Using Bio-inline Reactor to Evaluate Sanitizer Efficacy in Removing Dual-species Biofilms Formed by Escherichia coli O157:H7 and Listeria monocytogenes.

The efficacy of a sanitizer in biofilm removal may be influenced by a combination of factors such as sanitizer exposure time and concentration, bacterial species, surface topography, and shear stresses. We employed an inline biofilm reactor to investigate the interactions of these variables on biofilm removal with chlorine. The CDC bioreactor was used to grow E. coli O157:H7 and L. monocytogenes biofilms as a single species or with Ralstonia insidiosa as a dual-species biofilm on stainless steel, PTFE, and EPDM coupons at shear stresses 0.368 and 2.462 N/m2 for 48 hours. Coupons were retrieved from a CDC bioreactor and placed in an inline biofilm reactor and 100, 200, or 500 ppm of chlorine was supplied for 1- and 4 min. Bacterial populations in the biofilms were quantified pre- and posttreatment by plating on selective media. After chlorine treatment, reduction (Log CFU/cm2) in pathogen populations obtained from three replicates was analyzed for statistical significance. A 1-min chlorine treatment (500 ppm), on dual-species E. coli O157:H7 biofilms grown at high shear stress of 2.462 N/m2 resulted in significant E. coli O157:H7 reductions on SS 316L (2.79 log CFU/cm2) and PTFE (1.76 log CFU/cm2). Similar trend was also observed for biofilm removal after a 4-min chlorine treatment. Single species E. coli O157:H7 biofilms exhibited higher resistance to chlorine when biofilms were developed at high shear stress. The effect of chlorine in L. monocytogenes removal from dual-species biofilms was dependent primarily on the shear stress at which they were formed rather than the surface topography of materials. Besides surface topography, shear stresses at which biofilms were formed also influenced the effect of sanitizer. The removal of E. coli O157:H7 biofilms from EPDM material may require critical interventions due to difficulty in removing this pathogen. The inline biofilm reactor is a novel tool to evaluate the efficacy of a sanitizer in bacterial biofilm removal.

Inactivation of Listeria monocytogenes and Salmonella spp. During Cooking of Country Ham and Fate of L. monocytogenes and Staphylococcus aureus During Storage of Country Ham Slices.

Thermal inactivation studies were undertaken on Listeria monocytogenes and Salmonella spp. inoculated on the surface of country ham. Hams (average = ca. 3.4 ± 0.5 kg each; average = ca. ≥18% shrinkage) were used as provided by the processor (i.e., "salted hams"), desalted in tap water (i.e., "desalted hams"), or dried for an additional period (i.e., "extra-dried hams"). Hams were surface inoculated (ca. 9.5 log CFU/ham) with a multistrain cocktail of L. monocytogenes or Salmonella spp. and cooked within a bag ina circulating water bath to an internal temperature of 130°F (54.4°C) instantaneous, 145°F (62.8°C) and held for 4 min, 153°F (67.2°C) and held for 34 s, or 160°F (71.1°C) instantaneous. Regardless of ham type, all four time and temperature combinations tested herein delivered a ≥6.7-log reduction of cells of L. monocytogenes or Salmonella spp. Differences in product pH, moisture content, or aw did not have an appreciable impact on the thermal inactivation of L. monocytogenes or Salmonella spp. on country ham. In addition, shelf-life studies were undertaken using slices of "salted" country ham that were surface inoculated (ca. 5.5 log CFU/slice) with a multistrain cocktail of L. monocytogenes or Staphylococcus aureus and then stored at 20°C. Levels of S. aureus increased by ca. ≤1.4 log CFU/slice during storage for 90 days, whereas levels of L. monocytogenes remained relatively unchanged (≤0.2 log CFU/slice increase). Our data validated that cooking parameters elaborated in the U.S. Department of Agriculture's Food Safety and Inspection Service Cooking Guideline for Meat and Poultry Products (Revised Appendix A) are sufficient to deliver significant reductions (ca. ≥6.8 log CFU/ham) in levels of L.monocytogenes and Salmonella spp. on country ham. In addition, in the event of postprocessing contamination, country ham may support the outgrowth of S. aureus or survival of L. monocytogenes during storage at 20°C for 90 days.

Minimum Concentrations of Slow Pyrolysis Paper and Walnut Hull Cyclone Biochars Needed to Inactivate Escherichia coli O157:H7 in Soil.

