A multi-tiered approach to assess fecal pollution in an urban watershed: Bacterial and viral indicators and sediment microbial communities.

Development of effective pollution mitigation strategies require an understanding of the pollution sources and factors influencing fecal pollution loading. Fecal contamination of Turkey Creek in Gulfport, Mississippi, one of the nation's most endangered creeks, was studied through a multi-tiered approach. Over a period of approximately two years, four stations across the watershed were analyzed for nutrients, enumeration of E. coli, male-specific coliphages and bioinformatic analysis of sediment microbial communities. The results demonstrated that two stations, one adjacent to a lift station and one just upstream from the wastewater-treatment plant, were the most impacted. The station adjacent to land containing a few livestock was the least impaired. While genotyping of male-specific coliphage viruses generally revealed a mixed viral signature (human and other animals), fecal contamination at the station near the wastewater treatment plant exhibited predominant impact by municipal sewage. Fecal indicator loadings were positively associated with antecedent rainfall for three of four stations. No associations were noted between fecal indicator loadings and any of the nutrients. Taxonomic signatures of creek sediment were unique to each sample station, but the sediment microbial community did overlap somewhat following major rain events. No presence of Escherichia coli (E. coli) or enterococci were found in the sediment. At some of the stations it was evident that rainfall was not always the primary driver of fecal transport. Repeated monitoring and analysis of a variety of parameters presented in this study determined that point and non-point sources of fecal pollution varied spatially in association with treated and/or untreated sewage.

Humoral and Mucosal Immune Responses to Human Norovirus in the Elderly.

BACKGROUND: Most information on mucosal and systemic immune response to norovirus infection is derived from human challenge studies, birth cohort studies, or vaccine trials in healthy adults. However, few data are available on immune responses to norovirus in the elderly. METHODS: To study the mucosal and systemic immune response against norovirus, 43 long-term care facilities were enrolled prospectively in 2010-2014. Baseline saliva samples from 17 facilities, cases and controls up to day 84 from 10 outbreaks, as well as acute and convalescent sera were collected. RESULTS: Norovirus-specific immunoglobulin A (IgA) levels in baseline saliva samples were low and increased in both symptomatic patients and asymptomatic shedders at day 5 after onset during outbreaks. Receiver operating characteristics analysis correctly assigned prior norovirus infection in 23 (92%) of 25 participants. Cases and asymptomatic shedders showed seroconversion for IgG (80%), IgA (78%), and blockade antibodies (87%). Salivary IgA levels strongly correlated with increased convalescent serum IgA titers and blockade antibodies. CONCLUSIONS: Salivary IgA levels strongly correlated with serum IgA titers and blockade antibodies and remained elevated 3 months after a norovirus outbreak. A single salivary sample collected on day 14 could be used to identify recent infection in a suspected outbreak or to monitor population salivary IgA.

Epidemiologic, Virologic, and Host Genetic Factors of Norovirus Outbreaks in Long-term Care Facilities.

BACKGROUND: In the Unites States, long-term care facilities (LTCFs) are the most common setting for norovirus outbreaks. These outbreaks provide a unique opportunity to better characterize the viral and host characteristics of norovirus disease. METHODS: We enrolled 43 LTCFs prospectively to study the epidemiology, virology, and genetic host factors of naturally occurring norovirus outbreaks. Acute and convalescent stool, serum, and saliva samples from cases, exposed and nonexposed controls were collected. Norovirus infection was confirmed using quantitative polymerase chain reaction testing of stool samples or 4-fold increase in serum antibody titers. The presence of histo-blood group antigens (secretor, ABO, and Lewis type) was determined in saliva. RESULTS: Sixty-two cases, 34 exposed controls, and 18 nonexposed controls from 10 norovirus outbreaks were enrolled. Forty-six percent of acute, 27% of convalescent case, and 11% of control stool samples tested norovirus positive. Outbreak genotypes were GII.4 (Den Haag, n = 3; New Orleans, n = 4; and Sydney, n = 2) and GI.1 (n = 1). Viral load in GII.4 Sydney outbreaks was significantly higher than in outbreaks caused by other genotypes; cases and controls shed similar amounts of virus. Forty-seven percent of cases shed virus for ≥ 21 days. Symptomatic infections with GII.4 Den Haag and GII.4 New Orleans were detected among nonsecretor individuals. CONCLUSIONS: Almost half of all symptomatic individuals shed virus for at least 21 days. Viral load was highest in GII.4 viruses that most recently emerged; these viruses also infect the nonsecretor population. These findings will help to guide development of targeted prevention and control measures in the elderly.

