Internalization of murine norovirus 1 by Lactuca sativa during irrigation.

Romaine lettuce (Lactuca sativa) was grown hydroponically or in soil and challenged with murine norovirus 1 (MNV) under two conditions: one mimicking a severe one-time contamination event and another mimicking a lower level of contamination occurring over time. In each condition, lettuce was challenged with MNV delivered at the roots. In the first case, contamination occurred on day one with 5 × 10(8) reverse transcriptase quantitative PCR (RT-qPCR) U/ml MNV in nutrient buffer, and irrigation water was replaced with virus-free buffer every day for another 4 days. In the second case, contamination with 5 × 10(5) RT-qPCR U/ml MNV (freshly prepared) occurred every day for 5 days. Virus had a tendency to adsorb to soil particles, with a small portion suspended in nutrient buffer; e.g., ∼8 log RT-qPCR U/g MNV was detected in soil during 5 days of challenge with virus inoculums of 5 × 10(8) RT-qPCR U/ml at day one, but <6 log was found in nutrient buffer on days 3 and 5. For hydroponically grown lettuce, ∼3.4 log RT-qPCR U of viral RNA/50 mg of plant tissue was detected in some lettuce leaf samples after 5 days at high MNV inoculums, significantly higher than the internalized virus concentration (∼2.6 log) at low inoculums (P < 0.05). For lettuce grown in soil, approximately 2 log RT-qPCR U of viral RNA/50 mg of plant tissue was detected in lettuce with both high and low inoculums, showing no significant difference. For viral infectivity, infectious MNV was found in lettuce samples challenged with high virus inoculums grown hydroponically and in soil but not in lettuce grown with low virus inoculums. Lettuce grown hydroponically was further incubated in 99% and 70% relative humidities (RH) to evaluate plant transpiration relative to virus uptake. More lettuce samples were found positive for MNV at a significantly higher transpiration rate at 70% RH, indicating that transpiration might play an important role in virus internalization into L. sativa.

Survival of murine norovirus and hepatitis A virus in different types of manure and biosolids.

Noroviruses and hepatitis A virus (HAV) are common causes of foodborne disease. They are usually shed in feces and have been found in sewage water, biosolids, and animal manures. With the wide application of manure and biosolids on agricultural lands, there is an increasing interest in investigating virus survival in manure and biosolids. In this study, Murine norovirus-1 (MNV) and HAV were inoculated into different types of animal manure and three types of differently treated biosolids at 20 degrees C and 4 degrees C for up to 60 days. Both HAV and MNV viral genomes degraded immediately in high pH biosolids type 2 and 3 at time zero. For other types of manure and biosolids, HAV RNA was significantly reduced in biosolids type 1 and in liquid dairy manure (DM) after 60 days stored at 20 degrees C, but was stable in all types of manure and biosolids type 1 at 4 degrees C. MNV RNA was unstable in pelletized poultry litter and biosolids type 1 at 20 degrees C, and less stable in liquid DM at both temperatures. For MNV infectivity, there was no significant difference among pelletized poultry litter, alum-treated poultry litter, raw poultry litter, and swine manure at either 20 degrees C or 4 degrees C after 60 days of storage. However, HAV stored in swine manure and raw poultry litter had significantly higher infectivity levels than HAV stored in alum-treated poultry litter at both 20 degrees C and 4 degrees C. Overall, both viruses were inactivated rapidly in alkaline pH biosolids and unstable in liquid DM, but alum added in poultry litter had different effects on the two viruses: alum inactivated some HAV at both temperatures but had no effect on MNV.

Manure- and biosolids-resident murine norovirus 1 attachment to and internalization by Romaine lettuce.