Antimicrobial properties of biochar have been attributed to its ability to inactivate foodborne pathogens in soil, to varying degrees. High concentrations of biochar have reduced E. coli O157:H7 in soil and dairy manure compost, based on alkaline pH. Preliminary studies evaluating 31 different biochars determined that two slow pyrolysis biochars (paper biochar and walnut hull cyclone biochar) were the most effective at inactivating E. coli in soil. A study was conducted to determine the lowest percentages of paper and walnut hull cyclone biochars needed to reduce E. coli O157:H7 in soil. A model soil was adjusted to 17.75% moisture, and the two types of biochar were added at concentrations of 1.0, 1.5, 2.0, 2.5, 3.5, 4.5, 5.5, and 6.5%. Nontoxigenic E. coli O157:H7 were inoculated into soil at 6.84 log CFU/g and stored for up to 6 weeks at 21°C. Mean E. coli O157:H7 counts were 6.01-6.86 log CFU/g at all weeks between 1 and 6 in soil-only positive control samples. Populations in all soil amended with 1.0 and 1.5% of either type of biochar (as well as 2.0% of the walnut hull biochar) resulted in ≤0.68 log reductions at week 6, when compared with positive controls. All other concentrations (i.e., ≥2.0% paper and ≥2.5% walnut hull) inactivated ≥2.7 log at all weeks between 1 and 6 (p < 0.05). At the end of 6 weeks, E. coli O157:H7 declined by 2.84 log in 2.0% paper biochar samples, while concentrations of between 2.5 and 6.5% paper biochar completely inactivated E. coli O157:H7, as determined by spiral plating, at weeks 5 and 6. In contrast, 2.0% walnut hull biochar lowered populations by only 0.38 log at week 6, although 2.5-6.5% concentrations of walnut hull biochar resulted in complete inactivation at all weeks between 3 and 6, as assessed by spiral plating. In summary, ≥2.5% paper or walnut hull biochar reduced ≥5.0 log of E. coli O157:H7 during the 6-week storage period, which we attribute to high soil alkalinity. Amended at a 2.5% concentration, the pH of soil with paper or walnut hull biochar was 10.67 and 10.06, respectively. Results from this study may assist growers in the use of alkaline biochar for inactivating E. coli O157:H7 in soil.

Large Iodine Variability in Retail Cows' Milk in the U.S.: A Follow-Up Study among Different Retail Outlets.

In a previous study, large variability in iodine content was found among samples of store brand retail milk at a single time point in a sampling taken from 24 nationwide U.S. locations for the USDA FoodData Central database, but the sampling plan was not designed to detect differences among locations. This follow-up study was carried out to evaluate iodine levels in retail milk across the U.S. over time. Milk samples (2% fat) were collected bimonthly in fourteen locations for one year and analyzed in duplicate. Control materials were used to support accuracy of results and ensure precision across analytical batches. The overall mean and standard error (SE) for iodine concentration were 82.5 (7.0) µg/240 mL serving, which was comparable to the previous national mean [85.0 (5.5) µg/240 mL]. A similar wide range among individual samples was detected (27.9-282 µg/240 mL). For some locations, the mean iodine concentration differed significantly from others, and differed from the national average by amounts ranging from -47 µg to +37 µg per serving. The between-sample range within location was large for some (up to 229 µg/serving) and minimal for others (as little as 13.2 µg/serving). These findings suggest iodine intake from some retail milk supplies could be over- or underestimated relative to the national average, even if the national average is suitable for population-wide intake estimates.

Fate of Listeria monocytogenes, Salmonella spp., and Shiga Toxin-Producing Escherichia coli on Slices of an All-Beef Soppressata during Storage.

Cells of Listeria monocytogenes, Salmonella spp., or Shiga toxin-producing Escherichia coli (STEC) were inoculated (ca. 4.0 log CFU/slice) onto slices (ca. 4 g each slice) of an all-beef soppressata (ca. pH 5.05 and aw 0.85). The storage of vacuum-sealed slices of inoculated soppressata at 4 °C or 20 °C for 90 days resulted in reductions of all three pathogens by ca. 2.2 to 3.1 or ca. ≥3.3 log CFU/slice, respectively. When pathogen levels decreased to below detection (≤1.18 log CFU/slice) by direct plating, it was possible to recover each of the target pathogens by enrichment, albeit more frequently from slices stored at 4 °C (p < 0.05) compared to 20 °C. In summary, the slices of the commercially produced beef soppressata selected for this study did not provide a favorable environment for either survival or outgrowth of surface-inoculated cells of L. monocytogenes, Salmonella spp., or STEC during storage.

Viability of Listeria monocytogenes and Salmonella spp. on Slices of a German-Style Bologna Containing Blends of Organic Acid Salts During Storage at 4 or 12°C.

