Glass Wool Filters for Concentrating Waterborne Viruses and Agricultural Zoonotic Pathogens

Bacterial indicators are used to indicate increased health risk from pathogens and to make beach closure and advisory decisions; however, beaches are seldom monitored for the pathogens themselves. Studies of sources and types of pathogens at beaches are needed to improve estimates of swimming-associated health risks. It would be advantageous and cost-effective, especially for studies conducted on a regional scale, to use a method that can simultaneously filter and concentrate all classes of pathogens from the large volumes of water needed to detect pathogens. In seven recovery experiments, stock cultures of viruses and protozoa were seeded into 10-liter lake water samples, and concentrations of naturally occurring bacterial indicators were used to determine recoveries. For the five filtration methods tested, the highest median recoveries were as follows: glass wool for adenovirus (4.7%); NanoCeram for enterovirus (14.5%) and MS2 coliphage (84%); continuous-flow centrifugation (CFC) plus Virocap (CFC؉ViroCap) for Escherichia coli (68.3%) and Cryptosporidium (54%); automatic ultrafiltration (UF) for norovirus GII (2.4%); and dead-end UF for Enterococcus faecalis (80.5%), avian influenza virus (0.02%), and Giardia (57%). In evaluating filter performance in terms of both recovery and variability, the automatic UF resulted in the highest recovery while maintaining low variability for all nine microorganisms. The automatic UF was used to demonstrate that filtration can be scaled up to field deployment and the collection of 200-liter lake water samples.

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Comparison of Filters for Concentrating Microbial Indicators and Pathogens in Lake Water Samples

Francy

Stelzer

Brady

et al. 2013

“…Compared to detection of AIV via molecular methods, isolation of viable virus from natural lake water has occurred even less frequently (4,11). Low rates of AIV detection and isolation from natural surface water, combined with recent advances to concentrate large volumes of water for detection of AIV (14)(15)(16), demonstrate that even amid favorable conditions (i.e., high concentrations of aquatic birds and active viral shedding), AIV likely exists at low concentrations in water. As such, any factor that acts to increase virus concentrations and facilitate ingestion by birds could play an important role in transmission.…”

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confidence: 99%

Accumulation and Inactivation of Avian Influenza Virus by the Filter-Feeding Invertebrate Daphnia magna

Meixell

Borchardt

Spencer

2013

Self Cite

The principal mode of avian influenza A virus (AIV) transmission among wild birds is thought to occur via an indirect fecal-oral route, whereby individuals are exposed to virus from the environment through contact with virus-contaminated water. AIV can remain viable for an extended time in water; however, little is known regarding the influence of the biotic community (i.e., aquatic invertebrates) on virus persistence and infectivity in aquatic environments. We conducted laboratory experiments to investigate the ability of an aquatic filter-feeding invertebrate, Daphnia magna, to accumulate virus from AIV-dosed water under the hypothesis that they represent a potential vector of AIV to waterfowl hosts. We placed live daphnids in test tubes dosed with low-pathogenicity AIV (H3N8 subtype isolated from a wild duck) and sampled Daphnia tissue and the surrounding water using reverse transcription-quantitative PCR (RT-qPCR) at 3-to 120-min intervals for up to 960 min following dosing. Concentrations of viral RNA averaged 3 times higher in Daphnia tissue than the surrounding water shortly after viral exposure, but concentrations decreased exponentially through time for both. Extracts from Daphnia tissue were negative for AIV by cell culture, whereas AIV remained viable in water without Daphnia present. Our results suggest daphnids can accumulate AIV RNA and effectively remove virus particles from water. Although concentrations of viral RNA were consistently higher in Daphnia tissue than the water, additional research is needed on the time scale of AIV inactivation after Daphnia ingestion to fully elucidate Daphnia's role as a potential vector of AIV infection to aquatic birds.

“…This method of determining the denominator of the recovery calculation does not invalidate the results and has been used by other researchers in the field (2, 3, 4), including Borchardt (4). In fact, we were not able to find references, other than those associated with Borchardt et al (5,6), in which the concentration of the seed was determined in a seeded negative final concentrate created by filtering unseeded water with the same source and volume as the recovery test.Although for some experiments the recovery trials were done over several days, we took great pains to collect new samples for each trial day and ensure that water quality did not change over the course of the trial (no rain event, high waves, etc.). We also do not believe that mixing seeded and naturally occurring microorganisms makes the filter comparisons uninterpretable, as Borchardt et al suggest.…”

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confidence: 85%

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confidence: 94%

Reply to “Ranking Filter Methods for Concentrating Pathogens in Lake Water”

Bushon¹,

Francy²,

Gallardo³

et al. 2013

A ccurately comparing filtration methods is indeed difficult. Our method (1) and the method described by Borchardt et al. for determining recoveries are both acceptable approaches; however, each is designed to achieve a different research goal. Our study was designed to compare recoveries of multiple microorganisms in surface-water samples. Because, in practice, water-matrix effects come into play throughout filtration, concentration, and detection processes, we felt it important to incorporate those effects into the recovery results.In our study, the concentrations of microorganisms were measured prior to seeding the test sample. The concentrations of the seed organisms were determined in the absence of any matrix effect from the water sample. This method of determining the denominator of the recovery calculation does not invalidate the results and has been used by other researchers in the field (2, 3, 4), including Borchardt (4). In fact, we were not able to find references, other than those associated with Borchardt et al. (5,6), in which the concentration of the seed was determined in a seeded negative final concentrate created by filtering unseeded water with the same source and volume as the recovery test.Although for some experiments the recovery trials were done over several days, we took great pains to collect new samples for each trial day and ensure that water quality did not change over the course of the trial (no rain event, high waves, etc.). We also do not believe that mixing seeded and naturally occurring microorganisms makes the filter comparisons uninterpretable, as Borchardt et al. suggest. All the filters in our trials were treated the same in that they had all had the same matrix effects and the same microorganisms (seeded and naturally occurring). Although there were more trials for enterococci and Escherichia coli for the glass wool and automatic ultrafiltration (UF) (because these filters were used for unseeded controls) and fewer trials for protozoan pathogens for the NanoCeram, we do not believe that the numbers of trials need to be exactly the same to validate our results.We assume that the matrix effect and inhibition comments by Borchardt et al. are in regard to virus analyses since they refer to quantitative PCR (qPCR) determinations of the seed and qPCR inhibition values (which were only measured for viruses). Inhibition of qPCR was measured for each sample in our study. Instead of using hepatitis G virus (HGV) armored RNA as an inhibition control and assuming that inhibition of HGV is similar to that of other viruses, as was done by Lambertini et al. (5), we chose to seed a subsample of the final unseeded concentrate with the actual DNA and RNA viral targets. Multiple dilutions of these control samples were analyzed which permitted us to assess qPCR inhibition and choose an appropriate dilution of test sample to analyze.We do not disagree that the variability rank score (RCV) is an ad hoc measure and that filtration methods with better recoveries are more resistant to shifts in th...