Ricardo De Leon

Comparison of In Vitro Cell Culture and a Mouse Assay for Measuring Infectivity of Cryptosporidium parvum

ABSTRACT In vitro cell cultures were compared to neonatal mice for measuring the infectivity of five genotype 2 isolates of Cryptosporidium parvum. Oocyst doses were enumerated by flow cytometry and delivered to animals and cell monolayers by using standardized procedures. Each dose of oocysts was inoculated into up to nine replicates of 9 to 12 mice or 6 to 10 cell culture wells. Infections were detected by hematoxylin and eosin staining in CD-1 mice, by reverse transcriptase PCR in HCT-8 and Caco-2 cells, and by immunofluorescence microscopy in Madin-Darby canine kidney (MDCK) cells. Infectivity was expressed as a logistic transformation of the proportion of animals or cell culture wells that developed infection at each dose. In most instances, the slopes of the dose-response curves were not significantly different when we compared the infectivity models for each isolate. The 50% infective doses for the different isolates varied depending on the method of calculation but were in the range from 16 to 347 oocysts for CD-1 mice and in the ranges from 27 to 106, 31 to 629, and 13 to 18 oocysts for HCT-8, Caco-2, and MDCK cells, respectively. The average standard deviations for the percentages of infectivity for all replicates of all isolates were 13.9, 11.5, 13.2, and 10.7% for CD-1 mice, HCT-8 cells, Caco-2 cells, and MDCK cells, respectively, demonstrating that the levels of variability were similar in all assays. There was a good correlation between the average infectivity for HCT-8 cells and the results for CD-1 mice across all isolates for untreated oocysts (r = 0.85, n = 25) and for oocysts exposed to ozone and UV light (r = 0.89, n = 29). This study demonstrated that in vitro cell culture was equivalent to the “gold standard,” mouse infectivity, for measuring the infectivity of C. parvum and should therefore be considered a practical and accurate alternative for assessing oocyst infectivity and inactivation. However, the high levels of variability displayed by all assays indicated that infectivity and disinfection experiments should be limited to discerning relatively large differences.

Diversity of nitrifying bacteria in full-scale chloraminated distribution systems.

Chloramination for secondary disinfection of drinking water often promotes the growth of nitrifying bacteria in the distribution system due to the ammonia introduced by chloramine formation and decay. This study involved the application of molecular biology techniques to explore the types of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) present in several full-scale chloraminated systems. The results of AOB community characterization indicated the ubiquitous detection of representatives from the Nitrosomonas genus, with Nitrosospira constituting a negligible or small fraction of the AOB community in all but one sample. Cloning and sequencing demonstrated the presence of AOB representatives within the Nitrosomonas oligotropha cluster, a phylogenetic subgroup of AOB from which isolates demonstrate a high affinity for ammonia. For the NOB communities, Nitrospira were detected in most of the samples, while Nitrobacter were only detected in a few samples. These results provide insight into the types of AOB responsible for nitrification episodes in full-scale chloraminated systems, which should help direct future studies aimed at characterizing relevant AOB growth and inactivation properties. Furthermore, the detection of NOB in most of the samples suggests a need to evaluate the contribution of biological nitrite oxidation relative to chemical oxidation in these systems.

UV Inactivation of Cryptosporidium hominis as Measured in Cell Culture

ABSTRACT The Cryptosporidium spp. UV disinfection studies conducted to date have used Cryptosporidium parvum oocysts. However, Cryptosporidium hominis predominates in human cryptosporidiosis infections, so there is a critical need to assess the efficacy of UV disinfection of C. hominis. This study utilized cell culture-based methods to demonstrate that C. hominis oocysts displayed similar levels of infectivity and had the same sensitivity to UV light as C. parvum. Therefore, the water industry can be confident about extrapolating C. parvum UV disinfection data to C. hominis oocysts.

Quantitation of Cryptosporidium parvum infection in cell culture using a colorimetric in situ hybridization assay.

Abstract A quantitative colorimetric in situ hybridization assay was developed for detecting Cryptosporidium parvum infection in cell cultures using a digoxigenin-labeled probe targeting 18S rRNA. Intra-cellular developmental stages of C. parvum such as trophozoites and meronts were clearly discerned by light microscopy as localized areas of dark purple/black precipitate against a colorless background. Infections developed focally and the term infectious focus was applied to each cluster of developmental stages. There were no significant differences in the number of infectious foci following 24 h or 48 h incubation. However, 24 h and 48 h dose response curves were significantly different when infectivity was measured as the number of developmental stages per monolayer, with an average of 5.3-fold more stages following 48 h incubation. When infectivity was expressed as the number of infectious foci per inoculum oocyst converted to a percentage, it was demonstrated that the rate of infection decreased with increasing oocyst age. Oocysts of the Iowa isolate that were 7–10 days old demonstrated 7.8 ± 2.4% infectivity (mean ± standard deviation) compared to 4.2 ± 0.8% for 21–28 day-old oocysts and 1.4 ± 1.3% for 42–70 day-old oocysts. The assay also detected infection with other genotype 2 oocysts and a genoptye 1 isolate. This assay provides a direct quantitative approach for measuring C. parvum infectivity in cell culture.