Viability of cells of Listeria monocytogenes or Salmonella spp. was quantified on slices of a German-style bologna manufactured by a local butcher to contain no added antimicrobials or to include 0.9% or 1.3% of a blend of potassium acetate and sodium diacetate (K-Ace) or 2.5% of a blend of potassium lactate and sodium diacetate (K-Lac) as ingredients. After slicing (ca. 7.1 cm L by 6.7 cm W, ca. 0.5 cm thick, ca. 22.4 g each), a single slice of bologna was placed into a nylon-polyethylene bag and surface inoculated with 250 µL per side of a five-strain mixture of either cells of L. monocytogenes or Salmonella spp. to achieve an initial level of ca. 3.5-4.0 log CFU/slice. The packages were vacuum-sealed and then stored at 4 or 12°C for 90 and 30 days, respectively. Without antimicrobials added to the formulation, L. monocytogenes numbers increased by ca. 5.4 and 6.0 log CFU/slice at both 4 and 12°C during the entire 90- and 30-day storage period, respectively. Likewise, levels of Salmonella also increased by ca. 6.0 log CFU/slice at 12°C in the absence of added antimicrobials; however, levels of this pathogen decreased by ca. 1.7 log CFU/slice after 90 days at 4°C. With the inclusion of 0.9% or 1.3% K-Ace or 2.5% K-Lac in the bologna formulation, levels of L. monocytogenes decreased by ca. ≤0.7 log CFU/slice after 90 days at 4°C, whereas levels of Salmonella decreased by ca. 1.6-2.3 log CFU/slice. After 30 days at 12°C, levels of L. monocytogenes increased by ca. ≤3.4 log CFU/slice on product containing 0.9% K-Ace or 2.5% K-Lac but remained relatively unchanged on slices formulated with 1.3% K-Ace. For Salmonella, in the presence of 0.9% or 1.3% K-Ace or 2.5% K-Lac, pathogen levels decreased by ca. ≤0.7 log CFU/slice at 12°C after 30 days. Our data validate that the inclusion of K-Ace (0.9% or 1.3%) or K-Lac (2.5%) as ingredients is effective for controlling L. monocytogenes and Salmonella on slices of bologna during refrigerated storage.

Molecular Analysis and Sex-specific Response of the Hepcidin Gene in Yellow Perch (Perca Flavescens) Following Lipopolysaccharide Challenge.

Hepcidin antimicrobial peptide (hamp) is active in teleosts against invading pathogens and plays important roles in the stress and immune responses of finfish. The response of hamp gene was studied in yellow perch (yp) (Perca flavescens) challenged with lipopolysaccharides to understand if this immunity response is sex-specifically different. The cloned hamp gene consists of an open-reading frame of 273 bp and encodes a deduced protein of 90 amino acids (a.a.), which includes a signal peptide of 24 a.a., a pro-domain of 40 a.a. and a mature peptide of 26 a.a. Yp hamp involves 8 cysteine residues with 4 disulfide bonds, and a protein with an internal alpha helix flanked with C- and N-terminal random coils was modeling predicted. RT-qPCR was used to analyze the relative abundances (RAs) of hamp mRNA in the livers of juvenile female and male yellow perch challenged with lipopolysaccharide. The expression levels of hamp were significantly elevated by 3 h (RA = 7.3) and then peaked by 6 h (RA = 29.4) post-treatment in females but the peak was delayed to 12 h (RA = 65.4) post-treatment in males. The peak mRNA level of challenged males was shown 7.6-fold higher than females. The post-treatment responses in both genders decreased to their lowest levels by 24 h and 48 h. Overall, female perch had an earlier but less-sensitive response to the lipopolysaccharide challenge than male.

Annual and Seasonal Changes in Parasitism Rates by Hadronotus Pennsylvanicus (Hymenoptera: Scelionidae) on the Squash Bug Anasa Tristis (Hemiptera: Coreidae) in Squash Fields: Implications for Augmentative Releases.

This study evaluated parasitism rates by Hadronotus pennsylvanicus (Ashmead) (Hymenoptera: Scelionidae) on the squash bug Anasa tristis DeGeer (Hemiptera: Coreidae) over a six-year period in squash fields in Maryland. From 2016-2021, 2226 wild squash bug egg masses were collected, 2180 (98.0%) A. tristis egg masses and 46 (2.0%) A. armigera egg masses. The mean (±SE) parasitism rate was 10.9 ± 0.16%. Yearly parasitism rates were significantly different with rates in 2017 and 2018 that were significantly lower than in 2019, 2020, and 2021. The significant difference in parasitism rates based on planting date was primarily due to the high parasitism rate observed in 2021. These results suggest that the use of augmentative releases early in the season could result in effective control by increasing parasitism earlier in the season and by causing the parasitism rate in the field to peak at a higher number late in the season.

