For Debate: Impotence in Farm Workers using Toxic Chemicals

Four out of five members of a team of farmworkers who had been using various herbicides and pesticides in intensive agriculture became impotent. Sexual function recovered after further contact with the chemicals was stopped and hormone therapy had been given, though in one case this took about a year. We have not been able to incriminate one particular substance, but with the circumstantial evidence and the lack of any other obvious cause it seems likely that the impotence was due to the toxic effects of one or more of the chemicals being used.     

Identification of Cryptosporidium spp. Oocysts in United Kingdom Noncarbonated Natural Mineral Waters and Drinking Waters by Using a Modified Nested PCR-Restriction Fragment Length Polymorphism Assay

We describe a nested PCR-restriction fragment length polymorphism (RFLP) method for detecting low densities of Cryptosporidium spp. oocysts in natural mineral waters and drinking waters. Oocysts were recovered from seeded 1-liter volumes of mineral water by filtration through polycarbonate membranes and from drinking waters by filtration, immunomagnetizable separation, and filter entrapment, followed by direct extraction of DNA. The DNA was released from polycarbonate filter-entrapped oocysts by disruption in lysis buffer by using 15 cycles of freeze-thawing (1 min in liquid nitrogen and 1 min at 65°C), followed by proteinase K digestion. Amplicons were readily detected from two to five intact oocysts on ethidium bromide-stained gels. DNA extracted from Cryptosporidium parvum oocysts, C. muris (RN 66), C. baileyi (Belgium strain, LB 19), human-derived C. meleagridis, C. felis (DNA from oocysts isolated from a cat), and C. andersoni was used to demonstrate species identity by PCR-RFLP after simultaneous digestion with the restriction enzymes DraI and VspI. Discrimination between C. andersoni and C. muris isolates was confirmed by a separate, subsequent digestion with DdeI. Of 14 drinking water samples tested, 12 were found to be positive by microscopy, 8 were found to be positive by direct PCR, and 14 were found to be positive by using a nested PCR. The Cryptosporidium species detected in these finished water samples was C. parvum genotype 1. This method consistently and routinely detected >5 oocysts per sample.     

Identification of Cryptosporidium Species and Genotypes in Scottish Raw and Drinking Waters during a One-Year Monitoring Period

