Survival of adenovirus types 2 and 41 in surface and ground waters measured by a plaque assay.

To manage artificial recharge systems, it is necessary to understand the inactivation process of microorganisms within aquifers so that requirements regarding storage times and treatment strategies for ground and surface waters can be developed and modeled to improve water management practices. This study was designed to investigate the survival of representative adenoviruses in surface- and groundwaters using a cell culture plaque assay with human lung carcinoma cells (A549) to enumerate surviving viruses. Adenovirus types 2 (Ad2) and 41 (Ad41) were seeded into 50 mL of three sterilized surface waters and groundwaters, and incubated at 10 and 19 °C for up to 301 days. Concentrations of Ad2 and Ad41 were relatively stable in all waters at 10 °C for at least 160 days and in some instances up to 301 days. At 19 °C, virus concentrations were reduced by 99.99% (4 log) after 301 days in surface water. There was approximately 90% (1 log) reduction of both viruses at 19 °C after 160 days of incubation in groundwater samples. There was no overall difference in survival kinetics in surface waters compared to groundwaters. The relatively high stability and long-term survival of adenoviruses in environmental waters at elevated temperatures should be considered in risk assessment models and drinking water management strategies.

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

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.

Polymorphisms in the beta-tubulin gene of Cryptosporidium parvum differentiate between isolates based on animal host but not geographic origin.

Polymerase chain reaction primers were designed to target a region of the Cryptosporidium parvum beta-tubulin gene spanning an intron. Amplification products contained 11 polymorphic positions, representing a sequence divergence of 1.8%, which discriminated between isolates of C. parvum found solely in humans (genotype 1) and those found in humans and animals (genotype 2). Seven of the polymorphic sites were located outside of the intron and the polymorphism between isolates was readily demonstrated by HaeIII restriction digestion. However, all of the sequences from genotype 1 human-derived oocysts isolated in the United States and Australia were conserved. Also, there were no sequence differences between bovine isolates obtained from both continents. Therefore, isolates could not be differentiated based on geographic source of origin.

Evaluation of immunomagnetic separation for recovery of infectious Cryptosporidium parvum oocysts from environmental samples.

Two commercial immunomagnetic separation (IMS) kits for Cryptosporidium were compared for recovery of oocysts from environmental samples. Oocyst recovery efficiencies with the Dynal and Crypto-Scan kits ranged from 62 to 100% and 34 to 74%, respectively, for seeded environmental water concentrates (turbidity of 210 to 11,480 nephelometric turbidity units). Recovery efficiencies were dependent on the mechanism of agitation during the magnetic capture procedure. An assay combining in vitro cell culture and reverse transcriptase PCR demonstrated that oocysts recovered by IMS retained their infectivity.

An assay combining cell culture with reverse transcriptase PCR to detect and determine the infectivity of waterborne Cryptosporidium parvum.

The presence of Cryptosporidium in drinking water supplies is a significant problem faced by the water industry. Although a variety of methods exist for the detection of waterborne oocysts, water utilities currently have no way of assessing the infectivity of detected oocysts and consequently are unable to accurately determine the risks posed to public health by waterborne Cryptosporidium. In this paper, the development of an infectivity assay for waterborne Cryptosporidium parvum is described. Oocysts were inoculated onto monolayers of Caco-2 cells and grown on microscope slides, and infections were detected by C. parvum specific reverse transcriptase PCR of extracted mRNA, targeting the heat shock protein 70 (hsp70) gene. A single infectious oocyst was detected by this experimental procedure. The use of concentrated samples obtained from 250 liters of finished water had no observable effect on the integrity of cell monolayers or on the infectivity of oocysts seeded into the concentrate. Intracellular developmental stages of the parasite were also detected by using fluorescently labeled antibodies. One pair of PCR primers targeting the hsp70 gene was specific for C. parvum, while a second pair recognized all species of Cryptosporidium tested. The C. parvum-specific primers amplified DNA from 1 to 10 oocysts used to seed 65 to 100 liters of concentrated environmental water samples and were compatible with multiplex PCR for the simultaneous detection of C. parvum and Giardia lambia. This paper confirms the utility of PCR for the detection of waterborne C. parvum and, most importantly, demonstrates the potential of an in vitro infectivity assay.

