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A study conducted to better estimate the annual rate of environmental loading of Cryptosporidium spp. by colonies of California ground squirrels, to better map the distribution of genotypes for the host species is described. Sufficient numbers of oocysts were not available to conduct a mouse infectivity trial, as Sbey03a is a rare type of genotypes in the sample.

Cell culture models implicate increased nitric oxide (NO) synthesis as a cause of mucosal hyperpermeability in intestinal epithelial infection. NO may also mediate a multitude of subepithelial events, including activation of cyclooxygenases. We examined whether NO promotes barrier function via prostaglandin synthesis using Cryptosporidium parvum-infected ileal epithelium in residence with an intact submucosa. Expression of NO synthase (NOS) isoforms was examined by real-time RT-PCR of ileal mucosa from control and C. parvum-infected piglets. The isoforms mediating and mechanism of NO action on barrier function were assessed by measuring transepithelial resistance (TER) and eicosanoid synthesis by ileal mucosa mounted in Ussing chambers in the presence of selective and nonselective NOS inhibitors and after rescue with exogenous prostaglandins. C. parvum infection results in induction of mucosal inducible NOS (iNOS), increased synthesis of NO and PG[E.sub.2], and increased mucosal permeability. Nonselective inhibition of NOS ([N.sup.G]-nitro-L-arginine methyl ester) inhibited prostaglandin synthesis, resulting in further increases in paracellular permeability. Baseline permeability was restored in the absence of NO by exogenous PG[E.sub.2]. Selective inhibition of iNOS [L-[N.sup.6]-(1-iminoethyl)-L-lysine] accounted for ~50% of NOS-dependent PG[E.sub.2] synthesis and TER. Using an entire intestinal mucosa, we have demonstrated for the first time that NO serves as a proximal mediator of PG[E.sub.2] synthesis and barrier function in C. parvum infection. Expression of iNOS by infected mucosa was without detriment to overall barrier function and may serve to promote clearance of infected enterocytes. Cryptosporidium parvum; permeability

All gene-specific transcriptional activators initiate gene transcriptions by binding to promoter sequences and recruiting general transcription factors including TATA-binding protein (TBP) to upstream of targeted genes. Some of them require multiprotein bridging factors (MBFs); for example, the type 1 MBF (MBF1) which interconnects the gene activator with TBP. In this study, the properties of a previously cloned type 1 multiprotein bridging factor (CpMBF1) and a newly identified TBP (CpTBP1) from the apicomplexan Cryptosporidium parvum were investigated. Genes encoding both proteins were differentially expressed as determined by semi-quantitative RT-PCRs during the parasite life cycle, but in different patterns. The highest level of expression of CpMBF1 was in the well-developed intracellular parasites, whereas that of CpTBP1 was found in intact oocysts and late intracellular stages, possibly correlated with the formation of oocysts. Both CpMBF1 and CpTBP1 were expressed as maltose-binding protein fusion proteins. The function of CpTBP1 was confirmed by its ability to bind a biotinylated DNA oligonucleotide containing TATA consensus sequence. The interaction between CpMBF1 and CpTBP1 was also observed by an electrophoretic mobility shift assay. Since little is known about the regulation and control of gene activity in C. parvum, this study may point to a new direction for the study of gene activation associated with the development of the complex life cycle of this parasite

Replication protein A (RPA) is a heterotrimeric complex of single-stranded DNA-binding proteins that play multiple roles in eukaryotic DNA metabolism. The RPA complex is typically composed of heterologous proteins (termed RPA1, RPA2 and RPA3) in animals, plants and fungi, which possess different functions. Previously, two distinct, short-type RPA large subunits (CpRPA1 and CpRPA1B) from the apicomplexan parasite Cryptosporidium parvum were characterized. Here are reported the identification and characterization of a putative middle RPA subunit (CpRPA2) from this unicellular organism. Although the CpRPA2 gene encodes a predicted 40.1 kDa peptide, which is larger than other RPA2 subunits characterized to date, Western blot analysis of oocyst preparations detected a native CpRPA2 protein with a molecular mass of approximately 32 kDa, suggesting that CpRPA2 might undergo post-translational cleavage or the gene was translated at an alternative start codon. Immunofluorescence microscopy using a rabbit anti-CpRPA2 antibody revealed that CpRPA2 protein was mainly distributed in the cytosol (rather than the nuclei) of C. parvum sporozoites. Semi-quantitative RT-PCR data indicated that CpRPA2 was differentially expressed in a tissue culture model with highest expression in intracellular parasites infecting HOT-8 cells for 36 and 60 h. Sequence comparison suggests that RPA2 is a group of poorly conserved proteins. Nonetheless, functional analyses of recombinant proteins confirmed that CpRPA2 is a single-stranded DNA-binding protein and that it could serve as an in vitro phosphorylation target by a DNA-dependent protein kinase. The minimal length of poly(dT) required for CpRPA2 binding is 17 nucleotides, and the DNA-binding capability was inhibited by phosphorylation in vitro. These observations provide additional evidence on the divergence of RPA proteins between C. parvum and host, implying that the parasite DNA replication machinery could be explored as a chemotherapeutic target.