Correction: Solano-Aguilar et al. Fruit and Vegetable Supplemented Diet Modulates the Pig Transcriptome and Microbiome after a Two-Week Feeding Intervention. Nutrients 2021, 13, 4350.

There was an error in the original publication [...].

Viability of Shiga Toxin-Producing Escherichia coli, Salmonella spp., and Listeria monocytogenes during Preparation and Storage of Fuet, a Traditional Dry-Cured Spanish Pork Sausage.

ABSTRACT: The primary objective of this study was to monitor viability of Shiga toxin-producing Escherichia coli (STEC), Salmonella spp., and Listeria monocytogenes during preparation and storage of fuet. Regarding methodology, coarse-ground pork (ca. 35% fat) was mixed with salt (2.5%), dextrose (0.3%), starter culture (ca. 7.0 log CFU/g), celery powder (0.5%), and ground black pepper (0.3%) and then separately inoculated with a multistrain cocktail (ca. 7.0 log CFU/g) of each pathogen. The batter was stuffed into a ca. 42-mm natural swine casing and fermented at 23 ± 2°C and ca. 95% ± 4% relative humidity to ≤pH 5.3 (≤48 h). Sausages were then dried at 12 ± 2°C and ca. 80% ± 4% relative humidity to a water activity (aw) of 0.89 (within 33 days) or aw 0.86 (within 60 days). A portion of each batch of fuet was subjected to high-pressure processing (HPP; 600 MPa for 3 min) before chubs were vacuum packaged and stored for 30 days at 20 ± 2°C. The results revealed that pathogen numbers remained relatively unchanged after fermentation (≤0.35 log CFU/g reduction), whereas reductions of ca. 0.8 to 3.2 log CFU/g were achieved after drying fuet to aw 0.89 or 0.86. Regardless of whether fuet was or was not pressure treated, additional reductions of ca. 2.2 to ≥5.3 log CFU/g after drying were achieved following 30 days of storage at 20°C. For non-HPP-treated fuet dried to aw 0.89 and stored for 30 days at 20°C, total reductions of ≥5.3 log CFU/g in levels of STEC or Salmonella spp. were achieved, whereas levels of L. monocytogenes were reduced by ca. 3.6 log CFU/g. Total reductions of ≥5.3 log CFU/g in levels of all three pathogens were achieved after drying non-HPP-treated fuet to aw 0.86. For fuet dried to aw 0.89 or 0.86, that were pressure treated and then stored for 30 days at 20°C, total reductions of >6.2 log CFU/g in levels of all three pathogens were achieved. In conclusion, the processing parameters tested herein, with or without application of HPP, validated that reductions of ≥2.0 or ≥5.0 log CFU/g in levels of STEC, Salmonella spp., and L. monocytogenes were achieved during preparation and storage of fuet.

Fruit and Vegetable Supplemented Diet Modulates the Pig Transcriptome and Microbiome after a Two-Week Feeding Intervention.

A study was conducted to determine the effects of a diet supplemented with fruits and vegetables (FV) on the host whole blood cell (WBC) transcriptome and the composition and function of the intestinal microbiome. Nine six-week-old pigs were fed a pig grower diet alone or supplemented with lyophilized FV equivalent to half the daily recommended amount prescribed for humans by the Dietary Guideline for Americans (DGA) for two weeks. Host transcriptome changes in the WBC were evaluated by RNA sequencing. Isolated DNA from the fecal microbiome was used for 16S rDNA taxonomic analysis and prediction of metabolomic function. Feeding an FV-supplemented diet to pigs induced differential expression of several genes associated with an increase in B-cell development and differentiation and the regulation of cellular movement, inflammatory response, and cell-to-cell signaling. Linear discriminant analysis effect size (LEfSe) in fecal microbiome samples showed differential increases in genera from Lachnospiraceae and Ruminococcaceae families within the order Clostridiales and Erysipelotrichaceae family with a predicted reduction in rgpE-glucosyltransferase protein associated with lipopolysaccharide biosynthesis in pigs fed the FV-supplemented diet. These results suggest that feeding an FV-supplemented diet for two weeks modulated markers of cellular inflammatory and immune function in the WBC transcriptome and the composition of the intestinal microbiome by increasing the abundance of bacterial taxa that have been associated with improved intestinal health.

Spatial Heterogeneity of Sympatric Tick Species and Tick-Borne Pathogens Emphasizes the Need for Surveillance for Effective Tick Control.