Desulfovibrio profundus sp. nov., a novel barophilic sulfate-reducing bacterium from deep sediment layers in the Japan Sea.

Several strains of a strictly anaerobic, vibrio-shaped or sigmoid, sulfate-reducing bacterium were isolated from deep marine sediments (depth, 80 and 500 m) obtained from the Japan Sea (Ocean Drilling Program Leg 128, site 798B). This bacterium was identified as a member of the genus Desulfovibrio on the basis of the presence of desulfoviridin and characteristic phospholipid fatty acids (iso 17:1 omega 7 and iso 15:0), the small number of growth substrates utilized (lactate, pyruvate, and hydrogen), and 16S rRNA gene sequence analysis data. Based on data for 16S rRNA sequences (1,369 bp), all of the Japan Sea strains were identical to each other and were most closely related to Desulfovibrio salexigens and less closely related to Desulfovibrio desulfuricans (levels of similarity, 91 and 89.6%, respectively). There were, however, considerable phenotypic differences (in temperatures, pressures, and salinities tolerated, growth substrates, and electron donors) between the Japan Sea isolates and the type strains of previously described desulfovibrios, as well as important differences among the Japan Sea isolates. The Japan Sea isolates were active (with sulfide production) over a wide temperature range (15 to 65 degrees C) and a wide sodium chloride concentration range (0.2 to 10%) (moderate halophile), and they were barophiles that were active at pressures up to about 40 MPa (400 atm). The optimum pressures for activity corresponded to the calculated pressures at the depths from which the organisms were isolated (for isolates obtained at depths of 80 and 500 m the optimum activities occurred at 10 and 15 MPa, respectively [100 and 150 atm, respectively]). This confirms that the organisms came from deep sediments and indicates that they are well-adapted for deep sediment conditions, which is consistent with other characteristics (utilization of hydrogen, fermentation, and utilization of ferric iron and organic sulfonates as electron acceptors). We propose that Japan Sea isolate 500-1 is the type strain of a new species, Desulfovibrio profundus.

Comparison of primers and optimization of PCR conditions for detection of Cryptosporidium parvum and Giardia lamblia in water.

Eight pairs of published PCR primers were evaluated for the specific detection of Cryptosporidium parvum and Giardia lamblia in water. Detection sensitivities ranged from 1 to 10 oocysts or cysts for purified preparations and 5 to 50 oocysts or cysts for seeded environmental water samples. Maximum sensitivity was achieved with two successive rounds of amplification and hybridization, with oligonucleotide probes detected by chemiluminescence. Primer annealing temperatures and MgCl2 concentrations were optimized, and the specificities of the primer pairs were determined with closely related species. Some of the primers were species specific, while others were only genus specific. Multiplex PCR for the simultaneous detection of Cryptosporidium and Giardia was demonstrated with primers amplifying 256- and 163-bp products from the 18S rRNA gene of Cryptosporidium and the heat shock protein gene of Giardia, respectively. The results demonstrate the potential utility of PCR for the detection of pathogenic protozoa in water but emphasize the necessity of continued development.

DNA extraction for 16S rRNA gene analysis to determine genetic diversity in deep sediment communities.

A protocol was devised which permitted the extraction of DNA from deep marine sediments up to 503 m below the sea floor. These sediments have been laid down over the last 3 million years. 16S rRNA gene sequences were amplified from the DNA by the polymerase chain reaction. The details of the successful extraction and polymerase chain reaction methodology varied between samples from different depths. This emphasizes the attention to detail required to allow the diversity of bacteria in these deep sediments to be studied.

A simple technique for electroelution of DNA from environmental samples.

A novel method was developed for the extraction of DNA from environmental material. Soil or sediment samples were encapsulated in agarose blocks and, following treatment with lysis reagents, the DNA was extracted by electroelution.

