Molecular Detection of Campylobacter spp. and Fecal Indicator Bacteria during the Northern Migration of Sandhill Cranes (Grus canadensis) at the Central Platte River

The risk to human health of the annual sandhill crane (Grus canadensis) migration through Nebraska, which is thought to be a major source of fecal pollution of the central Platte River, is unknown. To better understand potential risks, the presence of Campylobacter species and three fecal indicator bacterial groups (Enterococcus spp., Escherichia coli, and Bacteroidetes) was assayed by PCR from crane excreta and water samples collected during their stopover at the Platte River, Nebraska, in 2010. Genus-specific PCR assays and sequence analyses identified Campylobacter jejuni as the predominant Campylobacter species in sandhill crane excreta. Campylobacter spp. were detected in 48% of crane excreta, 24% of water samples, and 11% of sediment samples. The estimated densities of Enterococcus spp. were highest in excreta samples (mean, 4.6 × 10⁸ cell equivalents [CE]/g), while water samples contained higher levels of Bacteroidetes (mean, 5.1 × 10⁵ CE/100 ml). Enterococcus spp., E. coli, and Campylobacter spp. were significantly increased in river water and sediments during the crane migration period, with Enterococcus sp. densities (∼3.3 × 10⁵ CE/g) 2 to 4 orders of magnitude higher than those of Bacteroidetes (4.9 × 10³ CE/g), E. coli (2.2 × 10³ CE/g), and Campylobacter spp. (37 CE/g). Sequencing data for the 16S rRNA gene and Campylobacter species-specific PCR assays indicated that C. jejuni was the major Campylobacter species present in water, sediments, and crane excreta. Overall, migration appeared to result in a significant, but temporary, change in water quality in spring, when there may be a C. jejuni health hazard associated with water and crops visited by the migrating birds.     

Accumulation and fate of microorganisms and microspheres in biofilms formed in a pilot-scale water distribution system

The accumulation and fate of model microbial "pathogens" within a drinking-water distribution system was investigated in naturally grown biofilms formed in a novel pilot-scale water distribution system provided with chlorinated and UV-treated water. Biofilms were exposed to 1-mum hydrophilic and hydrophobic microspheres, Salmonella bacteriophages 28B, and Legionella pneumophila bacteria, and their fate was monitored over a 38-day period. The accumulation of model pathogens was generally independent of the biofilm cell density and was shown to be dependent on particle surface properties, where hydrophilic spheres accumulated to a larger extent than hydrophobic ones. A higher accumulation of culturable legionellae was measured in the chlorinated system compared to the UV-treated system with increasing residence time. The fate of spheres and fluorescence in situ hybridization-positive legionellae was similar and independent of the primary disinfectant applied and water residence time. The more rapid loss of culturable legionellae compared to the fluorescence in situ hybridization-positive legionellae was attributed to a loss in culturability rather than physical desorption. Loss of bacteriophage 28B plaque-forming ability together with erosion may have affected their fate within biofilms in the pilot-scale distribution system. The current study has demonstrated that desorption was one of the primary mechanisms affecting the loss of microspheres, legionellae, and bacteriophage from biofilms within a pilot-scale distribution system as well as disinfection and biological grazing. In general, two primary disinfection regimens (chlorination and UV treatment) were not shown to have a measurable impact on the accumulation and fate of model microbial pathogens within a water distribution system.     

Environmental inactivation of Cryptosporidium oocysts in catchment soils

AIMS: To generate field-relevant inactivation rates for Cryptosporidium oocysts in soil that may serve as parameter values in models to predict the terrestrial fate and transport of oocysts in catchments. METHODS AND RESULTS: The inactivation of Cryptosporidium oocysts in closed soil microcosms over time was monitored using fluorescence in situ hybridization (FISH) as an estimate of oocyst 'viability'. Inactivation rates for Cryptosporidium in two soils were determined under a range of temperature, moisture and biotic status regimes. Temperature and soil type emerged as significantly influential factors (P < 0.05) for Cryptosporidium inactivation. In particular, temperatures as high as 35 degrees C may result in enhanced inactivation. CONCLUSIONS: When modelling the fate of Cryptosporidium oocysts in catchment soils, the use of inactivation rates that are appropriate for the specific catchment climate and soil types is essential. FISH was considered cost-effective and appropriate for determining oocyst inactivation rates in soil. SIGNIFICANCE AND IMPACT OF THE STUDY: Previous models for predicting the fate of pathogens in catchments have either made nonvalidated assumptions regarding inactivation of Cryptosporidium in the terrestrial environment or have not considered it at all. Field-relevant inactivation data are presented, with significant implications for the management of catchments in warm temperate and tropical environments.     

