Browsing by Author "Kniel, Kalmia E."

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    Effect of hydrogen peroxide and other protease inhibitors on Cryptosporidium parvum excystation and in vitro development
    Kniel, Kalmia E.; Sumner, Susan S.; Pierson, Merle D.; Zajac, Anne M.; Hackney, Cameron Raj; Fayer, Ronald; Lindsay, David S. (American Society of Parasitology, 2004-08)
    This study was undertaken to observe the effects of hydrogen peroxide on Cryptosporidium parvum oocysts with respect to protease activity in comparison to known protease inhibitors. In assessing the possible mechanisms of action of hydrogen peroxide, treatment effectiveness was analyzed using 3 assays and the potential roles of proteases and cations were considered. Treatment of C. parvum oocysts with hydrogen peroxide inhibited protease activity up to 50% compared with untreated controls. Treatment of oocysts with chemicals that affect sulfhydryls, including N-ethylmaleimide and dithiolthreitol, inhibited protease activity by > 90%. Treatment of oocysts with these chemicals, along with the protease inhibitors, phenylmethylsulfonyl fluoride (PMSF), ethylenediamine-tetraacetic acid, and cystatin, inhibited protease activity as well as in vitro excystation and infection in a cell culture assay. Several mechanisms may result in the successful inhibition of infection and excystation by hydrogen peroxide treatment, including: oxidation of oocyst wall proteins or lipids, chelating of cations necessary for infection, or hydroxyl radical-induced DNA damage to sporozoites, or both.
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    Effect of Metabolic Enzymes on Amylopectin Content and Infectivity of Cryptosporidium parvum
    Hartman, Angela Danielle (Virginia Tech, 2006-11-20)
    Amylopectin granules in Apicomplexan protozoa are hypothesized to be used as an energy source to aid the parasites in surviving in the environment allow latent stages to excyst and release infective stages, allow them to be mobile, invade host cells, and to continue their life cycle. The objective of this project was to determine if parasite glycolytic enzymes: alpha-amylase, amyloglucosidase, enolase, lactate dehydrogenase, and phosphorylase could be used to decrease amylopectin stores and subsequently infectivity of Cryptosporidium parvum oocysts/sporozoites in both fresh oocysts and stored oocysts. In addition, glycolytic enzymes and substrates: glucose, glucose-1-phosphate, and glycogen synthase were investigated to determine if they can be used to increase amylopectin stores and thus increase infectivity to aid in detection/storage of oocysts. Oocysts of Cryptosporidium parvum were suspended in 1mg/ml glycolytic enzymes or substrates (except glucose - 0.05M and glycogen synthase - 1U/ml) and electroporated. Oocysts were incubated at 37°C for one hour to allow treatments to react with amylopectin followed by incubation on HCT-8 cells for 24 hours for infection. Real-time PCR and immunohistochemistry were performed to determine the effect of the enzymes on infectivity. An amylopectin assay and excystation assay was performed to determine if the enzymes degraded amylopectin and if decreased amylopectin reduced excystation. Alpha amylase and amyloglucosidase had the greatest impact on reducing both amylopectin and infectivity of fresh oocysts with reductions of 99.5% and 99.1% in infective oocysts, respectively (P<0.05). These results suggest that amylopectin may be an important factor in infection, although further research is needed. In stored oocysts, enzymes significantly reduced amylopectin content but not infectivity. In fresh oocysts, amylopectin content was correlated to excystation and infectivity with a decrease in amylopectin correlating to decreased excystation and infectivity. In contrast, there was no direct correlation for stored oocysts. When glucose, glucose-1-phosphate, or glycogen synthase was used to increase infectivity, results show that glycogen synthase had little effect, but glucose and glucose-1-phosphate significantly increased amylopectin content, excystation, and infectivity. In conclusion, amylopectin may be an important polysaccharide store of Cryptosporidium parasites to cause infection by allowing excystation of the oocysts to release infective sporozoites.
