Pathogen Cryptosporidium Parvum Research Articles

Efficacy of Vegetated Buffer Strips for Retaining Cryptosporidium parvum

Overland and shallow subsurface hydrologic transport of pathogenic Cryptosporidium parvum oocysts from cattle feces into surface drinking water supplies is a major concern on annual grasslands in California's central and southern Sierra Nevada foothills. Soil boxes (0.5 m wide x 1.1 m long x 0.3 m deep) were used to evaluate the ability of grass vegetated buffer strips to retain 2 x 10(8) spiked C. parvum oocysts in 200-g fecal deposits during simulated rainfall intensities of 30 to 47.5 mm/h over 2 h. Buffers were comprised of Ahwahnee sandy loam (coarse-loamy, mixed, active, thermic Mollic Haploxeralfs; 78:18:4 sand to silt to clay ratio; dry bulk density = 1.4 g/cm(3)) set at 5 to 20% land slope, and >/=95% grass cover (grass stubble height = 10 cm; biomass = 900 kg/ha dry weight). Total number of oocysts discharged from each soil box (combined overland and subsurface flow) during the 120-min simulation ranged from 1.5 x 10(6) to 23.9 x 10(6) oocysts. Observed overall mean log(10) reduction of total C. parvum flux per meter of vegetated buffer was 1.44, 1.19, and 1.18 for buffers at 5, 12, and 20% land slope, respectively. Rainfall application rate (mm/h) was strongly associated with oocyst flux from these vegetated buffers, resulting in a decrease of 2 to 4% in the log(10) reduction per meter buffer for every additional mm/h applied to the soil box. These results support the use of strategically placed vegetated buffers as one of several management strategies that can reduce the risk of waterborne C. parvum attributable to extensive cattle grazing on annual grassland watersheds.

Strength in diversity.

A diverse set of genomes is described in this month's column, all of which are involved at some level in interactions with a human host. These range from the eukaryotic intracellular pathogen Cryptosporidium parvum, through the prokaryotic opportunistic pathogens Bacillus cereus and Leptospira interrogans, to the gut commensal Lactobacillus johnsonii. The genomes of these organisms display a wide range of adaptive responses to the challenges of survival in the mammalian host and many other environmental niches.

Population Structures and the Role of Genetic Exchange in the Zoonotic Pathogen Cryptosporidium parvum

Population Structures and the Role of Genetic Exchange in the Zoonotic Pathogen Cryptosporidium parvum

Population structures and the role of genetic exchange in the zoonotic pathogen Cryptosporidium parvum.

Apicomplexan protozoan parasites include some of the most globally important human and animal pathogens, all of which have obligatory sexual cycles in their definitive hosts. Despite their importance and the relevance of understanding the population genetic structure and role of genetic exchange in generating diversity, population genetic analysis has largely been restricted to Plasmodium spp. and Toxoplasma gondii. These species show a considerable diversity of population structure suggesting different strategies for transmission and survival in mammalian hosts. We have undertaken a population genetic analysis of a further apicomplexan species (Cryptosporidium parvum) to extend our understanding of the diversity of genetic structures and test whether it has a clonal population structure. Nothing is known about the population structure of this parasite. We have analyzed 180 parasite isolates from both humans and cattle derived from a single discrete geographical area, using three minisatellite and four microsatellite markers that define 38 multilocus genotypes. Analysis of linkage disequilibria between pairs of loci combined with measures of genetic distance and similarity provides evidence that the sample comprises four genetically isolated populations. One group of human isolates consists primarily of two closely related multilocus genotypes (clonal), while the major subtypes of a second group, common to both humans and animals, show a panmictic population structure. The data provide an important step in understanding the role of genetic exchange in these parasites, which is an essential prerequisite for determining the value of multilocus genotyping for the analysis of sources of human infection as well as future molecular epidemiological studies.

Giardia and Cryptosporidium in pristine protected catchments in central eastern Australia.