We analyzed 1,042 Cryptosporidium oocyst-positive slides (456 from raw waters and 586 from drinking waters) of which 55.7% contained 1 or 2 oocysts, to determine species/genotypes present in Scottish waters. Two nested PCR-restriction fragment length polymorphism (RFLP) assays targeting different loci (1 and 2) of the hypervariable region of the 18S rRNA gene were used for species identification, and 62.4% of samples were amplified with at least one of the PCR assays. More samples (577 slides; 48.7% from raw water and 51.3% from drinking water) were amplified at locus 1 than at locus 2 (419 slides; 50.1% from raw water and 49.9% from drinking water). PCR at loci 1 and 2 amplified 45.4% and 31.7% of samples containing 1 or 2 oocysts, respectively. We detected both human-infectious and non-human-infectious species/genotype oocysts in Scottish raw and drinking waters. Cryptosporidium andersoni, Cryptosporidium parvum, and the Cryptosporidium cervine genotype (now Cryptosporidium ubiquitum) were most commonly detected in both raw and drinking waters, with C. ubiquitum being most common in drinking waters (12.5%) followed by C. parvum (4.2%) and C. andersoni (4.0%). Numerous samples (16.6% total; 18.9% from drinking water) contained mixtures of two or more species/genotypes, and we describe strategies for unraveling their identity. Repetitive analysis for discriminating mixtures proved useful, but both template concentration and PCR assay influenced outcomes. Five novel Cryptosporidium spp. (SW1 to SW5) were identified by RFLP/sequencing, and Cryptosporidium sp. SW1 was the fourth most common contaminant of Scottish drinking water (3%).The protozoan parasite Cryptosporidium has been implicated in numerous waterborne and food-borne outbreaks of cryptosporidiosis (3, 6, 16, 17, 18). Currently, there are 22 valid Cryptosporidium species: Cryptosporidium hominis, infecting mainly humans; C. parvum, in humans and numerous other mammals, including cattle; C. andersoni, C. bovis (previously bovine genotype B), and C. ryanae (previously deer-like genotype) in cattle; C. xiaoi (previously bovis-like genotype) in sheep; C. muris in mice; C. felis in cats; C. suis (previously pig genotype I) in pigs; C. wrairi in guinea pigs; C. canis in dogs; C. meleagridis and C. baileyi in birds; C. galli in finches and chickens; C. fayeri (previously marsupial genotype I) and C. macropodum (previously marsupial genotype II) in various species of marsupials; C. fragile in toads; C. varanii (previously C. saurophilum) in lizards and snakes; C. serpentis in snakes; C. scophthalmi and C. molnari in fish (20); and C. ubiquitum (previously Cryptosporidium cervine genotype) in a wide variety of host species, including white-tailed deer, sheep, cattle, goat, mouse, various species of rodents, and humans (4). In addition, there are over 60 Cryptosporidium genotypes, which differ significantly in their molecular sequences but, as yet, have not been ascribed species status (13, 29).Genetic analyses reveal that at least eight species (C. hominis, C. parvum, C. meleagridis, C. felis, C. canis, C. suis, C. muris, and C. ubiquitum) and seven Cryptosporidium genotypes (C. hominis monkey, C. andersoni-like, and Cryptosporidium chipmunk I, skunk, horse, rabbit, and pig genotype II) are associated with human disease (1, 9, 22), but C. parvum and C. hominis remain the most common species infecting humans. Environmental contamination with oocysts of Cryptosporidium species that are not infectious to susceptible human hosts contributes to the difficulties in assessing the risk to public health from waterborne oocysts.Oocysts occur at low densities in water (16, 17, 21), and molecular methods which can genotype small numbers of organisms reliably and reproducibly from water concentrates are required to determine which species occur, and with what frequency, in water. We used our standardized, maximized freezing and thawing method for DNA extraction (10) and our procedure for retrieving oocysts from Cryptosporidium water monitoring slides to maximize DNA extraction for PCR-restriction fragment length polymorphism (RFLP) analysis (11, 12, 19) in this study.We undertook a 1-year survey to identify the species and genotypes of Cryptosporidium oocysts detected in the Scottish Water (SW) Routine Cryptosporidium Monitoring Programme to gain information on the occurrence and diversity of Cryptosporidium oocysts in drinking water sources and drinking waters in order to determine predominant types in water catchment areas and monitor variations in oocyst population distribution over a 1-year period with a view to adding value to current assessments of risk to human health.     

Arsenic Concentrations in the Surface,Well, and Drinking Waters of the Hisarcik,Turkey, Area

Arsenic is one of the most important water pollutants because of its carcinogenicity. The association between arsenical poisoning and the development of internal malignancies and skin cancer is well known. The U.S. Environmental Protection Agency (USEPA) sets maximum contaminant level goals at zero for carcinogens. In this study are presented groundwater arsenic concentrations in the area of naturally rich boron sources of Turkey. Water samples were collected from the Hisarcik, Turkey, area, which has a large boron mine. An inductively coupled plasma/mass spectrometry method was used to analyze arsenic concentrations in water samples. The arsenic levels in water ranged from no detectable amounts to 3.00 mg As/L (mean: 0.46 ± 0.07SD). This mean As level exceeds by a factor of 10 the USEPA's current Maximum Contaminant Level of 0.05 mgAs/L. Some possible health problems associated with consumption of arsenic-contaminated water are discussed and public health interventions proposed.     

Evaluation of Toxic Risk Assessment of Arsenic in Male Subjects Through Drinking Water in Southern Sindh Pakistan

The arsenic (As) hazardous quotient was estimated based on concentration of As in drinking water and scalp hair of male subjects of two age groups (n = 360) consuming As contaminated water at different levels and non-contaminated drinking water. The total As concentrations in drinking water of less-exposed (LE) and high-exposed (HE) areas was found to be 3- to 30-fold higher than the permissible limit of the World Health Organization (2004) for drinking water, while the levels of As in drinking water of non-exposed (NE) areas was within the permissible limit. The levels of As in scalp hair samples of male subjects of two age groups belonging to NE, LE, and HE areas ranged from 0.01 to 0.27, 0.11–1.31, and 0.36–6.80 μg/g, respectively. A significant correlation between As contents of drinking water and As concentration in scalp hair was observed in sub-district Gambit (r = 0.825–0.852, p < 0.001) as compared to those subjects belonging to LE sub-district Thari Mirwah. A toxicity risk assessment provides a hazard quotient corresponding to <10 that indicates non-carcinogenic exposure risk of understudy areas.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> Diversity in Livestock Manures and Agriculturally Impacted Stream Waters