The attachment of murine norovirus 1 (MNV) in biosolids, swine manure, and dairy manure to Romaine lettuce and internalization of this virus were evaluated. The MNV in animal manures had behavior similar to that of pure MNV; however, MNV in biosolids had significantly higher levels of attachment and internalization than pure MNV or MNV in manures. The incubation time did not affect the attachment of MNV in biosolids or manure. Confocal microscopy was used to observe MNV on lettuce after SYBR gold-labeled MNV was added directly to lettuce or after lettuce was submersed in labeled virus. MNV was observed on the lettuce surface, inside open cuts, and occasionally within stomata. In general, lettuce pieces with a long cut on the edge and short cuts on the stem was more likely to contain internalized MNV than intact lettuce pieces, as observed by confocal microscopy; however, while the difference was visible, it was not statistically significant. This study showed that the presence of MNV in biosolids may increase the risk of fresh produce contamination and that the MNV in open cuts and stomata is likely to be protected from sanitization.

Engaging partners to initiate evaluation efforts: tactics used and lessons learned from the prevention research centers program.

BACKGROUND: The Centers for Disease Control and Prevention's (CDC's) Prevention Research Centers (PRC) Program underwent a 2-year evaluation planning project using a participatory process that allowed perspectives from the national community of PRC partners to be expressed and reflected in a national logic model. CONTEXT: The PRC Program recognized the challenge in developing a feasible, useable, and relevant evaluation process for a large, diverse program. To address the challenge, participatory and utilization-focused evaluation models were used. METHODS: Four tactics guided the evaluation planning process: 1) assessing stakeholders' communication needs and existing communication mechanisms and infrastructure; 2) using existing mechanisms and establishing others as needed to inform, educate, and request feedback; 3) listening to and using feedback received; and 4) obtaining adequate resources and building flexibility into the project plan to support multifaceted mechanisms for data collection. CONSEQUENCES: Participatory methods resulted in buy-in from stakeholders and the development of a national logic model. Benefits included CDC's use of the logic model for program planning and development of a national evaluation protocol and increased expectations among PRC partners for involvement. Challenges included the time, effort, and investment of program resources required for the participatory approach and the identification of whom to engage and when to engage them for feedback on project decisions. INTERPRETATION: By using a participatory and utilization-focused model, program partners positively influenced how CDC developed an evaluation plan. The tactics we used can guide the involvement of program stakeholders and help with decisions on appropriate methods and approaches for engaging partners.

Selection of a water-extractable phosphorus test for manures and biosolids as an indicator of runoff loss potential.

The correlation of runoff phosphorus (P) with water-extractable phosphorus (WEP) in land-applied manures and biosolids has spurred wide use of WEP as a water quality indicator. Land managers, planners, and researchers need a common WEP protocol to consistently use WEP in nutrient management. Our objectives were to (i) identify a common WEP protocol with sufficient accuracy and precision to be adopted by commercial testing laboratories and (ii) confirm that the common protocol is a reliable index of runoff P. Ten laboratories across North America evaluated alternative protocols with an array of manure and biosolids samples. A single laboratory analyzed all samples and conducted a separate runoff study with the manures and biosolids. Extraction ratio (solution:solids) was the most important factor affecting WEP, with WEP increasing from 10:1 to 100:1 and increasing from 100:1 to 200:1. When WEP was measured by a single laboratory, correlations with runoff P from packed soil boxes amended with manure and biosolids ranged from 0.79 to 0.92 across all protocol combinations (extraction ratio, filtration method, and P determination method). Correlations with P in runoff were slightly lower but significant when WEP was measured by the 10 labs (r=0.56-0.86). Based on laboratory repeatability and water quality evaluation criteria, we recommend the following common protocol: 100:1 extraction ratio; 1-h shaking and centrifuge 10 min at 1500xg (filter with Whatman #1 paper if necessary); and determining P by inductively coupled plasma-atomic emission spectrometry or colorimetric methods.

Using concept mapping to develop a logic model for the Prevention Research Centers Program.