We report the first confirmed records of the pathogenic protozoa Giardia lamblia and Cryptosporidium parvum from small remote streams in pristine protected areas in Australia, beyond the reach of urban or agricultural contamination.

Comparing Cryptosporidium AND MS2 Bioassays—implications for UV Reactor validation

The use and selection of microbial surrogates to validate ultraviolet (UV) reactor performance remain a challenge. Proper interpretation of bioassay results requires an appreciation of the relative UV dose–response of the surrogate and target organisms and the limitations surrogates impose. The aim of this study was to improve the understanding of MS2 coliphage's use as a bioassay surrogate for the waterborne pathogen Cryptosporidium parvum. Side‐by‐side MS2 and Cryptosporidium bioassays were performed to directly link MS2 and Cryptosporidium inactivation. At a flow rate of 757 L/min (200 gpm), the low‐pressure, high‐output UV reactor provided > 4.7‐log Cryptosporidium inactivation at an MS2 equivalent dose of 45 mJ/cm2. Although these data support the use of MS2 as a test organism to validate UV reactor performance, MS2 is a very conservative surrogate for Cryptosporidium. Use of a test organism with a more comparable UV dose–response would allow validation of much higher levels of log inactivation.

Environmental loading rates of the waterborne pathogenic protozoa Cryptosporidium parvum in certain domestic and wildlife species in California

Author(s): Atwill, Edward R.; Phillips, Ralph; Rulofson, Franz | Abstract: Waterborne transmission of the pathogenic protozoa Cryptosporidium parvum has emerged as an important public health concern. To develop focused strategies to minimize the risk of waterborne transmission of this parasite to humans or animals, a standardized methodology is needed for comparing environmental loading rates for different populations of vertebrate hosts for C. parvum. A reasonable approximation for an estimate of the environmental loading rate is to measure the prevalence of infection and the intensity of shedding using cross-sectional surveys of the mammalian population, and then multiplying by an estimate of fecal production. We applied this concept to a variety of livestock and wildlife species found throughout California. In general we found that regardless of age, striped skunks, coyotes, California ground squirrels, and yellow-bellied marmots were substantial sources of C. parvum oocysts. In contrast, only the young stock of beef and dairy cattle were substantial sources of oocysts; adult cattle appear to excrete only limited numbers oocysts relative to either calves or wildlife. Watershed management plans that endeavor to minimize contamination of drinking water with C. parvum need to focus on appropriate management of wildlife reservoirs of C. parvum in addition to the traditional concern of animal agriculture.

Experimental investigations and numerical modelling of Cryptosporidium parvum transport behaviour in aquifers

The purpose of this study was to improve understanding of the potential for transfer of the protozoan pathogen Cryptosporidium parvum through aquifers to drinking water wells. Therefore, the factors characterising this transport were experimentally determined. We have developed a continuously recirculating column assay. The latter allows small amounts of C. parvum oocysts to be manipulated providing as much protection as possible from the risks of contamination. As the analysis of oocyst samples is time consuming, a numerical model, simulating the transport phenomena of oocysts under the experimental conditions of assays, was developed to establish the whole experimental curve of results using a small number of experimental points. The comparisons drawn between analytic solutions, experimental results with tracer (NaCl solution) and numerical simulation were in good agreement. A continuously recirculating column assay was performed using oocysts in suspension (flow rate = 1.43 mL/min). Treated sand was used as previous experiments had shown that no adsorption occurs. We observed almost total filtration (99.85%). To check this result, an assay with an open column was carried out under the same conditions. We observed a filtration value of 97%. Consequently, we may say that the continuously recirculating column assay provides satisfactory results.