Biotic Interactions and Sunlight Affect Persistence of Fecal Indicator Bacteria and Microbial Source Tracking Genetic Markers in the Upper Mississippi River

The sanitary quality of recreational waters that may be impacted by sewage is assessed by enumerating fecal indicator bacteria (FIB) (Escherichia coli and enterococci); these organisms are found in the gastrointestinal tracts of humans and many other animals, and hence their presence provides no information about the pollution source. Microbial source tracking (MST) methods can discriminate between different pollution sources, providing critical information to water quality managers, but relatively little is known about factors influencing the decay of FIB and MST genetic markers following release into aquatic environments. An in situ mesocosm was deployed at a temperate recreational beach in the Mississippi River to evaluate the effects of ambient sunlight and biotic interactions (predation, competition, and viral lysis) on the decay of culture-based FIB, as well as molecularly based FIB (Entero1a and GenBac3) and human-associated MST genetic markers (HF183 and HumM2) measured by quantitative real-time PCR (qPCR). In general, culturable FIB decayed the fastest, while molecularly based FIB and human-associated genetic markers decayed more slowly. There was a strong correlation between the decay of molecularly based FIB and that of human-associated genetic markers (r², 0.96 to 0.98; P < 0.0001) but not between culturable FIB and any qPCR measurement. Overall, exposure to ambient sunlight may be an important factor in the early-stage decay dynamics but generally was not after continued exposure (i.e., after 120 h), when biotic interactions tended to be the only/major influential determinant of persistence.     

Diversity and Abundance of Zoonotic Pathogens and Indicators in Manures of Feedlot Cattle in Australia

The occurrence of 10 pathogens and three fecal indicators was assessed by quantitative PCR in manures of Australian feedlot cattle. Most samples tested positive for one or more pathogens. For the dominant pathogens Campylobacter jejuni, Listeria monocytogenes, Giardia spp., Cryptosporidium spp., and eaeA-positive Escherichia coli, 10² to 10⁷ genome copies g⁻¹ (dry weight) manure were recovered.More than 600,000 tons of feedlot cattle manure are generated each year in Australia, which raises concern for potential water, air, and soil contamination (21, 27). Hence, better monitoring and knowledge of the resulting risks are needed (5, 26). Most zoonotic pathogens associated with cattle are well described in the literature, especially those of major health significance, including the bacterial pathogens Campylobacter spp., Listeria monocytogenes, pathogenic Escherichia coli (particularly serotypes O157 and O111), Salmonella enterica, Yersinia spp., Leptospira spp., Coxiella burnetii, Mycobacterium avium subsp. paratuberculosis, and the parasitic protozoa Giardia lamblia and Cryptosporidium parvum (2, 21, 27). While studies of pathogen occurrence in manure are numerous, data suited to quantitatively estimating end user risks are still limited. Few surveys quantify multiple pathogens (11, 12, 14, 28), and none have concurrently measured all 10 above in cattle manure. A further constraint on risk assessment is that most data were generated in North America or Europe, where cli-mate and environment can differ markedly from Australian conditions.Addressing this knowledge gap now appears feasible, as real-time quantitative PCR (qPCR) can be used as an alternative to culture-based methods for quantifying environmental pathogens (7, 23, 29). Improvements in sample preparation and nucleic acid cleanup methods have largely overcome problems associated with the molecular biology-based analysis of fecal matter (22). Further, qPCR can detect stressed, damaged, and otherwise nonculturable cells persisting in a state of dormancy or indeed dead (15, 17, 29). The aim of this paper is to report on a quantitative survey of zoonotic pathogens and indicators in manures from Australian feedlot beef cattle.A total of 128 composited samples (five subsamples each) representing fresh feces (n = 32), pen manure (n = 32), harvested pen manure (n = 28), stockpiled manure (n = 23), composted manure (n = 6), and carcass compost (n = 7) were collected from five cattle feedlots in eastern Australia in the winter/summer of 2009 (13). All samples were assayed for the 10 key pathogens listed above and also fecal indicators (total coliforms, E. coli, and enterococci).     

Soil inactivation of DNA viruses in septic seepage

AIMS: To generate field-relevant inactivation data for incorporation into models to predict the likelihood of viral contamination of surface waters by septic seepage. METHODS AND RESULTS: Inactivation rates were determined for PRD1 bacteriophage and Adenovirus 2 in two catchment soils under a range of temperature, moisture and biotic status regimes. Inactivation rates presented for both viruses were significantly different at different temperatures and in different soil types (alpha = 0.05). Soil moisture generally did not significantly affect virus inactivation rate. Biotic status significantly affected inactivation rates of PRD1 in the loam soil but not the clay-loam soil. Adenovirus 2 was inactivated more rapidly in the loam soil than PRD1 bacteriophage. CONCLUSIONS: Virus inactivation rates incorporated into models should be appropriate for the climate/catchment in question with particular regard to soil type and temperature. Given that PRD1 is similar in size to adenoviruses, yet more conservative with regard to inactivation in soil, it may be a useful surrogate in studies of Adenovirus fate and transport. SIGNIFICANCE AND IMPACT OF THE STUDY: A better understanding of the factors that govern virus fate and transport in catchments would facilitate the design of barrier measures to prevent viral contamination of surface waters by septic seepage.