Development of a Rapid Detection Procedure for Ctyptosporidium, Using In Vitro Cell Culture Combined with PCR

A detection, viability, and infectivity assay was developed for Cryptosporidiurn parvum. Oocysts or excysted sporozoites were inoculated onto monolayers of CaCo‐2 cells grown on chamber slides. C. parvum infection was monitored by three methods: a) application of a fluorescein‐labeled anti‐sporozoite antibody; b) PCR of a heat‐shock protein gene fragment; and c) detection of mRNA from the heat‐shock protein gene by RT‐PCR.

Development of a molecular method for the detection of enteric viruses in oysters

Detection of enteric virus contamination of shellfish is limited by current methodology, which is time-consuming, tedious, and lacking in sensitivity due to reliance on cell culture infectivity. Alternative detection methods based on nucleic acid amplification have been hampered by high sample volumes and the presence of enzymatic inhibitors. The goal of this study was to develop methods to purify and concentrate intact virions from oyster extracts to a volume and quality compatible with viral genomic nucleic acid amplification by reverse transcriptase-polymerase chain reaction (RT-PCR). Fifty-gram oyster samples were homogenized and processed by standard adsorption-elution precipitation methodology and then seeded with 105 PFU of poliovirus 1 (PV1) or hepatitis A virus (HAV). Seeded viruses were concentrated by fluorocarbon extraction, polyethylene glycol (PEG) precipitation, chloroform extraction, and cetyltrimethyl ammonium bromide (CTAB) precipitation to a volume of 100 μl with removal of RT-PCR inhibitors. Virus recovery after elution of PEG precipitates was 50% for PVI and IS to 20% for HAV as evaluted by cell culture infectivity. The CTAB precipitation step yielded a concentrated sample which was directly compatible with RT-PCR reactions and capable of detecting about 100 placque=forming units (PFU) of PVl or HAV. When 50-g oyster extracts were seeded and processed by the entire concentration and purification scheme, direct RT-PCR detection of viral genomic RNA was possible at initial inoculum levels of 104 PFU of HAV and 103 PFU of PV1, with recoveries of 1 to 5% of seeded viruses.

Measuring Inactivation of Cryptosporidium Parvum by In Vitro Cell Culture

Publisher Summary Cell culture-based infectivity of C. parvum is a practical tool that can provide valuable information about the efficacy of disinfectants and the infectivity of oocysts in environmental waters. It is a widely applicable technique that supports the growth of the two primary genotypes of C. parvum and has been shown to be equivalent to a standard mouse assay. Therefore, in vitro cell culture-based assays should be regarded as practical alternatives to the use of animals for measuring the infectivity of C. parvum and assessing the efficacy of disinfectants. To fully assess the public health significance of C. parvum oocysts in water, it is necessary to know whether the oocysts in the environment are infectious. Additionally because of the resistance of C. parvum to standard chlorine disinfection, an increasingly important application of infectivity assays will be to determine the anticryptosporidial efficacy of alternative disinfectants such as ozone and Ultraviolet (UV) light. Human infectivity assays are not practical for use on a routine basis due to the difficulty in obtaining a sufficiently large number of study subjects and potential adverse health effects for volunteers. Mice are the most commonly used animals for C. parvum infectivity assays. The use of animals in scientific research raises ethical concerns, and animal-based assays can be expensive and time consuming with significant hidden costs, such as the maintenance of accredited facilities and license fees. Consequently, infectivity assays based on in vitro cell culture have been developed as an alternative to human- and animal-based assays.

Virulence factor-activity relationships: workshop summary

The concept or notion of virulence factor-activity relationships (VFAR) is an approach for identifying an analogous process to the use of qualitative structure-activity relationships (QSAR) for identifying new microbial contaminants. In QSAR, it is hypothesized that, for new chemical contaminants, their potential acute or chronic toxicity may be reasonably estimated on the basis of structural relationships to other known toxic contaminants. Thus the parallel that is being attempted for pathogenic microorganisms is that known virulence factors may be used as predictors for identifying undiscovered pathogens and microbial causes of emerging diseases. Advances in molecular biology, genomics and proteomics have led the Committee on Drinking Water Contaminants of the National Research Council, as requested by the EPA, to recommend the VFAR approach as a potentially more systematic and scientific process for the selection of microorganisms for inclusion in the Contaminant Candidate List (CCL).

Development of a method for poliovirus detection in freshwater clams

Abstract Consumption of shellfish has been implicated in more than 100 viral gastroenteritis and hepatitis A outbreaks in the United States. Methods for virus detection in marine shellfish have been recently developed. However recent interest in the establishment of freshwater clam fisheries requires the development of methods capable of detecting viral contamination in these species. In this study, freshwater clam homogenates were seeded with poliovirus and detection efficiencies were determined by the adsorption-elution method. Adsorption of poliovirus to clam homogenates was >99·9% at pH 4·5. Recovery efficiency using elution conditions of pH 9·5 and 8000 mg l −1 sodium chloride averaged 55%. Addition of 3% beef extract to the eluent increased overall recoveries to 83%. In summary, the recovery of seeded poliovirus from freshwater clams requires the same basic methodology used for marine species with minor modifications.