Interferon gamma induces enterocyte resistance against infection by the intracellular pathogen Cryptosporidium parvum

Interferon (IFN)-gamma plays an important role in the immunologic control of infection by the protozoan enteropathogen Cryptosporidium parvum. We tested the hypothesis that IFN-gamma may directly inhibit infection of enterocytes by this pathogen. HT-29, Caco-2, and H4 human enterocyte cell lines were grown in monolayers and incubated with IFN-gamma before exposure with C. parvum. IFN-gamma receptor expression in the cell lines was determined by Western blot analysis. IFN-gamma inhibited C. parvum infection of both HT-29 and Caco-2 cells but not H4 cells. Response to IFN-gamma was related to the expression of the IFN-gamma receptor in the respective cell lines. The effect of IFN-gamma was partially reversed by inhibition of the JAK/STAT signaling pathway. IFN-gamma mediated its action by at least 2 mechanisms: (1) inhibition of parasite invasion and (2) by modification of intracellular Fe(2+) concentration. No role for tryptophan starvation or nitric oxide synthase activity was found. TNF-alpha and IL-1beta also had anti-C. parvum activity but had no synergistic effect with IFN-gamma. IFN-gamma directly induces enterocyte resistance against C. parvum infection; this observation may have important consequences for our understanding of the mucosal immune response to invasive pathogens.

Identification of Cryptosporidium parvum dihydrofolate reductase inhibitors by complementation in Saccharomyces cerevisiae.

There is a pressing need for drugs effective against the opportunistic protozoan pathogen Cryptosporidium parvum. Folate metabolic enzymes and enzymes of the thymidylate cycle, particularly dihydrofolate reductase (DHFR), have been widely exploited as chemotherapeutic targets. Although many DHFR inhibitors have been synthesized, only a few have been tested against C. parvum. To expedite and facilitate the discovery of effective anti-Cryptosporidium antifolates, we have developed a rapid and facile method to screen potential inhibitors of C. parvum DHFR using the model eukaryote, Saccharomyces cerevisiae. We expressed the DHFR genes of C. parvum, Plasmodium falciparum, Toxoplasma gondii, Pneumocystis carinii, and humans in the same DHFR-deficient yeast strain and observed that each heterologous enzyme complemented the yeast DHFR deficiency. In this work we describe our use of the complementation system to screen known DHFR inhibitors and our discovery of several compounds that inhibited the growth of yeast reliant on the C. parvum enzyme. These same compounds were also potent or selective inhibitors of the purified recombinant C. parvum DHFR enzyme. Six novel lipophilic DHFR inhibitors potently inhibited the growth of yeast expressing C. parvum DHFR. However, the inhibition was nonselective, as these compounds also strongly inhibited the growth of yeast dependent on the human enzyme. Conversely, the antibacterial DHFR inhibitor trimethoprim and two close structural analogs were highly selective, but weak, inhibitors of yeast complemented by the C. parvum enzyme. Future chemical refinement of the potent and selective lead compounds identified in this study may allow the design of an efficacious antifolate drug for the treatment of cryptosporidiosis.

Criteria for evaluation of proposed protozoan detection methods

There has been a proliferation of techniques and methods reported for analysis of water samples to determine the presence of the protozoan pathogens Cryptosporidium parvum and Giardia lamblia. Many of the proposed methods are presented as complete procedures, which include sampling, processing, staining, or detection steps while other methods are not complete. Some proposed methods have been extensively tested in multi-laboratory settings, however, others are still in the developmental stage. A set of evaluation criteria has been developed to evaluate the many proposed methods. These criteria have been applied as an example, to an existing method. These criteria should be useful to individuals attempting to evaluate methods developed for detecting protozoa in water, and conversely, they should serve as a guideline for individuals interested in developing methods, allowing them to gather data with and about their methods, and present this data in a manner that is both logical and easily evaluated.

