Roberto A. Rodríguez

Trans–shell infection by pathogenic micro–organisms reduces the shelf life of non–incubated bird's eggs: a constraint on the onset of incubation?

Many birds initiate incubation before clutch completion, which results in asynchronous hatching. The ensuing within–brood size disparity often places later–hatched nestlings at a developmental disadvantage, but the functional significance of the timing of the onset of incubation is poorly understood. Early incubation may serve to maintain the viability of early–laid eggs, which declines over time owing to the putative effects of ambient temperature. An unexplored risk to egg viability is trans–shell infection by micro–organisms. We experimentally investigated the rate and magnitude of microbial trans–shell infection of the egg, and the relative effects of ambient temperature and micro–organisms on hatching success. We show that infection of egg contents is prevalent and occurs within the time required to lay a clutch. The probability of infection depends on the climatic conditions, the exposure period and the phylogenetic composition of the eggshell microbiota. We also demonstrate that microbial infection and ambient temperature act independently to reduce egg viability considerably. Our results suggest that these two factors could affect the onset of avian incubation in a wide range of environments.

Application of PCR-based methods to assess the infectivity of enteric viruses in environmental samples.

The advent of the PCR has greatly enhanced our ability to detect human enteric viral pathogens in the environment, including water, municipal wastes, sewage, food, air, and fomites ([2][1], [3][2], [59][3], [69][4], [79][5]). This is especially true for those viruses which do not grow in cell

Norovirus outbreak associated with undercooked oysters and secondary household transmission.

During December 2009, over 200 individuals reported gastrointestinal symptoms after dining at a North Carolina restaurant. An outbreak investigation included a case-control study of restaurant patrons, a secondary household transmission study, environmental assessment of the restaurant facilities and operations, and laboratory analysis of stool and food samples. Illness was primarily associated with consumption of steamed oysters (odds ratio 12, 95% confidence interval 4·8-28) and 20% (8/41 households) reported secondary cases, with a secondary attack rate of 14% among the 70 susceptible household contacts. Norovirus RNA was detected in 3/5 stool specimens from ill patrons; sequencing of RT-PCR products from two of these specimens identified identical genogroup II genotype 12 sequences. Final cooked temperatures of the steamed oysters were generally inadequate to inactivate norovirus, ranging from 21°C to 74°C. Undercooked contaminated oysters pose a similar risk for norovirus illness as raw oysters and household contacts are at risk for secondary infection.

Evaluation of positively charged alumina nanofibre cartridge filters for the primary concentration of noroviruses, adenoviruses and male-specific coliphages from seawater.

Aim:  To evaluate the electropositive, alumina nanofibre (NanoCeram) cartridge filter as a primary concentration method for recovering adenovirus, norovirus and male‐specific coliphages from natural seawater.

Reducing the effects of environmental inhibition in quantitative real-time PCR detection of adenovirus and norovirus in recreational seawaters

Quantitative real-time PCR (qPCR) is used commonly to detect adenovirus (Ads) and norovirus (Nvs) in recreational waters. However, qPCR detection may be limited by interference from inhibitory substances found in recreational waters. In previous work, viruses in Avalon and Doheny Beach water samples were concentrated by electropositive cartridge filtration and PEG precipitation, and high inhibition was found in the samples when using qPCR for detection of Ads and Nvs. Therefore, different approaches were evaluated for removal or blocking of inhibitory compounds that affect qPCR. Avalon and Doheny concentrates were spiked with known amounts of Ads 41 and Nvs GII, and spiked deionized water was used as a positive control. Modifications included gel chromatography with columns of Sephadex G-200/Chelex 100, different sample volumes for nucleic acid extraction, organic solvent extraction, and nucleic acid precipitation. The efficiency of each treatment varied according to sampling location and virus type. The best option for improved Nvs detection by reverse transcription-qPCR was to reduce the sample volume for nucleic acid purification. The best option for improving Ads detection in both beach samples was Sephadex/Chelex spin column chromatography. Chloroform extraction only improved virus detection in Doheny Beach samples but not in Avalon Beach samples. Observed differences in effective treatments between viruses may be related to the different PCR targets, amplification conditions, and enzymes used in each assay, and differences between beaches may be related to differences in PCR inhibitory environmental compounds at each location. The results suggest that methods for detecting viruses from marine beaches, including treatments for the removal of PCR inhibitory compounds, should be optimized for each sampling site and probably for each virus of interest.

Comparison of BGM and PLC/PRC/5 Cell Lines for Total Culturable Viral Assay of Treated Sewage

ABSTRACT The objective of this study was to compare PLC/PRF/5 and BGM cell lines for use in a total culturable viral assay (TCVA) of treated sewage effluents. Samples were collected before and after chlorination from an activated sludge wastewater treatment plant and from the effluent of a high-rate enhanced flocculation system, followed by UV light disinfection. Cell monolayers were observed for cytopathic effect (CPE) after two passages of 14 days each. Monolayers exhibiting viral CPE were tested for the presence of adenoviruses and enteroviruses by PCR or reverse transcription-PCR. Eight percent of the samples exhibited CPE on BGM cells, and 57% showed CPE on PLC/PRF/5 cells. Only enteroviruses were detected on the BGM cells, while 30% and 52% of the samples were positive for enteroviruses and adenoviruses, respectively, on the PLC/PRF/5 cells. Thirty percent of the samples were positive for both adenoviruses and enteroviruses in chlorinated activated sludge effluent. Thirty percent of the samples were positive for adenoviruses in the UV treatment effluent, but no enteroviruses were detected. In conclusion, the PLC/PRF/5 cells were more susceptible than BGM cells to viruses found in treated sewage. The use of BGM cells for TCVA may underestimate viral concentration in sewage effluent samples. The PLC/PRF/5 cells were more susceptible to adenoviruses, which is important in the evaluation of UV disinfection systems because adenoviruses are highly resistant to UV inactivation.

