Rebecca M. Hoffman

Effect of pathogen concentrations on removal of Cryptosporidium and Giardia by conventional drinking water treatment

The presence of waterborne enteric pathogens in municipal water supplies contributes risk to public health. To evaluate the removal of these pathogens in drinking water treatment processes, previous researchers have spiked raw waters with up to 10(6) pathogens/L in order to reliably detect the pathogens in treated water. These spike doses are 6-8 orders of magnitude higher than pathogen concentrations routinely observed in practice. In the present study, experiments were conducted with different sampling methods (i.e., grab versus continuous sampling) and initial pathogen concentrations ranging from 10(1) to 10(6) pathogens/L. Results showed that Cryptosporidium oocyst and Giardia cyst removal across conventional treatment were dependent on initial pathogen concentrations, with lower pathogen removals observed when lower initial pathogen spike doses were used. In addition, higher raw water turbidity appeared to result in higher log removal for both Cryptosporidium oocysts and Giardia cysts.

Detection and Resolution of Cryptosporidium Species and Species Mixtures by Genus-Specific Nested PCR-Restriction Fragment Length Polymorphism Analysis, Direct Sequencing, and Cloning

ABSTRACT Molecular methods incorporating nested PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene of Cryptosporidium species were validated to assess performance based on limit of detection (LoD) and for detecting and resolving mixtures of species and genotypes within a single sample. The 95% LoD was determined for seven species (Cryptosporidium hominis, C. parvum, C. felis, C. meleagridis, C. ubiquitum, C. muris, and C. andersoni) and ranged from 7 to 11 plasmid template copies with overlapping 95% confidence limits. The LoD values for genomic DNA from oocysts on microscope slides were 7 and 10 template copies for C. andersoni and C. parvum, respectively. The repetitive nested PCR-RFLP slide protocol had an LoD of 4 oocysts per slide. When templates of two species were mixed in equal ratios in the nested PCR-RFLP reaction mixture, there was no amplification bias toward one species over another. At high ratios of template mixtures (>1:10), there was a reduction or loss of detection of the less abundant species by RFLP analysis, most likely due to heteroduplex formation in the later cycles of the PCR. Replicate nested PCR was successful at resolving many mixtures of Cryptosporidium at template concentrations near or below the LoD. The cloning of nested PCR products resulted in 17% of the cloned sequences being recombinants of the two original templates. Limiting-dilution nested PCR followed by the sequencing of PCR products resulted in no sequence anomalies, suggesting that this method is an effective and accurate way to study the species diversity of Cryptosporidium, particularly for environmental water samples, in which mixtures of parasites are common.

Identification and Characterization of Two Subpopulations of Encephalitozoon intestinalis

ABSTRACT Microsporidia are obligate intracellular protozoa that have been shown to be pathogenic to most living creatures. The development of in vitro cell culture propagation methods has provided researchers with large numbers of spores and facilitated the study of these organisms. Here, we describe heterogeneity within cell culture-propagated Encephalitozoon intestinalis suspensions. Flow cytometer histograms depicting the log side scatter and forward-angle light scatter of spores from nine suspensions produced over 12 months consistently showed two populations differing in size. The suspensions were composed primarily of the smaller-spore subpopulation (76.4% ± 5.1%). The presence of two subpopulations was confirmed by microscopic examination and image analysis (P < 0.001). Small subpopulation spores were noninfectious in rabbit kidney (RK13) cell culture infectivity assays, while the large spores were infectious when inocula included ≥25 spores. The small spores stained brilliantly with fluorescein isothiocyanate-conjugated monoclonal antibody against Encephalitozoon genus spore wall antigen, while the large spores stained poorly. There was no difference in staining intensities using commercial (MicroSporFA) and experimental polyclonal antibodies. Vital-dye (DAPI [4′,6′-diamidino-2-phenylindole], propidium iodide, or SYTOX Green) staining showed the spores of the small subpopulation to be permeable to all vital dyes tested, while spores of the large subpopulation were not permeable in the absence of ethanol pretreatment. PCR using primers directed to the 16S rRNA or β-tubulin genes and subsequent sequence analysis confirmed both subpopulations as E. intestinalis. Our data suggest that existing cell culture propagation methods produce two types of spores differing in infectivity, and the presence of these noninfective spores in purified spore suspensions should be considered when designing disinfection and drug treatment studies.

Microbes Continue to be a Detection Challenge (PDF)

Federal Public quality ical quality regulation Health began standards in Service 1914 of drinking set for when bacteriologdrinking the water US Federal quality began in 1914 when the US Public Health Service set bacteriologica quality standards for dr king water. By consensus or fate JOURNAL. AWWA began publishing that same year. continues to grow within the United States, the number of organisms capable of initiating disease in humans through a waterborne route is expected to rise. Between 1971 and 1988, the Centers for Disease Control and Prevention (CDC) reported more than 687 outbreaks of disease associated with drinking water but recognizes that this number is likely to be a gross underestimation. In fact, CDC estiIdentification of water-