[Avian cytogenetics goes functional] Third report on chicken genes and chromosomes 2015

High-density gridded libraries of large-insert clones using bacterial artificial chromosome (BAC) and other vectors are essential tools for genetic and genomic research in chicken and other avian species... Taken together, these studies demonstrate that applications of large-insert clones and BAC libraries derived from birds are, and will continue to be, effective tools to aid high-throughput and state-of-the-art genomic efforts and the important biological insight that arises from them

Evaluation of White Rot Fungus to Control Growth of Escherichia coli in Cattle Manure

Biological soil amendments of animal origin, such as aqueous dairy manure, may be contaminated with microbial pathogens that can subsequently result in contaminated soil, water runoff, and crops. Multiple mitigation strategies are being evaluated to reduce these risks. Inclusion of filamentous fungus in a biofiltration system to inactivate pathogenic bacteria in aqueous dairy manure prior to land application is explored in this study as a preharvest preventative method. Of the microbes used to remediate biologically contaminated sites, ligninolytic white-rot fungi have been previously studied for their ability to degrade a wide variety of toxic or persistent environmental contaminants. Reduction of two E. coli strains (E. coli TVS355 and E. coli O157:H7 4407) was evaluated in aqueous dairy manure and PBS and in the presence of white-rot fungi Pleurotus ostreatus on three different nutrient sources (woodchips (WC), spent mushroom compost (SMC), and reticulated polyurethane foam (RPF)). Overall, E. coli TVS355 was more persistent in aqueous dairy manure and PBS, surviving for 50 days in the presence of P. ostreatus, with a final concentration of 4 log CFU/g in aqueous manure and 7 log CFU/g in PBS. However, greater (p < 0.0001) reduction of E. coli O157:H7 was observed, surviving for 50 days at an average of 4 log CFU/g in aqueous dairy manure and an average of 3 log CFU/g in PBS. Therefore, P. ostreatus has the potential to result in bacterial decay, with potential reduction observed in E. coli O157:H7. The RPF matrix showed positive results as a potential model for a nutrient limiting resource for P. ostreatus and could be the key to increased bacterial reductions if resulting in ligninolytic activity in order to seek other nutrient sources

Microgravity and evasion of plant innate immunity by human bacterial pathogens

Abstract Spaceflight microgravity and modeled-microgravity analogs (MMA) broadly alter gene expression and physiology in both pathogens and plants. Research elucidating plant and bacterial responses to normal gravity or microgravity has shown the involvement of both physiological and molecular mechanisms. Under true and simulated microgravity, plants display differential expression of pathogen-defense genes while human bacterial pathogens exhibit increased virulence, antibiotic resistance, stress tolerance, and reduced LD50 in animal hosts. Human bacterial pathogens including Salmonella enterica and E. coli act as cross-kingdom foodborne pathogens by evading and suppressing the innate immunity of plants for colonization of intracellular spaces. It is unknown if evasion and colonization of plants by human pathogens occurs under microgravity and if there is increased infection capability as demonstrated using animal hosts. Understanding the relationship between microgravity, plant immunity, and human pathogens could prevent potentially deadly outbreaks of foodborne disease during spaceflight. This review will summarize (1) alterations to the virulency of human pathogens under microgravity and MMA, (2) alterations to plant physiology and gene expression under microgravity and MMA, (3) suppression and evasion of plant immunity by human pathogens under normal gravity, (4) studies of plant-microbe interactions under microgravity and MMA. A conclusion suggests future study of interactions between plants and human pathogens under microgravity is beneficial to human safety, and an investment in humanity’s long and short-term space travel goals

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 × 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

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

An evaluation of the virulence and adherence properties of avian pathogenic Escherichia coli

Avian pathogenic E. coli (APEC) cause disease primarly in poultry; however, the link between APEC and infections in humans is questionable. In this current study, a total of 100 APEC strains isolated from chickens in Delmarva were evaluated for the presence of virulence genes to investigate their zoonotic potential in humans. A total of 28 isolates possessed one Enterohaemorrhagic E. coli (EHEC) virulence factor each and 87 isolates possessed up to 5 extraintestinal pathogenic E. coli (ExPEC) virulence factors. Five APEC isolates exhibited stronger attachment to chicken breast than both human E. coli outbreak strains tested. Ten APEC isolates exhibited stronger attachment to human epithelial cells (HCT-8) than both E. coli outbreak strains. While the APEC isolates in this study were not found to possess all the virulence genes necessary to cause clinical illness in humans, their potential to acquire these genes in the environment as well as their ability to attach to food surfaces and human cells warrants further attention