Escherichia coli (E. coli) isolate diversity enhances the likelihood of survival, spread, and/or transmission of the organism among environments. Understanding the ecology of this important organism is requisite for development of more accurate protocols for monitoring and regulatory purposes. In this study, E. coli diversity, gene profiles and transport properties of isolates from different livestock and water sources were evaluated. Strain diversity was evaluated by BOX-PCR, phylotyping, and profiling for 15 genes associated with adhesion, toxin production, iron acquisition or capsular synthesis. Attachment efficiencies were calculated for 17 isolates following transport through saturated porous media. Richness of genotype profiles for livestock isolates was relatively low (25, 12, and 11 for swine, poultry and dairy, respectively) compared to those from stream-water (115 and 126 from dry or wet weather events, respectively). Attachment efficiencies varied by an order of magnitude (0.039–0.44) and the isolate with the highest attachment efficiency possessed the largest suite of targeted genes including those for adherence (iha, agn43, and fimH), surface exclusion (traT) and the siderophore iroN _E.coli . Variation in E. coli isolates based on temporal and ecological source was found to translate to equally broad ranges in transport efficiency underscoring the large degree of genotypic and phenotypic variation that exists among E. coli isolates. The impact of this diversity on genetic exchange and the concomitant effect on the organisms’ fate and transport under in situ environmental conditions warrant further investigation. These factors also require careful consideration for purposes of modeling, source tracking, and risk assessment.     

A Dilution Technique For The Direct Measurement Of Viral Production: A Comparison In Stratified And Tidally Mixed Coastal Waters

The abundance of heterotrophic bacteria and viruses, as well as rates of viral production and virus-mediated mortality, were measured in Discovery Passage and the Strait of Georgia (British Columbia, Canada) along a gradient of tidal mixing ranging from well mixed to stratified. The abundances of bacteria and viruses were approximately 10(6) and 10(7) mL(-1), respectively, independent of mixing regime. Viral production estimates, monitored by a dilution technique, demonstrated that new viruses were produced at rates of 10(6) and 10(7) mL(-1)h(-1) across the different mixing regimes. Using an estimated burst size of 50 viruses per lytic event, ca. 19 to 27% of the standing stock of bacteria at the stratified stations and 46 to 137% at the deep-mixed stations were removed by viruses. The results suggest that mixing of stratified waters during tidal exchange enhances virus-mediated bacterial lysis. Consequently, viral lysis recycled a greater proportion of the organic carbon required for bacterial growth under non-steady-state compared to steady-state conditions.     

Quantitative PCR Method for Enumeration of Cells of Cryptic Species of the Toxic Marine Dinoflagellate Ostreopsis spp. in Coastal Waters of Japan

Monitoring of harmful algal bloom (HAB) species in coastal waters is important for assessment of environmental impacts associated with HABs. Co-occurrence of multiple cryptic species such as toxic dinoflagellate Ostreopsis species make reliable microscopic identification difficult, so the employment of molecular tools is often necessary. Here we developed new qPCR method by which cells of cryptic species can be enumerated based on actual gene number of target species. The qPCR assay targets the LSU rDNA of Ostreopsis spp. from Japan. First, we constructed standard curves with a linearized plasmid containing the target rDNA. We then determined the number of rDNA copies per cell of target species from a single cell isolated from environmental samples using the qPCR assay. Differences in the DNA recovery efficiency was calculated by adding exogenous plasmid to a portion of the sample lysate before and after DNA extraction followed by qPCR. Then, the number of cells of each species was calculated by division of the total number of rDNA copies of each species in the samples by the number of rDNA copies per cell. To test our procedure, we determined the total number of rDNA copies using environmental samples containing no target cells but spiked with cultured cells of several species of Ostreopsis. The numbers estimated by the qPCR method closely approximated total numbers of cells added. Finally, the numbers of cells of target species in environmental samples containing cryptic species were enumerated by the qPCR method and the total numbers also closely approximated the microscopy cell counts. We developed a qPCR method that provides accurate enumeration of each cryptic species in environments. This method is expected to be a powerful tool for monitoring the various HAB species that occur as cryptic species in coastal waters.