Manure presents one of the greatest challenges to livestock (dairy and beef cattle, swine, poultry, equine, sheep, llamas, etc.) operations in the Chesapeake Bay Watershed, serving both as resource and liability. The Chesapeake Bay is threatened by excessive nutrient loadings, and, according to the US Environmental Protection Agency (USEPA), manure is the source of 18% of the nitrogen and 27% of the phosphorus entering the Chesapeake Bay annually (figure 1) (Chesapeake Bay Program 2010). Developing economical, practical, and effective manure management options for livestock producers will not only contribute to the restoration of the Chesapeake Bay, but will also provide a model for other areas where water quality and livestock production objectives must be balanced.

ABSTRACT 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 × 108 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 × 105 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 × 108 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.

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.

Human adenovirus (Ads) 40 and 41 are important human enteric pathogens and they are second to rotaviruses as etiological agents of pediatric gastroenteritis. Ads are prevalent in environment and are shed in the feces of infected humans and have been found to accumulate in sludge at ~107 genomic copies/L. Human Ads were also found in sewage samples from animal slaughter houses, probably originating from human contamination. As a result, fresh fruits and vegetables may be contaminated by contact with Ad contaminated water and manures/biosolids. The objective of this study is to understand factors controlling the survival of Ads in agriculture systems. One ml of human Ads 41 (~6.7x105 viral particles (VP)/ml) was added into 2 g biosoilds, or raw poultry litter (RPL), incubated at 20 or 4 °C and analyzed every 10 days. One-step quantitative real-time PCR (qPCR) was used to quantify Ad genomic copies in manure/biosolids. The Most Probable Number Method (MPN) based on one-step RT-PCR results of Ad infected cells was used to detect the infectivity of MNV in manure/biosolids. After 60 days incubation, there was no significant reduction of Ad genomic copies in either biosolids or RPL at 20 or 4 °C, showing that the virus was very stable in these environments. However, only 3 log infectious unit (IU)/ml Ads were detected after 10 days incubation at 20 °C, and after 20 days the infectious virus was below the detection limit (36 IU/ml); and at 4 °C, there was still 2 log IU/ml Ads infectious after 50 days, demonstrating that maintaining of infectivity in biosolids was temperature dependent. In RPL, there was ~3 log IU/ml infectious Ads after 60 days for both 20 and 4 °C. Thus the survival of Ads is dependent upon biosolid type, treatment, and storage conditions. Impact Statement:

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.

Human Adenovirus 41 (Ad41) is an important human enteric pathogen and widely prevalent in the environment. The aim of this study was to assess the survival of Ad41 based on genome stability and infectivity in different types of manure and three types of biosolids. For viral survival studies, Ad41 was added to pelletized poultry litter (PL), alum-treated poultry litter (AL), raw poultry litter (RPL), liquid dairy manure (DM), swine manure (SM), and three types of biosolids 1, 2, 3. All samples were stored at 20 or 4°C and analyzed every 10 days for up to 60 days. Quantification PCR (qPCR) standard curves were generated for PL, AL, biosolids 1, and DM to measure the number of viral genomic copies remaining in the samples. To study the infectivity, all contaminated manure/biosolids samples were added to mammalian cell culture and viral mRNA was detected using one-step RT–PCR. Overall, Ad41 viral genomes were stable at both 20 and 4°C and there was no significant loss of viral DNA after 60 days in PL, AL, biosolids type 1, and DM. However, infectivity was lost almost immediately in high pH biosolids type 2 and 3, and infectivity decreased quickly in DM, with estimated T90 of 4.3 and 8.7 days at 20 and 4°C, respectively. Ad41 had ~1.9 log loss of infectivity after added in SM and biosolids type 1 at day 0, and estimated T90 was 12.5 and 28.6 days for biosolids type 1, and 19.1 and 51.0 days for SM at 20 and 4°C, respectively. Ad41 maintained infectivity in all three poultry litter, and after 60 days incubation, there were significantly more infectious virus in PL, AL, and RPL than biosolids 1, SM, and DM at 20°C.

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.