Sensitivity of Escherichia albertii, a Potential Food-Borne Pathogen, to Food Preservation Treatments▿

Escherichia albertii is a potential food-borne pathogen because of its documented ability to cause diarrheal disease by producing attachment and effacement lesions. Its tolerances to heat (56°C), acid (pH 3.0), and pressure (500 MPa [5 min]) were evaluated and found to be significantly less than those of wild-type E. coli O157:H7

Recovery of SARS-CoV-2 from Wastewater Using Centrifugal Ultrafiltration

The COVID-19 pandemic is a global crisis and continues to impact communities as the disease spreads. Clinical testing alone provides a snapshot of infected individuals but is costly and difficult to perform logistically across whole populations. The virus which causes COVID-19, SARS-CoV-2, is shed in human feces and urine and can be detected in human waste. SARS-CoV-2 can be shed in high concentrations (&gt;107 genomic copies/mL) due to its ability to replicate in the gastrointestinal tract of humans through attachment to the angiotensin-converting enzyme 2 (ACE-2) receptors there. Monitoring wastewater for SARS-CoV-2, alongside clinical testing, can more accurately represent the spread of disease within a community. This protocol describes a reliable and efficacious method to recover SARS-CoV-2 in wastewater, quantify genomic RNA levels, and evaluate concentration fluctuations over time. Using this protocol, viral levels as low as 10 genomic copies/mL were successfully detected from 30 mL of wastewater in more than seven-hundred samples collected between August 2020 and March 2021. Through the adaptation of traditional enteric virus methods used in food safety research, targets have been reliably detected with no inhibition of detection (RT-qPCR) observed in any sample processed. This protocol is currently used for surveillance of wastewater systems across New Castle County, Delaware

Zero-valent iron sand filtration reduces concentrations of virus-like particles and modifies virome community composition in reclaimed water used for agricultural irrigation

Abstract Objective Zero-valent iron sand filtration can remove multiple contaminants, including some types of pathogenic bacteria, from contaminated water. However, its efficacy at removing complex viral populations, such as those found in reclaimed water used for agricultural irrigation, has not been fully evaluated. Therefore, this study utilized metagenomic sequencing and epifluorescent microscopy to enumerate and characterize viral populations found in reclaimed water and zero-valent iron-sand filtered reclaimed water sampled three times during a larger greenhouse study. Results Zero-valent iron-sand filtered reclaimed water samples had significantly less virus-like particles than reclaimed water samples at all collection dates, with the reclaimed water averaging between 108 and 109 and the zero-valent iron-sand filtered reclaimed water averaging between 106 and 107 virus-like particles per mL. In addition, for both sample types, viral metagenomes (viromes) were dominated by bacteriophages of the order Caudovirales, largely Siphoviridae, and genes related to DNA metabolism. However, the proportion of sequences homologous to bacteria, as well as the abundance of genes possibly originating from a bacterial host, was higher in the viromes of zero-valent iron-sand filtered reclaimed water samples. Overall, zero-valent iron-sand filtered reclaimed water had a lower total concentration of virus-like particles and a different virome community composition compared to unfiltered reclaimed water

Human Norovirus Surrogates Persist in Nontraditional Sources of Irrigation Water in Excess of 100 Days

Human norovirus (HuNoV) has been implicated as the leading cause of foodborne illness worldwide. The ability of HuNoV to persist in water can significantly impact food safety as agriculture and processing water could serve as vehicles of virus transmission. This study focused on the persistence and infectivity of the HuNoV surrogate viruses, murine norovirus (MNV), and Tulane virus (TV), after prolonged storage in diverse environmental water types currently used for agricultural irrigation. In this study, vegetable processing water (VW), brackish tidal surface water (SW), municipal reclaimed water (RW), and pond water (PW) were inoculated with each virus in a 1:10 v/v ratio containing virus at 3.5–4.5 logPFU/mL and stored at 16°C for 100 days. This time and temperature combination was chosen to mimic growing and harvest conditions in the mid-Atlantic area of the United States. Samples were then assayed for the presence of viral RNA using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) approximately weekly throughout the study. Persistence of MNV and TV was not significantly different (p > 0.05) from one another in any water sample (n = 7) or the control (HBSS). However, there was variability observed in viral persistence across water samples with significant differences observed between several water samples. The presence of intact viral capsids enclosing the genomes of MNV and TV were evaluated by an RNase assay coupled with RT-qPCR on specific timepoints and determined to be intact up to and at 100 days after inoculation. TV was also shown to remain infectious in a cell culture assay (TCID50) up to 100 days of incubation. These findings are significant in that the potential for not only detection of enteric viruses can occur long after a contamination event occurs but these viruses may also remain infectious