INTRODUCTION: Concept mapping is a structured conceptualization process that provides a visual representation of relationships among ideas. Concept mapping was used to develop a logic model for the Centers for Disease Control and Prevention's Prevention Research Centers Program, which has a large and diverse group of stakeholders throughout the United States. No published studies have used concept mapping to develop a logic model for a national program. METHODS: Two logic models were constructed using the data from the concept mapping process and program documents: one for the national level and one for the local level. Concept mapping involved three phases: 1) developing questions to generate ideas about the program's purpose and function, 2) gathering input from 145 national stakeholders and 135 local stakeholders and sorting ideas into themes, and 3) using multivariate statistical analyses to generate concept maps. Logic models were refined using feedback received from stakeholders at regional meetings and conferences and from a structured feedback tool. RESULTS: The national concept map consisted of 9 clusters with 88 statements; the local concept map consisted of 11 clusters with 75 statements. Clusters were categorized into three logic model components: inputs, activities, and outcomes. Based on feedback, two draft logic models were combined and finalized into one for the Prevention Research Centers Program. CONCLUSION: Concept mapping provides a valuable data source, establishes a common view of a program, and identifies inputs, activities, and outcomes in a logic model. Our concept mapping process resulted in a logic model that is meaningful for stakeholders, incorporates input from the program's partners, and establishes important program expectations. Our methods may be beneficial for other programs that are developing logic models for evaluation planning.

Serum IgG to heat shock proteins and Porphyromonas gingivalis antigens in diabetic patients with periodontitis.

BACKGROUND: Past studies have reported a correlation between the presence and severity of periodontitis and serum antibody titers to species-specific antigens of Porphyromonas gingivalis or to cross-reactive antigens, such as lipopolysaccharide (LPS) and heat shock proteins (HSP), shared between P. gingivalis and other bacteria. Our recent study of periodontal treatment outcome in insulin-dependent (type 1) diabetes mellitus patients with severe periodontitis (IDDMI/periodontitis) resulted in two key findings: 1. serum glutamic acid decarboxylase autoantibody (GAD65 Ab) levels were significantly associated with periodontal pocket depth change (PDC) and 2. serum IgG titers to P. gingivalis cells were positively associated with GAD65 Ab level in seropositive (GAD65 Ab +) patients. We have therefore hypothesized that profiles of serum autoantibody levels and IgG titers, to P. gingivalis-specific antigens may be useful in assessing risk for refractory periodontitis in such patients. AIM: To determine whether PDC resulting from non-surgical periodontal treatment can be predicted using profiles of baseline IgG titers to P. gingivalis-specific antigens, human HSP, and GAD65. METHODS: PDC was assessed two months after non-surgical periodontal treatment of 7 GAD65 Ab + and 11 GAD65 AbIDDM/periodontitis patients. Pretreatment titers to GAD65, recombinant human heat shock proteins (HSP90, HSP70, and HSP60), and various P. gingivalis antigens were measured using radioligand precipitation or enzyme-linked immunosorbent (ELISA) assays and compared to the same measurements for 154 recent-onset IDDM patients and 46 non-diabetic controls. RESULTS: Median titers (ELISA units) to HSP90 and HSP70 were significantly higher than non-diabetic controls for GAD65 Ab + (p degrees = 0.002) and GAD65 Ab- (p = 0.034) IDDM/periodontitis patients, respectively. Multivariate regression analysis indicated significant partial correlation of PDC with log-transformed titers to HSP90 (r = - 0.62, p = 0.008), HSP70 (r = + 0.62, p = 0.009), GAD65 (r = - 0.60, p = 0.01) and P. gingivalis LPS (r = - 0.5 1, p = 0.04). Furthermore, hierarchical clustering of baseline profiles of log-transformed HSP90, HSP70, and GAD65 Ab titers sorted patients into two distinct clusters with significantly different median PDC (1.45 min, n = 10 vs. 0.65 min, n = 8; p = 0.016, Mann-Whitney). CONCLUSION: Pretreatment profiles of serum antibody titers to HSP90, HSP70, GAD65, and P. gingivalis LPS may be useful for predicting which patients with IDDM/periodontitis will have a poor response to non-surgical periodontal therapy.