The genome of Cryptosporidium parvum contains a relatively small number of introns, which includes the [beta]-tubulin gene with only a single intron. Recently, it was observed that the intron was not removed from some of the [beta]-tubulin transcripts in the late life cycle stages cultured in vitro. Although normally spliced [beta]-tubulin mRNA was detected in all parasite intracellular stages by RT-PCR (e.g. HCT-8 or Caco-2 cells infected with C. parvum for 12-72 h), at 48-72 h post-infection unprocessed [beta]-tubulin transcripts containing intact introns started to appear in parasite mRNA within infected host cells. The intron-containing transcripts could be detected by fluorescence in situ hybridization (FISH) using an intron-specific probe. The intron-containing [beta]-tubulin transcripts appeared unique to the in vitro-cultured C. parvum, since they were not detected in parasite-infected calves at 72 h. As yet, it is unclear whether the late life cycle stages of C. parvum are partially deficient in intron-splicing or the intron-splicing processes have merely slowed, both of which would allow the detection of intron-containing transcripts. Another possible explanation is that the decay in transcript processing might simply be due to the onset of parasite death. Nonetheless, the appearance of intron-containing transcripts coincides with the arrest of C. parvum development in vitro. This unusual observation prompts speculation that the abnormal intron-splicing of [beta]-tubulin transcripts may be one of the factors preventing complete development of this parasite in vitro. Furthermore, the presence of both processed and unprocessed introns in [beta]-tubulin transcripts in vitro may provide a venue for studying overall mechanisms for intron-splicing in this parasite.

The five member filters consisting of Pall Life Sciences Envirochek (EC) standard filtration, Envirochek high-volume (EC-HV) membrane filters, the Millipore flatbed membrane filter, Sartorius flatbed membrane filter (SMF) and Filta-Max (FM) depth filter were evaluated for studying their effectiveness in the recovery of Crytosporidium parvum oocysts and Giardia lamblia cysts. Results indicate that the Giardia lamblia cysts were recovered at a higher rate as compared to the Crytosporidium parvum oocysts.

Cryptosporidium Oocysts that are present in animal fecal deposits are linked to increases in pathogen concentration in streams and reservoirs. Dispersion and transportation of Cryptosporidium oocysts from fecal pats were studied under simulated rainfall conditions and results are discussed and tabulated.

The genetic diversity of cryptosporidium in reptiles was analyzed using PCR-restriction fragment length polymorphism and sequence analysis of a small subunit rRNA gene. In the study samples of snakes, lizards and tortoises were analyzed and results suggest extensive genetic diversity.

Results demonstrate that about 32.2% of the oocyst-positive raw and 41% of oocyst-positive final water samples do not contain 4',6'-Diamidino-2-phenylindole-positive nuclei. This method based on the presence of one or more fluorescent nuclei in a positive sample provides an enhanced fluorogenic detection method.

Many ducks carry Cryptosporidium and Giardia species but not C. parvum or G. lamblia. Of 69 fecal samples taken from wild ducks in New Mexico, 49% tested positive for Cryptosporidium and 28% tested positive for Giardia. C. parvum and G. lamblia are the species most often associated with illness in humans.

Increased water potential of soil can increase the rate of degradation of Cryptosporidium parvum oocysts at all temperatures. This is important because many animals infected with the parasite may excrete the oocysts on soil in watersheds that supply drinking water.

Several sample preparation methods for extracting high-quality DNA for PCR were evaluated. For Escherichia coli, Xtra Amp tube, Isocode paper, Instagene matrix, and PrepMan matrix methods were all suitable. For Cryptosporidium parvum, Xtra Amp and Instagene methods were suitable.

Capturing Cryptosporidium parvum oocysts in water using the cellulose acetate membrane dissolution filtration system can render the oocysts non-infective. This would make it difficult for researchers to determine the viability of the oocysts.

Monchromatic, low-pressure UV radiation can inactivate Cryptosporidium oocysts and the oocysts do not seem to be capable of repairing the CNA damage. Cryptosporidium is resistant to other forms of disinfection, including chemical disinfection.

