scholarly journals Inactivation of Cryptosporidium parvumOocysts by Ammonia

1998 ◽  
Vol 64 (2) ◽  
pp. 784-788 ◽  
Author(s):  
Michael B. Jenkins ◽  
Dwight D. Bowman ◽  
William C. Ghiorse
Keyword(s):  
Cryptosporidium Parvum ◽  
Continuous Flow ◽  
Free Ammonia ◽  
Infected Calf ◽  
Alkaline Ph ◽  
Wild Type ◽  
Dye Permeability ◽  
Permeability Assay ◽  
Soil And Water

ABSTRACT 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.

Survival ofCryptosporidium parvumoocysts in source separated human urine

1999 ◽  
Vol 45 (9) ◽  
pp. 740-746 ◽  
Author(s):  
Caroline E Höglund ◽  
Thor Axel B Stenström
Keyword(s):  
Cryptosporidium Parvum ◽  
Human Urine ◽  
Ph Effects ◽  
Primary Methods ◽  
Dye Permeability ◽  
Cryptosporidium Parvum Oocysts ◽  
Microbial Risks ◽  
Permeability Assay ◽  
Made In

The survival of Cryptosporidium parvum in source separated urine was investigated as part of a broader study on microbial risks associated with the reuse of human urine for sustainable agriculture. A dye permeability assay and in vitro excystation were the primary methods used to assess viability. In the collected urine most of the nitrogen is present as ammonia and the pH is generally around 9. Parallel investigations were made in buffers to compare possible toxic effects of urine to actual pH effects. Oocysts in the untreated urine were inactivated below the detection limit (1/300) within 63 days. This inactivation rate was significantly higher (P < 0.01) than in urine adjusted to pH 5 or 7 according to the dye permeability assay. The corresponding difference between different pH values was not seen in buffers, suggesting that the antiprotozoan effect of urine was mediated by other factors besides pH. The Swedish practice of storing urine for six months before its use thus appears satisfactory for the inactivation of Cryptosporidium oocysts.Key words: Cryptosporidium parvum, oocysts, human urine, survival, source separation.

scholarly journals Improving the Stability and Activity of Arginine Decarboxylase at Alkaline pH for the Production of Agmatine

2021 ◽  
Vol 1 ◽  
Author(s):  
Eun Young Hong ◽  
Sun-Gu Lee ◽  
Hyungdon Yun ◽  
Byung-Gee Kim
Keyword(s):  
Disulfide Bond ◽  
Specific Activity ◽  
Catalytic Efficiency ◽  
Arginine Decarboxylase ◽  
Saturation Mutagenesis ◽  
Alkaline Ph ◽  
Wild Type ◽  
Active Site Residues ◽  
Cellular Mechanisms

Agmatine, involved in various modulatory actions in cellular mechanisms, is produced from arginine (Arg) by decarboxylation reaction using arginine decarboxylase (ADC, EC 4.1.1.19). The major obstacle of using wild-type Escherichia coli ADC (ADCes) in agmatine production is its sharp activity loss and instability at alkaline pH. Here, to overcome this problem, a new disulfide bond was rationally introduced in the decameric interface region of the enzyme. Among the mutants generated, W16C/D43C increased both thermostability and activity. The half-life (T1/2) of W16C/D43C at pH 8.0 and 60°C was 560 min, which was 280-fold longer than that of the wild-type, and the specific activity at pH 8.0 also increased 2.1-fold. Site-saturation mutagenesis was subsequently performed at the active site residues of ADCes using the disulfide-bond mutant (W16C/D43C) as a template. The best variant W16C/D43C/I258A displayed a 4.4-fold increase in the catalytic efficiency when compared with the wild-type. The final mutant (W16C/D43C/I258A) was successfully applied to in vitro synthesis of agmatine with an improved yield and productivity (&gt;89.0% yield based on 100 mM of Arg within 5  h).

scholarly journals Hyperencapsulated mucoid pneumococcal isolates from patients with cystic fibrosis have increased biofilm density and persistence in vivo

2018 ◽  
Vol 76 (7) ◽  
Author(s):  
Evida A Dennis ◽  
Mamie T Coats ◽  
Sarah Griffin ◽  
Bing Pang ◽  
David E Briles ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Continuous Flow ◽  
Biofilm Formation ◽  
Capsular Polysaccharides ◽  
Wild Type ◽  
Pneumococcal Colonization ◽  
Phase Variants

AbstractMucoid bacteria, predominately Pseudomonas aeruginosa, are commonly associated with decline in pulmonary function in children with cystic fibrosis (CF), and are thought to persist at least in part due to a greater propensity toward forming biofilms. We isolated a higher frequency of mucoid Streptococcus pneumoniae (Sp) expressing high levels of capsular polysaccharides from sputa from children with CF, compared to those without CF. We compared biofilm formation and maturation by mucoid and non-mucoid isolates of Sp collected from children with and without CF. Non-mucoid Sp serotype 19A and 19F isolates had significantly higher levels of biofilm initiation and adherence to CF epithelial cells than did serotype 3 isolates. However, strains expressing high levels of capsule had significantly greater biofilm maturation, as evidenced by increased density and thickness in static and continuous flow assays via confocal microscopy. Finally, using a serotype 3 Sp strain, we showed that highly encapsulated mucoid phase variants predominate during late adherence and better colonize CFTR–/– as compared to wild-type mice in respiratory infection studies. These findings indicate that overexpression of capsule can enhance the development of mature pneumococcal biofilms in vitro, and may contribute to pneumococcal colonization in CF lung disease.