Statistical Analyses: Possible Reasons for Unreliability of Source Tracking Efforts

ABSTRACT Analyses for the presence of indicator organisms provide information on the microbiological quality of water. Indicator organisms recommended by the United States Environmental Protection Agency for monitoring the microbiological quality of water include Escherichia coli, a thermotolerant coliform found in the feces of warm-blooded animals. These bacteria can also be isolated from environmental sources such as the recreational and pristine waters of tropical rain forests in the absence of fecal contamination. In the present study, E. coli isolates were compared to E. coli K12 (ATCC 29425) by restriction fragment length polymorphism using pulsed-field gel electrophoresis. Theoretically, genomic DNA patterns generated by PFGE are highly specific for the different isolates of an organism and can be used to identify variability between environmental and fecal isolates. Our results indicate a different band pattern for almost every one of the E. coli isolates analyzed. Cluster analysis did not show any relations between isolates and their source of origin. Only the discriminant function analysis grouped the samples with the source of origin. The discrepancy observed between the cluster analysis and discriminant function analysis relies on their mathematical basis. Our validation analyses indicate the presence of an artifact (i.e., grouping of environmental versus fecal samples as a product of the statistical analyses used and not as a result of separation in terms of source of origin) in the classification results; therefore, the large genetic heterogeneity observed in these E. coli populations makes the grouping of isolates by source rather difficult, if not impossible.

Inactivation of adenovirus using low-dose UV/H2O2 advanced oxidation.

Adenovirus has consistently been observed to be the most resistant known pathogen to disinfection by ultraviolet light. This has had an impact on regulations set by the United States Environmental Protection Agency regarding the use of UV disinfection for virus inactivation in groundwater and surface water. In this study, enhancement of UV inactivation of adenovirus was evaluated when hydrogen peroxide was added to create an advanced oxidation process (AOP). While 4 log reduction of adenovirus was determined to require a UV dose (UV fluence) of about 200 mJ/cm(2) from a low pressure (LP) UV source (emitting at 253.7 nm), addition of 10 mg/L H(2)O(2) achieved 4 log inactivation at a dose of 120 mJ/cm(2). DNA damage was assessed using a novel nested PCR approach, and similar levels of DNA damage between the two different treatments were noted, suggesting the AOP enhancement in inactivation was not due to additional DNA damage. Hydroxyl radicals produced in the advanced oxidation process are likely able to damage parts of the virus not targeted by LPUV, such as attachment proteins, enhancing the UV-induced inactivation. The AOP-enhanced inactivation potential was modeled in three natural waters. This research sheds light on the inactivation mechanisms of viruses with ultraviolet light and in the presence of hydroxyl radicals and provides a practical means to enhance inactivation of this UV-resistant virus.

Wavelength dependent UV inactivation and DNA damage of adenovirus as measured by cell culture infectivity and long range quantitative PCR.

Adenovirus is regarded as the most resistant pathogen to ultraviolet (UV) disinfection due to its demonstrated resistance to monochromatic, low-pressure (LP) UV irradiation at 254 nm. This resistance has resulted in high UV dose requirements for all viruses in regulations set by the United States Environmental Protection Agency. Polychromatic, medium-pressure (MP) UV irradiation has been shown to be much more effective than 254 nm, although the mechanisms of polychromatic UV inactivation are not completely understood. This research analyzes the wavelength-specific effects of UV light on adenovirus type 2 by analyzing in parallel the reduction in viral infectivity and damage to the viral genome. A tunable laser from the National Institute of Standards and Technology was used to isolate single UV wavelengths. Cell culture infectivity and PCR were employed to quantify the adenoviral inactivation rates using narrow bands of irradiation (<1 nm) at 10 nm intervals between 210 and 290 nm. The inactivation rate corresponding to adenoviral genome damage matched the inactivation rate of adenovirus infectivity at 253.7 nm, 270 nm, 280 nm, and 290 nm, suggesting that damage to the viral DNA was primarily responsible for loss of infectivity at those wavelengths. At 260 nm, more damage to the nucleic acid was observed than reduction in viral infectivity. At 240 nm and below, the reduction of viral infectivity was significantly greater than the reduction of DNA amplification, suggesting that UV damage to a viral component other than DNA contributed to the loss of infectivity at those wavelengths. Inactivation rates were used to develop a detailed spectral sensitivity or action spectrum of adenovirus 2. This research has significant implications for the water treatment industry with regard to polychromatic inactivation of viruses and the development of novel wavelength-specific UV disinfection technologies.

Long-range quantitative PCR for determining inactivation of adenovirus 2 by ultraviolet light.

An extra‐long‐range quantitative PCR (LR‐qPCR) method was developed for estimating genome damage to adenovirus 2 caused by UV irradiation. The objective was to use LR‐qPCR as a rapid method to determine adenovirus UV inactivation.