George D. Di Giovanni

Cell culture assays in various formats have been used to study the infectivity of Cryptosporidium spp. as well as to determine the infectivity of naturally occurring oocysts in water. Currently, cell culture assays for infectious Cryptosporidium spp. in water have largely been limited to practice in research laboratories. One obstacle to the routine use of Cryptosporidium cell culture assays for the analysis of water samples is the coordination of water sample collection and processing with readiness of cell culture monolayers. For most Cryptosporidium cell culture assays, monolayers are allowed to develop for 24 to 48 h to reach 80 to 100% confluence prior to inoculation. In this study, we used immunofluorescent assay microscopy to evaluate freshly confluent (2-day-old) and aged (8- to 67-day-old) HCT-8 cell monolayers for their ability to support Cryptosporidium parvum infection. HCT-8 monolayers as old as 67 days were clearly shown to support infection. In two of three experiments, aged monolayers (8- to 11-day-old and 11- to 22-day-old, respectively) developed the same number of C. parvum clusters of infection as freshly confluent monolayers. Results suggest that it may be possible to use cell monolayers from freshly confluent to 3 weeks old on hand for infectivity assays without having to schedule sample processing to coincide with development of freshly confluent monolayers. This would make Cryptosporidium cell culture assays much more feasible for water quality and utility laboratories.

Aims: Four bacterial source tracking (BST) methods, enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC‐PCR), automated ribotyping using HindIII, Kirby‐Bauer antibiotic resistance analysis (KB‐ARA) and pulsed‐field gel electrophoresis (PFGE) were directly compared using the same collection of Escherichia coli isolates. The data sets from each BST method and from composite methods were compared for library accuracy and their ability to identify water isolates. Methods and Results: Potential sources of faecal pollution were identified by watershed sanitary surveys. Domestic sewage and faecal samples from pets, cattle, avian livestock, other nonavian livestock, avian wildlife and nonavian wildlife sources were collected for isolation of E. coli. A total of 2275 E. coli isolates from 813 source samples were screened using ERIC‐PCR to exclude clones and to maximize library diversity, resulting in 883 isolates from 745 samples selected for the library. The selected isolates were further analysed using automated ribotyping with HindIII, KB‐ARA and PFGE. A total of 555 E. coli isolates obtained from 412 water samples were analysed by the four BST methods. A composite data set of the four BST methods gave the highest rates of correct classification (RCCs) with the fewest unidentified isolates than any single method alone. RCCs for the four‐method composite data set and a seven‐way split of source classes ranged from 22% for avian livestock to 83% for domestic sewage. Two‐method composite data sets were also found to be better than individual methods, having RCCs similar to the four‐method composite and identification of the same major sources of faecal pollution. Conclusions: The use of BST composite data sets may be more beneficial than the use of single methods. Significance and Impact of the Study: This is one of the first comprehensive comparisons using composite data from several BST methods. While the four‐method approach provided the most desirable BST results, the use of two‐method composite data sets may yield comparable BST results while providing for cost, labour and time savings.

A field study in the Juarez Valley of Mexico was performed to investigate the potential transmission of Cryptosporidium and Giardia to sheep livestock grazing on forage irrigated with reclaimed wastewater, and the potential for disease transmission back to humans. United States Environmental Protection Agency Method 1623 immunofluorescent assay (IFA) revealed high levels of pathogens in reclaimed wastewater, with 183 to >7000 Giardia cysts and 9 – 762 Cryptosporidium oocysts detected per litre. Infectious Cryptosporidium were detected in the reclaimed wastewater using the cell culture focus detection method (FDM). Polymerase chain reaction (PCR) analyses revealed reclaimed wastewater contained the C. parvum bovine (zoonotic) genotype, human-specific C. hominis subgenotypes, and G. lamblia (syn. G. duodenalis, G. intestinalis) Assemblage A genotypes (A2 and A3). Despite high levels of Cryptosporidium and Giardia in the reclaimed wastewater, these pathogens were rarely found on the forage plants, possibly due to environmental attenuation. Sheep fecal specimens were positive for only livestock-associated G. lamblia Assemblage E genotypes. Therefore, in this field study, there was no evidence of zooanthroponotic transmission of Cryptosporidium or Giardia.

A quantitative TaqMan PCR method was developed for assessing the Cryptosporidium parvum infection of in vitro cultivated human ileocecal adenocarcinoma (HCT-8) cell cultures. This method, termed cell culture quantitative sequence detection (CC-QSD), has numerous applications, several of which are presented. CC-QSD was used to investigate parasite infection in cell culture over time, the effects of oocyst treatment on infectivity and infectivity assessment of different C. parvum isolates. CC-QSD revealed that cell culture infection at 24 and 48 h postinoculation was approximately 20 and 60%, respectively, of the endpoint 72-h postinoculation infection. Evaluation of three different lots of C. parvum Iowa isolate oocysts revealed that the mean infection of 0.1 N HCl-treated oocysts was only 36% of the infection obtained with oocysts treated with acidified Hanks' balanced salt solution containing 1% trypsin. CC-QSD comparison of the C. parvum Iowa and TAMU isolates revealed significantly higher levels of infection for the TAMU isolate, which agrees with and supports previous human, animal, and cell culture studies. CC-QSD has the potential to aid in the optimization of Cryptosporidium cell culture methods and facilitate quantitative evaluation of cell culture infectivity experiments.