Effects of ozonation and chlorination on viability and infectivity of Cryptosporidium parvum oocysts

2000 ◽  
Vol 41 (7) ◽  
pp. 39-46 ◽  
Author(s):  
T. Hirata ◽  
D. Chikuma ◽  
A. Shimura ◽  
A. Hashimoto ◽  
N. Motoyama ◽  
...  
Keyword(s):  
Cryptosporidium Parvum ◽  
Experimental Studies ◽  
Free Chlorine ◽  
Log Reduction ◽  
Cryptosporidium Parvum Oocysts ◽  
Permeability Assay

Experimental studies on ozonation and chlorination were conducted to determine capacity for inactivating Cryptosporidium parvum oocysts in batch modes at pH 7, 20°C. In both experiments, the log reduction of animal infectivity was linear and clearly decreased as disinfectant CT product increased. However, the curve of reduction in viability determined by both in vitro excystation assay and DAPI/PI permeability assay exhibited a shoulder. The CT products of ozone per 1 log reduction in infectivity were 3 mg middot min/L for 0.5 mg/L and 1.5 mg · min/L for 0.3 mg/L, while viability determined by in vitro excystation was reduced by only 0.2 logs for the CT product of 3 mg · min/L. In the chlorination experiment, thereduction of animal infectivity was up to 3 logs for the CT product of 2,700 mg middot; min/L, while reduction of viability was smaller at 0.16 logs in in vitro excystation and 0.04 logs in DAPI/PI permeability (in PI exclusion) for the same CT product. The CT product of free chlorine per 1 log reduction in infectivity was estimated to be in the range of 800 to 900 mg · min/L.

Development of ATP assay as a surrogate indicator of viability of Cryptosporidium parvum oocysts

2000 ◽  
Vol 41 (7) ◽  
pp. 181-188 ◽  
Author(s):  
I. Somiya ◽  
S. Fujii ◽  
N. Kishimoto ◽  
R-H. Kim
Keyword(s):  
Linear Relationship ◽  
Cryptosporidium Parvum ◽  
Simple Procedure ◽  
Ozone Treatment ◽  
Atp Assay ◽  
Cryptosporidium Oocysts ◽  
Batch Condition ◽  
Cryptosporidium Parvum Oocysts ◽  
Permeability Assay

Adenosine Triphosphate (ATP) determination was applied to evaluate viability of Cryptosporidium oocysts. Three pretreatment methods, such as incubation in acidified Hanks balanced salt solution (HBSS), excystation and sonication were investigated for ATP extraction from oocysts. Incubation in acidified HBSS was insufficient to extract ATP from oocysts, but a linear relationship between the number of oocysts and the concentration of ATP extracted was observed in the test of excystation and sonication treatments. Sonicationtreatment was able to extract ATP from oocysts more rapidly and precisely than excystation treatment. ATP amount per oocyst by sonication treatment (ATPs) was evaluated to be 2.9×10–8 μg on average, andits detection limit was 500 oocysts/100 μl. Ozone treatment experiments were conducted in batch condition to evaluate differences among ATP concentrations extracted, in vitro excystation and DAPI/PI permeability assays. ATPs assay was observed to have a linear relationship with DAPI/PI permeability assay (R2=0.98). As a result, ATP assay is applicable as a surrogate indicator of the viability of C. parvum, and is superior to in vitro excystation and DAPI/PI permeability assay, because of its rapid, accurate and simple procedure.

scholarly journals Transport of Cryptosporidium parvum Oocysts in Soil Columns following Applications of Raw and Separated Liquid Slurries

2012 ◽  
Vol 78 (17) ◽  
pp. 5994-6000 ◽  
Author(s):  
Heidi H. Petersen ◽  
Heidi L. Enemark ◽  
Annette Olsen ◽  
M. G. Mostofa Amin ◽  
Anders Dalsgaard
Keyword(s):  
Cryptosporidium Parvum ◽  
Agricultural Land ◽  
Soil Columns ◽  
Content Type ◽  
Dye Permeability ◽  
Cryptosporidium Parvum Oocysts ◽  
Liquid Slurry ◽  
The Vertical Distribution ◽  
Intact Soil ◽  
Permeability Assay

ABSTRACTThe potential for the transport of viableCryptosporidium parvumoocysts through soil to land drains and groundwater was studied using simulated rainfall and intact soil columns which were applied raw slurry or separated liquid slurry. Following irrigation and weekly samplings over a 4-week period,C. parvumoocysts were detected from all soil columns regardless of slurry type and application method, although recovery rates were low (<1%). Soil columns with injected liquid slurry leached 73 and 90% more oocysts compared to columns with injected and surface-applied raw slurries, respectively. Among leachate samples containing oocysts, 44/72 samples yielded viable oocysts as determined by a dye permeability assay (DAPI [4′,6′-diamidino-2-phenylindole]/propidium iodide) with the majority (41%) of viable oocysts found in leachate from soil columns with added liquid slurry. The number of viable oocysts was positively correlated (r= 0.63) with the total number of oocysts found. Destructively sampling of the soil columns showed that type of slurry and irrigation played a role in the vertical distribution of oocysts, with more oocysts recovered from soil columns added liquid slurry irrespective of the irrigation status. Further studies are needed to determine the effectiveness of different slurry separation technologies to remove oocysts and other pathogens, as well as whether the application of separated liquid slurry to agricultural land may represent higher risks for groundwater contamination compared to application of raw slurry.

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