No description available.

No description available.

No description available.

M.S.

Enteroviruses affect a broad segment of the population throughout the world and have been suspected to play a major role in waterborne disease for quite some time. The presence of these viruses in drinking water supplies constitutes a major health risk to the population because of their low infectious dose. The monitoring and study of these viruses in the environment have been limited by the current standard detection methodologies. Nucleic acid probe hybridization is a new and effective approach for the study and detection of these viruses in the environment. An important step in the detection of viruses in concentrated water samples by nucleic acid probes is the isolation of the viral genome from the water sample for hybridization. Previously, a series of time consuming organic extract ions was used to isolate viral RNA. This study reports the development of an alternative method for the isolation and preservation of viral RNA in environmental samples. Briefly, the sample is heated in the presence of an RNase inhibitor, and then applied to a hybridization membrane. This procedure has greatly reduced the time and difficulty of the assay while maintaining sensitivity and increasing consistency. This study reports the development and modification of a nonradioactive labeling system for the detection of viruses in water. Nonradioactive labels such as biotin offer several advantages over radioactive labels including unlimited shelf life, reduced cost and time of assay, and elimination of the radiation hazard. However, radioactive labels are generally the more sensitive method of detection. By combining direct and indirect labeling strategies, the sensitivity of this nonradioactive assay has been increased ten-fold. This assay can detect as little as 100 plaque forming units of poliovirus, only one order of magnitude less sensitive than radiolabeled probes. This assay is also ten-fold less sensitive than radiolabeled probes for the detection of enteroviruses in water samples. Nonradioactive probes offer a safe, inexpensive alternative to radiolabeled probes and tissue culture for the detection of viruses in the environment when ultrasensitivity is not required.

Rotaviruses are one of the most significant causes of diarrheal disease in the world. Their presence in groundwater and drinking water supplies constitutes a health risk to the population. The study of rotaviruses in the environment has been hampered by the lack of accessible and consistent detection methodologies. Gene probes and other molecular techniques are a novel approach for the detection of these viruses in water. The feasibility of these new techniques for the detection and study of rotaviruses in the environment has been assessed using the simian SA-11 and the culturable human Wa rotavirus strains as models. Two general approaches have been undertaken consisting of hybridization of probes with genomic RNA and hybridization with mRNA produced by the virion-incorporated transcriptase. Hybridization of gene probes with genomic dsRNA of rotaviruses in environmental concentrates resulted in the detection of 10 4 immunofoci of Wa rotavirus. In vitro transcription serves as an amplification method with sensitivity 100- to 1000-fold greater than when probing for genomic RNA. The sensitivity obtained in Wa-seeded distilled water and environmental concentrates after in vitro transcription is 2 and 20 immunofoci, respectively. Proteins in environmental concentrates decrease the efficiency of probe hybridization by 10-100 fold. Also, transcriptase-inhibiting factors found in environmental samples decrease the production of mRNA. Both proteins and transcriptase-inhibiting factors can be reduced significantly with Sephadex G-200 columns. Passage of environmental concentrate through Sephadex G-200 spun columns, followed by in vitro transcription, was used to detect rotaviruses in environmental samples. Rotaviruses were detected by this combination of techniques in eight of 20 sewage samples, one of 16 tap water samples, five of 32 ground water samples, and two of nine surface water samples. Only one of 17 samples which tested positive with Wa cDNA 4 was positive for non-specific probe binding. The probing of rotavirus mRNA, amplified by the virion-incorporated transcriptase, is a practical and feasible method for monitoring these viruses in the environment.

Contamination of recreational and shellfish waters with fecal pollution is a major water quality issue with associated economic impacts and human health risks. Reliable fecal source identification and rapid, quantitative analyses are essential components of risk assessment. Enteric bacteria that are endemic to specific hosts have a potential role as public health indicators of fecal pollution. Building on previous work to discriminate ruminant and human fecal contamination, we cloned class Bacteroidetes 16S rRNA genes from pig, elk, dog, cat, and seagull fecal DNAs. Unique restriction patterns were identified among clones from each of the host species using Terminal Restriction Fragment Length Polymorphisms (T-RFLP). Clones exhibiting unique patterns were sequenced and analyzed phylogenetically, along with human, horse, and cattle sequences recovered from previous work. The analysis revealed both endemic and cosmopolitan (global) host distributions. The sequence data were used to identify host-specific genetic markers for pig and horse feces, and to design PCR primers that identify these sources of fecal pollution in water. There was a high degree of sequence overlap among the fecal Bacteroidetes of wild and domestic ruminants, and among human, domestic pet, and seagull Bacteroidetes. We compared fecal Bacteroidetes rRNA genes from these hosts using subtractive hybridization, a method that identifies differences between closely related genomes or gene sequences. A Bacteroidetes rDNA marker that distinguishes elk and cow feces was identified, as well as a host-specific marker for dog fecal Bacteroidetes. The four newly designed PCR primers were tested for specificity and sensitivity, and the dog primer was successfully used, along with the human and ruminant-specific primers, in a collaborative study comparing fecal source tracking methods. We also developed a real time Taq nuclease assay for quantification of fecal Bacteroidetes 16S rDNA, and compared it with an EPA-approved enumeration method for the current standard public health indicator, Escherichia coli, in serial dilutions of sewage primary influent. There was a strong, positive correlation between the methods, and the Taq nuclease assay was sensitive and much more rapid than the E. coli assay. PCR source identification and enumeration of fecal Bacteroidetes 16S rDNA show promise for application in a health risk-based analysis of fecal pollution. / Graduation date: 2005

published_or_final_version / abstract / toc / Civil Engineering / Master / Master of Philosophy

No description available.

A bench study was conducted to compare two membrane filter techniques for their efficiency in recovering fecal coliforms exposed to selected heavy metals. The effects on recovery by increasing time and metal concentration were also examined. The recovery methods employed included the standard membrane filter technique (S-MF) and a modified membrane filter technique (M-MF)~ which consisted of a lactose agar overlay and a five-hour preincubation at 35 C. The heavy metals Cd, Cr, Pb, and Zn were examined, each at two concentrations, to evaluate their effect on the recovery of the I. coli test organism, after exposure for 6, 24, and 48 hours. A statistical analysis of the data found the recoveries obtained by the M-MF to be significantly greater (.0001 level) than those of the S-MF, in all cases. Time was also found to significantly effect recovery, with recoveries decreasing as time increased. A significant difference was also found between the effects of the heavy metals tested and the concentrations of metals was likewise found to significantly effect recovery, with decreased recoveries being obtained at the high concentration of each metal. / Master of Science