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    Evaluation of chemical treatments and ozone on the viability of Cryptosporidium parvum oocysts in fruit juices
    Kniel, Kalmia E. (Virginia Tech, 2002-03-28)
    Cryptosporidium parvum is a protozoan parasite historically associated with waterborne and more recently foodborne outbreaks of diarrheal illness. Contamination of certain foods, such as unpasteurized apple cider, with infective oocysts may occur as oocysts are shed in the feces of common ruminants like cattle and deer that graze in and around orchards. Cryptosporidiosis can result in a severe illness for previously healthy individuals and a life-threatening illness in immunocompromised individuals. Disease occurs after the ingestion of small infective oocysts (4 to 5 mm in size). The relatively thick membrane of the oocysts allows them to be resistant to chlorine and many other environmental pressures, making oocysts difficult to inactivate. In this study, alternative treatments to pasteurization were evaluated for their ability to inhibit C. parvum oocyst viability in fruit juices. Oocyst viability was analyzed with a cell culture infectivity assay, using a human illeocecal cell line (HCT-8) that is most similar to human infection. The percent inhibition of infection by each treatment was determined along with the corresponding log reduction for the treatments found to be most effective. Infection by treated oocysts was compared to that of control untreated oocysts. Cell monolayers were infected with 10⁶ treated oocysts or a series of 10-fold dilutions. Parasitic life stages were visualized using an immunohistochemistry system and 100 microscope fields counted per monolayer. Organic acids and H₂O₂ were added on a wt/vol basis to apple cider, orange juice, and grape juices. Malic, citric, and tartaric acids at concentrations from 1%-5% inhibited C. parvum infectivity of HCT-8 cells by up to 88%. Concentrations ranging from 0.025%-3% H₂O₂ were evaluated where addition of 0.025% H₂O₂ to each juice resulted in a >5 log reduction of C. parvum infectivity as determined with an MPN-based cell culture infectivity assay. Treating apple cider, orange juice, and grape juice with ozone for a time period of 30 seconds up to 15 minutes at 6° and 22°C (0.9 g/L flow rate) inhibited C. parvum viability to > 90% as monitored in the cell culture assay. It is hypothesized that oocyst wall proteins that are necessary for infection are oxidized by the reactive oxygen species generated from the decomposition of the ozone and hydrogen peroxide treatments. These treatments or combinations thereof may offer potential alternatives to traditional pasteurization for fruit juices to successfully inhibit C. parvum viability.
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    Mechanisms Associated with Attachment of Escherichia coli O157:H7 to Lettuce Surfaces
    Boyer, Renee R. (Virginia Tech, 2006-04-07)
    Fresh produce is increasingly associated with foodborne outbreaks. In order to develop effective intervention and measures to reduce microbial risks, it is essential to attain a better understand the mechanisms of attachment of foodborne pathogens to fruits and vegetables. Using lettuce as a model, the attachment of Escherichia coli O157:H7 to produce surfaces was studied. Strains expressing various extracellular proteins (curli, O157-antigen, and intimin) known to influence attachment of E. coli to intestinal cells were evaluated for their physicochemical properties and ability to adhere to cut edge and whole leaf lettuce. Escherichia coli O157:H7 strains included: 0018, 43894 and 43895 (curli producing and non-producing); 86-24 (WT), F-12 (O157-antigen negative), pRFBE (O-antigen replaced on plasmid); and 86-24, 86-24Ã eae10 (intimin negative). The eleven strains were surveyed for their hydrophobicity and cell charge using hydrophobic interaction chromatography (HIC) and electrostatic interaction chromatography (ESIC) techniques. Iceberg lettuce squares (2 x 2 cm) were inoculated with E. coli O157:H7 strains separately (7.0 log CFU/square) and dried in a laminar flow hood. Lettuce was sampled before (unrinsed) and after being rinsed twice with sterile de-ionized water (rinsed). Strips (2 mm wide) of each cut edge of the lettuce were aseptically removed. Cut-edge and whole-leaf samples were homogenized and spiral plated onto Luria-Bertani agar, supplemented with nalidixic acid (50ppm), to assess levels of bacteria remaining on the lettuce leaf after rinsing. The rinse steps were not effective in significantly removing bacteria from lettuce (p>0.05). Curli-producing and non-producing strains preferentially attached to cut edge versus the whole leaf portions of lettuce (p<0.05); however the 86-24 strains showed no preference for attachment. With the exception of 0018 curli-producing and non-producing strains, presence/absence of extracellular proteins surveyed did not influence attachment of E. coli O157:H7 to either cut edge or whole leaf lettuce. There was significantly greater attachment of the curli-producing 0018 strain over the curli non-producing 0018 strain to cut and whole lettuce surfaces (p<0.05). Production of curli and O-polysaccharide significantly increased (p<0.05) the cell's overall hydrophobicity of the cell; however this did not affect attachment (p<0.05). The overall cell charge of all strains was negative; however, charge did not affect attachment of E. coli O157:H7 to lettuce. The presence of extracellular appendages (curli, O157-antigen, intimin) as well as hydrophobicity and cell charge properties had no affect on attachment of the cell to lettuce.