Electrochemical biosensor for detecting DNA sequences from the pathogenic protozoan Cryptosporidium parvum

An electrochemical hybridization biosensor was developed for the detection of short DNA fragments specific to the deadly waterborne pathogen Cryptosporidium. The sensor relies on the immobilization of a 38-mer oligonucleotide unique to the Cryptosporidium DNA onto the carbon-paste transducer, and employs a highly sensitive chronopotentiometric transduction mode for monitoring the hybridization event. Variables of the probe-immobilization, hybridization and indicator-detection steps are optimized to offer convenient quantitation of ng ml −1 levels of the Cryptosporidium DNA target, in connection with short (3–15 min) hybridization times. The suitability for direct detection of the spiked Cryptosporidium DNA target in untreated drinking and river water samples is demonstrated. Similar performance characteristics are observed at DNA-coated microfabricated thick-film carbon strips. This and similar developments hold great promise for field screening of Cryptosporidium and other microorganisms in environmental samples.

Use of a novel soil tilting table apparatus to demonstrate the horizontal and vertical movement of the protozoan pathogen Cryptosporidium parvum in soil

Use of a novel soil tilting table apparatus to demonstrate the horizontal and vertical movement of the protozoan pathogen Cryptosporidium parvum in soil

Use of a novel soil tilting table apparatus to demonstrate the horizontal and vertical movement of the protozoan pathogen Cryptosporidium parvum in soil

A novel greenhouse based soil tilting table apparatus was used to investigate the potential for movement of the protozoan pathogen Cryptosporidium parvum both through and across a low permeability soil following the application of contaminated livestock waste to land. Soil blocks supported at an angle of 7.5% by the soil table were inoculated at one end with oocyst seeded slurry and subsequently irrigated at regular intervals over a 70-day period. Movement of the pathogen in runoff was demonstrated for at least 21 days and in one case in excess of 70 days from the time of inoculation. Water was also lost following percolation down through the soil profile and significant numbers of oocysts were also lost via this route, average numbers leached decreasing from 8.36±0.56×106 at day 1 to 2.27±0.73×104 at day 70. At the end of the study cores were removed from the soil blocks to determine the location of oocysts remaining within the soil. Numbers decreased down through the soil profile and as the distance from the point of inoculation increased so that 70 cm from the point of inoculation no oocysts could be detected in the soil at any depth. This implies that oocysts contained in runoff stay in the aqueous phase and do not precipitate out onto the soil surface, suggesting that even if the distances travelled are increased there may still be a significant pollution threat.

Movement of the protozoan pathogen Cryptosporidium parvum through three contrasting soil types

The potential for transfer of the protozoan pathogen Cryptosporidium parvum through soil to land drains and, subsequently, water courses following the application of livestock waste to land was monitored in the laboratory using simulated rainfall and intact soil cores. Following irrigation over a 21-day period, Cryptosporidium parvum oocysts applied to the surface of soil cores (initial inoculum concentration 1×108 oocysts core−1) were detected, albeit in low numbers, in the leachates from clay loam and silty loam soils but not in that from a loamy sand soil. Variations in leaching patterns were recorded between replicate cores. At the end of the study soil cores were destructively sampled to establish the location of oocysts remaining within the soil. Distribution within cores was similar in all three soil types. The majority (72.8+-5.2%) of oocysts were found in the top 2 cm of soil, with numbers decreasing with increasing depth to 13.2±2.8%, 8.39±1.4%, and 5.36±1.4% at depths of 10, 20, and 30 cm, respectively.

Macrophage Activation and Innate Resistance to Infection in SCID Mice

Resistance to infection against a variety of pathogens requires the co-ordinated interaction of both the innate and acquired immune responses. Mice bearing the SCID mutation are devoid of T and B cells but retain elements of the innate immune system including natural killer (NK) cells, macrophages, granulocytes and complement proteins. Using the SCID model we have identified a T cell independent mechanism of macrophage activation mediated by the secretion of IFN-γ from NK cells. This process occurs in response to a variety of parasites and bacteria including Listeria monocytogenes, and IS strictly regulated both in vitro and in vivo by cytokines such as IL-2 and IL-10. Here we discuss the mechanisms of NK cell activation and regulation and describe a new model of opportunistic infection in SCID mice with the AIDS related pathogen Cryptosporidium parvum.