Some bacterial spores are more sensitive to chlorine dioxide than Cryptosporidium parvum and therefore should not be used as indicator organisms. In addition, oocyst purification methods may differ, as purified oocysts from three different supplies had different sensitivities to chlorine dioxide.

Some California ground squirrels shed over 50,000 Cryptosporidium parvum oocysts per gram of feces. Males shed greater numbers of oocysts than females, which could facilitate the dissemination of the organism because males are more likely to leave the family colony.

Researchers report the first known use of MALDI-TOF mass spectroscopy to analyze protozoal oocysts. They found reproducible patterns of spectral markers that could distinguish Cryptosporidium parvum from other Cryptosporidium species.

Studies suggest that there is no correlation between age or testosterone treatment and coccidia infection in Dark-eyed Junco males, indicating an absence of a tradeoff between immunological survival and reproduction. Research topics also include peak points for coccidia oocyst shedding in Junco feces.

Research has been conducted on the heat shock protein genes. The characterazation of these genes' nucleotide sequences has been carried out.

Research has been conducted on the Crystosporidium isolates' genetic and phylogenetic characterization. Results indicate that dog-derived Crystosporidium isolates have a distinct genotype.

Researchers from the University of Geneva, Switzerland investigated the ability of Rhodococcus opacus to reduce the concentrations of zinc in an external medium.

Results show that Cryptosporidium meleagridis is distinct from others in the family of coccidian parasites and is widely distributed with a broad host range. Data point out that birds are susceptible for these parasite.

Research was conducted to quantify infectious Cryptosporidium parvum oocysts using the most-probable-number (MPN) method of enumerating microorganisms. The approach involved creating a dilution series, incubating the samples for an adequate period, plating in replicate and scoring the presence of growth. Results provide evidence that the focus detection method-MPN approach is an excellent and reproducible assay for quantifying oocyst infectivity in vitro.

A direct immunofluorescence assay (DFA) was contrasted with an immunomagnetic separation (IMS) assay combined with immunofluorescent microscopy for their ability to detect low concentrations of Cryptosporidium parvum oocysts in adult bovine feces. IMS-DFA resulted in a 2-log-unit increase in sensitivity compared to DFA alone. The higher sensitivity of the IMS-DFA resulted from testing 2 g of fecal material instead of the 13 to 19 mg of fecal material tested in the DFA. The increased sensitivity was not attributable to a higher percent recovery.

A two-year study of the survival kinetics of oocysts in field settings on three watershed farms in New York, NY, is reported. Wastes from neonatal calves in these farms were being managed as part of dairy operations. A small-volume sentinel system was used to evaluate the effects of environmental stresses on survival of Cryptosporidium parvum oocysts in soil and animal wastes. Results suggested that manure piles containing C paevum oocysts can hasten inactivation and thus reduce the numbers of infective oocysts before wastes are spread in the soil environment.

A new infection caused by Cryptosporidium felis, aside from known infections with C muris and C parvum, was identfiied in a cow bred on the Bystra farm in northern Poland. The identfication was made on the basis of the size of the oocysts and analysis of the molecular sequence of the variable region of the small-subunit rRNA. This finding provides important insight into the complex host specificity and circulation in the environment of Cryptosporidium species.

Evidence from biological characteristics and host specificity studies show that species differentiation occurs in Cryptosporidium parasites. This observation was further supported by a phylogenetic analysis of the parasite genus Cryptosporidium wherein the small-subunit rRNA genes of Cryptosporidium parvum, Cryptosporidium muris, Cryptosporidium baileyi and Cryptosporidium serpentis were characterized. The Cryptosporidium species produced two clades, with Cryptosporidium baileyi and Cryptosporidium parvum belonging to one clade and Cryptosporidium muris and Cryptosporidium serpentis belonging to the other.

The viability of waterborne Cryptosporidium parvum oocysts can be assessed using beta-tubulin mRNA as marker. To prove this observation, rRNA and mRNA were investigated in a reverse transcription-PCR assay as potential markers of oocyst viability. mRNA levels decreased gradually after prolonged incubation of initially viable oocysts at room temperature, which coincided with the loss of oocyst infectivity to neonatal mice under study. The postmortem decay of two mRNA species shows that reverse transcription-PCR analysis can elucidate the viability of Cryptosporidium parvum oocysts.