Factors affecting conjugal transfer of plasmids encoding mercury resistance from pure cultures and mixed natural suspensions of epilithic bacteria.

Sixty-five pure cultures of epilithic bacteria were examined for their ability to transfer mercury resistance to Pseudomonas aeruginosa; five isolates transferred plasmids encoding mercury resistance with frequencies ranging from 8.4 x 10(-8) to 2.8 x 10(-3) per recipient. Two of the plasmids, pQM3 and pQM4, encoded narrow-spectrum mercury resistance, pQM3 also encoded streptomycin resistance, and both plasmids were broad host range. Maximum transfer frequencies of epilithic plasmids from pure cultures occurred over the range 10-25 degrees C at 3.5 g C l-1 and with donor to recipient ratios of 0.4-30. Transfer occurred over a range of pH values (pH 5.0-8.0) but the effect of pH was most significant at non-optimal temperature. Anaerobiosis inhibited transfer of one epilithic plasmid, pQM1, but not that of pQM3. Plasmids encoding mercury resistance were also transferred from mixed natural suspensions of epilithic bacteria (MNS) to Pseudomonas spp. on agar in the laboratory. Transfer from MNS occurred over a wide range of environmentally relevant conditions with maximum frequencies (2 x 10(-5) per recipient) after 24 h, at 25 degrees C, pH 5.5-8.0 and on a medium containing 10 g C l-1. The optimal initial cell density of MNS and recipient was 1.7 x 10(5) c.f.u. cm-2 and highest frequencies were obtained with donor to recipient ratios ranging from 1.2 x 10(-1) to 1.7 x 10(-3). Most of the plasmids (54%) from MNS transferred from their original P. aeruginosa transconjugants to a Pseudomonas putida strain, with frequencies ranging from 1.1 x 10(-6) to greater than 1.0 x 10(-1) per recipient. The majority (80%) of the plasmids were larger than 300 kb and all of these large plasmids encoded UV resistance in addition to mercury resistance. Twenty-one plasmids greater than 300 kb were analysed by restriction digests and were shown to be similar, with only minor structural alterations. One of these alterations was associated with the acquisition of streptomycin resistance. Overall, these results suggest that the epilithic bacteria examined possess the potential to transfer mercury resistance within the epilithon under a wide range of environmentally relevant conditions.

Occurrence, transfer and mobilization in epilithic strains of Acinetobacter of mercury-resistance plasmids capable of transformation.

A 7.8 kb plasmid (pQM17) encoding mercury resistance was isolated from two epilithic strains of Acinetobacter calcoaceticus. The plasmid had a broad host range when mobilized by RP1, transferring into Pseudomonas aeruginosa, P. putida, P. fluorescens, Escherichia coli, Proteus vulgaris and Chromobacterium sp. with frequencies ranging from 5.3 x 10(-9) to 4.6 x 10(-4) per recipient. The plasmid could be transferred into A. calcoaceticus BD413 using intact cells of donor and recipient bacteria (i.e. natural transformation) and there was a broad temperature optimum (14-37 degrees C) for transformation. Transformation was as efficient in liquid matings as on plates but there was no effect of pH in the range 5.6-7.9. Maximum transformation frequencies were obtained after 24 h on agar plates containing 3.5-10 g C 1-1 with donor to recipient ratios ranging from 6 to 415.

An accurate method for estimating sizes of small and large plasmids and DNA fragments by gel electrophoresis.

Several regression methods were tested for estimating the sizes of a wide range of plasmids (1.37-312 MDa) and restriction fragments (2.2-14.2 MDa) by agarose gel electrophoresis. The most accurate and least variable method was the multiple regression of log10 molecular size against log10 relative mobility and the reciprocal square root of the relative mobility. This method gave a good fit to all the data with low percentage errors of the molecular size estimates (less than or equal to 3.0 +/- 1.5%). It is suggested that with this method the molecular size of unknown plasmids can be accurately estimated using the plasmids from Escherichia coli V517 and E. coli IR713 as standards.