Design and assessment of a real time reverse transcription-PCR method to genotype single-stranded RNA male-specific coliphages (Family Leviviridae).

A real-time, reverse transcription-PCR (RT-qPCR) assay was developed to differentiate the four genogroups of male-specific ssRNA coliphages (FRNA) (family Leviviridae). As FRNA display a trend of source-specificity (human sewage or animal waste) at the genogroup level, this assay provides a tool to help identify the origin of fecal contamination. Primers and probes were designed using complete genomic sequences from 29 FRNA phages. The final selection of primer/probe sets were based on (i) ability to amplify a single, specific product, (ii) genogroup specificity, (iii) lack of cross-reactivity, and (iv) experimental reproducibility and sensitivity over a range of target concentrations. Assay time was reduced by using heat-released viral RNA rather than purified RNA. For quality assurance, a custom RNA molecule was employed as an internal, non-competitive control. The usefulness of this method to identify sources of fecal contamination was tested on a total of 49 FRNA phages isolated from various warm-blooded animals, sewage and combined sewage overflow. FRNA phages from animal wastes were genotyped as 86% I, 4% III Q-like and 9% IV. Two sewage isolates typed to genogroup I and combined sewage overflow isolates genotyped as 40% II and 52% III. Primer specificity designed from this comprehensive sequence database may better discriminate FRNA from different sources.

A reverse transcription-PCR assay to distinguish the four genogroups of male-specific (F+) RNA coliphages.

Goals of reducing fecal contamination in recreational, drinking, shellfishing and other waters and accurately assessing risk from exposure can best be attained if tools to distinguish between sources of pollution are available. The male-specific RNA coliphage (FRNA) genogroups display a trend of source specificity. Reverse transcription-PCR (RT-PCR) can be effectively used for genotyping if specific primer sets are designed to be capable of identifying all members within each genogroup. In this study genogroup-specific primer sets were designed using a minimum of 5 to a maximum of 10 complete phage genome sequences from strains in each genogroup. With these primers and employing a heat-release procedure that eliminated the need for RNA purification an RT-PCR method for genotype identification of FRNA phages was developed. The four genogroup-specific primer sets generated discrete PCR amplicon sizes from a variety of environmental FRNA phage strains. Limits of detection, cross-reactivity and/or non-specific binding to strains from other genogroups were evaluated.

Examining requirement for formation of functional Presenilin proteins and their processing events in vivo.

Presenilin (Psn) is a multipass transmembrane protein that functions as the catalytic subunit of gamma-secretase for mediating intramembrane cleavage of type 1 transmembrane proteins. Normally active Psn is in the form of a heterodimer composed by its N-terminal and C-terminal fragments that are generated from a Presenilinase-mediated endoproteolytic cleavage within its large cytosolic loop during assembly of the protease complex. Using the Psn forms that either bypass or disable Presenilinase-mediated endoproteolysis, and a Psn form that has most of the large cytosolic loop deleted, we have established an in vivo system to enable investigations of Psn functional domains in Drosophila. We show that the Presenilinase-mediated endoproteolytic event is not essential for producing Psn activity during animal development, and is regulated by integrity of the large cytosolic loop of Psn in Drosophila. The Psn transgenic flies described here could be applied to a broad range of studies on Psn functioning and its related gamma-secretase activity at any developmental stage.

Aph-1 is required to regulate Presenilin-mediated gamma-secretase activity and cell survival in Drosophila wing development.

Aph-1 is a multipass transmembrane protein and an essential component of the Presenilin (Psn)-mediated gamma-secretase complex. During protease assembly, Aph-1 stabilizes the newly synthesized Psn holoprotein to facilitate generation of the active form of Psn, which is a Psn-NTF/Psn-CTF heterodimer produced through a Presenilinase-initiated endoproteolytic cleavage of the Psn holoprotein. Although it is clear that loss of Aph-1 activity leads to failure of Psn heterodimer formation, little is understood about whether Aph-1 plays a role in regulating gamma-secretase activity in addition to assisting Psn maturation. Using various modified Psn forms that do not require endoproteolysis or have a large deletion of the cytosolic loop, we show that in Drosophila Aph-1 is still required for gamma-secretase activity independent of its role in promoting Psn endoproteolysis. In addition, our results indicate that Aph-1 is required to promote cell survival in the wing imaginal disc; aph-1 mutant cells are lost either through cell death or because of a defect in cell proliferation. This function of Aph-1 is independent of its role in regulating gamma-secretase activity, but possibly involves downregulating the activity of uncleaved Psn holoprotein.