The waterborne pathogen Cryptosporidium has been identified in surface drinking water supplies. Suspected sources of this pathogen include sewage and the feces of animals, particularly dairy calves. There are many dairy cattle and significant sewage effluent discharges in the Catskill-Delaware watershed that is part of the New York City water supply system. This water supply serves 8,000,000 customers with 5.8 x [10.sup.9] L (1.5 billion gal. of water daily). This paper is concerned with the movement and fate of pathogens from waste-water and dairy sources and the resulting raw water quality for New York City. Manure and Cryptosporidium oocysts are modeled as surface pollutants and assumed to move in response to runoff events in the six watershed-reservoir systems within the Catskill-Delaware watershed. Oocyst degradation in manure and in water is modeled with first-order kinetics. Rudimentary stream routing and reservoir modeling with a first-order decay function complete the fate and transport modeling of oocysts in the watercourse. Reported effluent discharge rates and oocyst concentrations in secondary treated sewage allow estimation of wastewater-derived oocyst contributions. This research highlights the importance of wastewater-derived oocysts, the need for expanded research into oocyst fate in streams and reservoirs, and the concentration of oocysts in sewage effluent.

Research was conducted to evaluate the effect of immunomagnetic separation (IMS) on oocyst infectivity by means of in vitro cell culture assay combined with mRNA extraction and reverse transcriptase PCR. Two IMS kits for Cryptosporidium parvum were compared for recovery of oocysts from environmental samples. Results provide evidence that oocysts that were recovered by IMS retained their infectivity.

The ultrastructure of the protozoon Cryptosporidium parvum's sporozoite was investigated through cryotechnique and energy-filtering transmission electron microscopy. The cryotechnique generated image thick sections of the parasite that preserved cell integrity. In the longitudinal section studied, the cell exhibited the typical conical-shaped apical region which contained most of the organelles. The sporozoite had a lone rhoptry having an organized lamellar body, an absent mitochondria or conventional Golgi apparatus.

Research was conducted to examine the effects of sample turbidity on the recovery of Cryptosporidium parvum oocysts using two immunomagnetic separation (IMS) kits. The objectives of the study was to identify an IMS kit that generated high purified oocyst suspensions, to determine turbidity levels and to use the IMS kit for the evaluation of the effects of age on the recovery of C. parvum oocysts. Results indicate that IMS is a promising alternative to flotation procedures for the recovery of oocysts from turbid water matrices.

Research was conducted to examine the sedimentation velocities of Cryptosporidium parvum oocysts and Giardia lamblia cysts using measurements of density, (oo)cyst size, and viscosity of the sedimentation medium. The objective was to observe whether the Stokes' law can describe the sedimentation kinetics of freely suspended cysts of G. lamblia and oocysts of C. parvum. Results indicate that particle attachment has an influence on the behavior of (oo)cysts in drinking water treatment processes and on their sedimentation in surface water.

Research was conducted to examine the effect of pretreatment and experimental conditions on the hydrophobicity and electrophoretic mobility of Cryptosporidium parvum oocysts. A further objective was to measure the effects of solution ionic strength on the hydrophobicity of polystyrene beads. Results indicate that oocysts purified using the EAPS method displayed an electrophoretic mobility that strongly depended on pH.

A simplified version of the randomly amplified polymorphic DNA technique was used to genetically characterize Cryptosporidium parvum. This approach involved the introduction of a genomic DNA isolation method that used in vitro excystation and specific immunomagnetic separation of Cryptosporidium parvum oocysts. Results indicated that it was effective in isolating an estuary C. parvum isolate from several bovine isolates.

Oysters in natural waters harbor infectious Cryptosporidium parvum oocysts. This was demonstrated in a study of the survival of oocysts of Cryptosporidium parvum in artificial seawater of various salinities and various temperatures. Those placed in artificial seawater at salinities of 10, 20 and 30 ppt at 10 degrees Centigrade and at 10 ppt at 20 degrees Centigrade were infectious after 12 weeks. Oocysts placed in seawater at 20 ppt and 30 ppt at 20 degrees Centigrade were infectious for eight and four weeks, respectively.

The natural production and release of free ammonia may have an impact on the viability of Cryptosporidium parvum oocysts in soil and water microhabitants. A study was conducted to determine how the survival of oocysts is affected by increased free ammonia concentrations and times of exposure. The findings indicate that the inactivation of oocysts in ammonia-containing environments may be increased by environmentally relevant concentrations of free ammonia.

A study was conducted on Asian freshwater clams exposed to water contaminated with infectious Cryptosporidium parvum oocysts. No oocysts were detected in the water 24 hours after the contamination event. The percentage of oocyst-containing clams varied from 20 to 100%, depending on the type of tissue examined and the technique used. Positive samples were obtained when gills and gastrointestinal tracts were processed with immunofluorescent antibody. Clam-released oocysts were surrounded by feces, no free oocysts or oocysts dissociated from fecal matter were observed.

