₂O gas was emitted at very high potential rates of 387±143 and 31±6 μmol N m⁻² h⁻¹ from the cyanobacterial and lichen crust, respectively, with N₂O accounting for 53–66% of the total emission of nitrogenous gases. Microsensor measurements revealed that N₂O was produced in the anoxic layer and thus apparently originated from incomplete denitrification. Using quantitative PCR, denitrification genes were detected in both the crusts and were expressed either in comparable (nirS) or slightly higher (narG) numbers in the cyanobacterial crusts. Although 99% of the nirS sequences in the cyanobacterial crust were affiliated to an uncultured denitrifying bacterium, 94% of these sequences were most closely affiliated to Paracoccus denitrificans in the lichen crust. Sequences of nosZ gene formed a distinct cluster that did not branch with known denitrifying bacteria. Our results demonstrate that nitrogen loss via denitrification is a dominant process in crusts from Oman, which leads to N₂O gas emission and potentially reduces desert soil fertility.     

Rainfall-Induced Release of Fecal Coliforms and Other Manure Constituents: Comparison and Modeling

Modeling release of fecal coliforms is an important component of fate and transport simulations related to environmental water quality. Manure constituents other than fecal coliforms may serve as natural tracers of fecal contamination provided that their release from manure to runoff is similar to the fecal coliform release. The objectives of this work were to compare release of fecal coliforms (FC), chloride (Cl⁻), organic carbon (OC), and water-soluble phosphorus (P) from dissolving manure and to assess the performance of three models in describing the observed release. Bovine manure was applied on 0.5- by 0.3-m bare and vegetated subplots with 20% slope on sandy loam and clay loam soils. Concentrations of Cl⁻, FC, OC, and P were measured in runoff collected from troughs at the edges of the subplots at 5-min intervals during 1-h rainfall simulations. The one-parametric exponential model and two-parametric Vadas-Kleinman-Sharpley model and Bradford-Schijven model were fitted to the data. The Bradford-Schijven model had uncorrelated parameters, one of which was linearly related to the irrigation rate, and another parameter reflected the presence or the absence of vegetation. Kinetics of the FC release from manure was similar to the release kinetics of P and OC. The Bradford-Schijven model is recommended to simulate the release of manure constituents.     

Development of a Method for Detection of Giardia duodenalis Cysts on Lettuce and for Simultaneous Analysis of Salad Products for the Presence of Giardia Cysts and Cryptosporidium Oocysts

We report a method for detecting Giardia duodenalis cysts on lettuce, which we subsequently use to examine salad products for the presence of Giardia cysts and Cryptosporidium oocysts. The method is based on four basic steps: extraction of cysts from the foodstuffs, concentration of the extract and separation of the cysts from food materials, staining of the cysts to allow their visualization, and identification of cysts by microscopy. The concentration and separation steps are performed by centrifugation, followed by immunomagnetic separation using proprietary kits. Cyst staining is also performed using proprietary reagents. The method recovered 46.0% ± 19.0% (n = 30) of artificially contaminating cysts in 30 g of lettuce. We tested the method on a variety of commercially available natural foods, which we also seeded with a commercially available internal control, immediately prior to concentration of the extract. Recoveries of the Texas Red-stained Giardia cyst and Cryptosporidium oocyst internal controls were 36.5% ± 14.3% and 36.2% ± 19.7% (n = 20), respectively. One natural food sample of organic watercress, spinach, and rocket salad contained one Giardia cyst 50 g⁻¹ of sample as an indigenous surface contaminant.     

Modeling Reduction of Uranium U(VI) under Variable Sulfate Concentrations by Sulfate-Reducing Bacteria

The kinetics for the reduction of sulfate alone and for concurrent uranium [U(VI)] and sulfate reduction, by mixed and pure cultures of sulfate-reducing bacteria (SRB) at 21 ± 3°C were studied. The mixed culture contained the SRB Desulfovibrio vulgaris along with a Clostridium sp. determined via 16S ribosomal DNA analysis. The pure culture was Desulfovibrio desulfuricans (ATCC 7757). A zero-order model best fit the data for the reduction of sulfate from 0.1 to 10 mM. A lag time occurred below cell concentrations of 0.1 mg (dry weight) of cells/ml. For the mixed culture, average values for the maximum specific reaction rate, V_max, ranged from 2.4 ± 0.2 μmol of sulfate/mg (dry weight) of SRB · h⁻¹) at 0.25 mM sulfate to 5.0 ± 1.1 μmol of sulfate/mg (dry weight) of SRB · h⁻¹ at 10 mM sulfate (average cell concentration, 0.52 mg [dry weight]/ml). For the pure culture, V_max was 1.6 ± 0.2 μmol of sulfate/mg (dry weight) of SRB · h⁻¹ at 1 mM sulfate (0.29 mg [dry weight] of cells/ml). When both electron acceptors were present, sulfate reduction remained zero order for both cultures, while uranium reduction was first order, with rate constants of 0.071 ± 0.003 mg (dry weight) of cells/ml · min⁻¹ for the mixed culture and 0.137 ± 0.016 mg (dry weight) of cells/ml · min⁻¹ (U₀ = 1 mM) for the D. desulfuricans culture. Both cultures exhibited a faster rate of uranium reduction in the presence of sulfate and no lag time until the onset of U reduction in contrast to U alone. This kinetics information can be used to design an SRB-dominated biotreatment scheme for the removal of U(VI) from an aqueous source.     

Continuous and Batch Production of Chloroperoxidase by Mycelial Pellets of Caldariomyces fumago in an Airlift Fermentor

Batch and continuous production of the extracellular heme glycoprotein chloroperoxidase (CPO) was studied with an airlift fermentor. We induced Caldariomyces fumago CMI 89362 to form pellets by transferring a small inoculum volume in preculture prior to growth in a 1-liter fermentor. Continuous replacement of the fructose-salts medium (dilution rate, 0.008 h⁻¹) supported continuous CPO formation at an average concentration of 128 ± 10 mg of CPO liter⁻¹ for 8 days. Optimum CPO production rates averaged 1.2 ± 0.1 mg of CPO h⁻¹ at dilution rates below 0.033 h⁻¹. Varying the carbohydrate content of the feed solution or the time of starting the feed did not significantly alter the amount of CPO produced. Batch fermentation in the airlift fermentor resulted in maximum CPO concentrations of 280 ± 80 mg of CPO liter⁻¹, although on two separate occasions CPO concentrations reached 400 to 450 mg liter⁻¹, which was double the amount obtained by free hyphae in shake flask culture.