A cross-sectional study was conducted on the risk factors and prevalence of Giardia sp cysts and Cryptosporidium parvum oocsyts in populations of feral pigs in western California. Results show that, of the 221 feral pigs studied, 12 were shedding Cryptosporidium oocysts and 17 were shedding Giardia sp cysts. Results also indicate that feral pigs may cause surface water protozoal contamination.

The capacity of dye permeability assay to identify Cryptosporidium parvum oocysts was examined by comparing the test to an in vitro excystation assay. The assays were used on sodium hypochlorite-permeabilized formaldehyde-fixed and untreated oocysts. Results show that dye permeability viability testing is not based on metabolic activity potential but on oocyst wall permeability. Results also verify the use of dye permeability assay in predicting oocyst infectivity.

An alternative disinfection system composed of an electrochemically produced mixed-oxidant solution was examined for inactivation of both Cryptosporidium parvum oocysts and Clostridium perfringens spores. The disinfection efficacy of the mixed-oxidant solution was compared to that of free chlorine. Findings showed that the mixed-oxidant solution was considerably more effective than free chlorine in inactivating both microorganisms. Free chlorine produced no quantifiable inactivation of C. parvum oocysts and C. perfringens spores.

Sampling the headwaters of tributaries for the parasitic protozoa Giardia and Cryptosporidium presents challenges, especially because calculated limits of detection are dependent on volumes of water filtered with a sampling apparatus. The design for sampling equipment analyzed here is useful for sampling waters of low to moderate sediment-generated turbidity and has been tested in the Catskill Mountains of New York State. It is highly portable and operates under gravity head. It can be left unattended, provided that conditions at sampled sites are favorable for completing collection of a sample. In all field applications, it met or exceeded federally recommended sample volumes. Tests with suspended sediment indicate that the sampler will filter at least 140 L (35 gal.) if sediment-generated turbidities are less than 140 NTU and elevation head is between 1-4 m. If turbidity at sites of interest is related primarily to suspended sediments, this testing method may be useful for raw water sampling.

Cryptosporidium parvum oocysts obtained from the feces of Peking ducks, innoculated with the oocysts, produces severe infection in neonatal mice. Histological sections of the ducks fail to reveal life-cycle stages of the cryptosporidia. The number of oocyst shells in the feces is less than that of intact oocysts. The number of intact oocyst shells decreases from day two to day five post inoculation. This indicates that the viability and infectivity of the oocysts is unaffected by passage through the duck's intestine.

Raw water samples from the black mountain irrigation district, (BMID), and the vernon irrigation district, (VID), contain Giardia cysts. The parasite contamination is higher for the BMID watershed than the VID because cattle can access the creeks in the former. The peak concentration of both parasites coincides with calving activity. The downstream creek of cattle ranch has more Giardia cysts and Cryptosporidium oocysts than the upstream creek. The fecal samples from the two geographically adjacent areas are Giardia positive but Cryptosporidium negative.

The low adhesion of oocysts of the parasite, Cryptosporidium parvum, to octane indicates that Cryptosporidium sp. have low hydrophobic properties. Analysis of hydrophobicity and zeta values indicates that pH and ionic strength affect the hydrophobicity. The maximum adhesion of the oocysts to octane is only 40%. The zeta potentials are pH-dependent and become less negative as pH drops.

The Cryptosporidium DNA recovery-PCR detection method effectively identifies the genomic DNA of the pathogenic protozoa Cryptosporidium in bovine feces. The test is more sensitive than the commercially used Color Vue-Cryptosporidium EIA test. The DNA is extracted by freezing and thawing dilute feces solutions followed by incubation and centrifugation. The PCR amplification involves the hybridization of the DNA with an internal oligonucleotide probe. No harmful wastes are produced during the procedure.

A polymerase chain reaction (PCR) protocol, that involves hybridization with a chemiluminescent probe, detects the oocysts of the parasite Cryptosporidium in environmental samples such as wastewater, surface water, and drinking water. The method is faster and more reliable than the generally used immunofluorescence method. Specimens are prepared for the PCR assay by pretreatment followed by a freeze-thaw procedure. Unlike the immunofluorescence assay, result of this PCR assay is not influenced by empty oocysts.

Some species of algae interfere with tests for the protozoan parasites Giardia and Cryptosporidium by emitting background fluorescence, but goat serum may eliminate this interference when it is added to the test mixture. Researchers tested 54 species of algae for interference with a fluorescence test for the parasites and found that 24 species emitted fluorescence. Goat serum prevented fluorescent emission from most of these species but did not affect the fluorescence of Giardia cysts and Cryptosporidium eggs.