Three tick species that can transmit pathogen causing disease are commonly found parasitizing people and animals in the mid-Atlantic United States: the blacklegged tick (Ixodes scapularis Say), the American dog tick (Dermacentor variabilis [Say]), and the lone star tick (Amblyomma americanum [L.]) (Acari: Ixodidae). The potential risk of pathogen transmission from tick bites acquired at schools in tick-endemic areas is a concern, as school-aged children are a high-risk group for tick-borne disease. Integrated pest management (IPM) is often required in school districts, and continued tick range expansion and population growth will likely necessitate IPM strategies to manage ticks on school grounds. However, an often-overlooked step of tick management is monitoring and assessment of local tick species assemblages to inform the selection of control methodologies. The purpose of this study was to evaluate tick species presence, abundance, and distribution and the prevalence of tick-borne pathogens in both questing ticks and those removed from rodent hosts on six school properties in Maryland. Overall, there was extensive heterogeneity in tick species dominance, abundance, and evenness across the field sites. A. americanum and I. scapularis were found on all sites in all years. Overall, A. americanum was the dominant tick species. D. variabilis was collected in limited numbers. Several pathogens were found in both questing ticks and those removed from rodent hosts, although prevalence of infection was not consistent between years. Borrelia burgdorferi, Ehrlichia chaffeensis, Ehrlichia ewingii, and Ehrlichia "Panola Mountain" were identified in questing ticks, and B. burgdorferi and Borrelia miyamotoi were detected in trapped Peromyscus spp. mice. B. burgdorferi was the dominant pathogen detected. The impact of tick diversity on IPM of ticks is discussed.

Repeated, drug-truncated infections with Ostertagia ostertagi elicit strong humoral and cell-mediated immune responses and confer partial protection in cattle.

Bovine ostertagiasis causes significant production losses to the cattle industry. Protective immunity induced by natural infection is slow to develop and anthelmintic resistance is rapidly developing. There is a need to advance alternatives for control of gastrointestinal nematode parasites. The present study investigated the effects of repeated, drug-truncated infections (rDTI) on development of protective immunity and attenuation of a challenge infection by O. ostertagi. Helminth-free calves were randomly assigned to either a rDTI or a control group (n = 5). The rDTI group received daily oral infections of 5000 Ostertagia L3 for 5 consecutive days, then were drug-treated on 14 and 15 days post infection (dpi), to attenuate O. ostertagi at the late fourth larval (L4) through young adult stages. DTI was repeated 3 weeks after the drug treatment. A total of 5 DTIs were administered to the DTI-treated animals. Non-DTI-treated, control animals received tap water as infection control. All animals were drug-treated at the same time. Animals were challenge-infected 4 weeks following the final round of rDTI. The results show that eggs per gram of feces (EPG) in the rDTI group were significantly reduced (P < 0.05) from 21 to 39 dpi, with an overall reduction in cumulative EPG. The control group exhibited reduced (P = 0.0564) average weight gains when compared to those of the rDTI group during weeks 4-5 post infection, a period coinciding with peak EPG output of control animals. Antigen-specific IgG, IgE and IgA responses were detected after the 2nd DTI, and stronger antibody recall responses were elicited by challenge infection. High levels of antigen-specific peripheral blood mononuclear cell (PBMC)/T cell proliferation to whole worm and excretory-secretory (ES) antigens were detected in rDTI-treated animals. These data indicate that partial protective immunity against ostertagiasis, involving cell-mediated and humoral responses, can be attained by rDTI which allowed for maximal antigen exposure from staggered parasitic developmental stages. The data suggest that rDTI can be used as a model to study host-parasite interactions and identify parasite antigens responsible for eliciting host protective immune responses.

Vaccination with Neospora caninum-cyclophilin and -profilin confers partial protection against experimental neosporosis-induced abortion in sheep.

The purpose of this study was to evaluate the protective efficacy of a vaccine consisting of recombinant Neospora caninum-cyclophilin (NcCyP) and -profilin (NcPro) in sheep. At 42 d and 21 d prior to mating, adult Dorset ewes were immunized with the rNcCyP-rNcPro vaccine (Group 1) or co-purifying non-recombinant (NR) control vaccine (Group 2). At 90 days post-mating, all immunized ewes and were challenged by intravenous injection with 106Nesopora caninum Illinois tachyzoites (NcTZ). Significant protection (P < 0.05) was observed in Group 1 with 9 out of 13 ewes giving birth to live-born lambs (69.2%), whereas all Group 2 ewes aborted (6/6). Neospora caninum was detected by PCR in both fetal and placental tissues from all Group 2 aborting ewes and in the placental tissues of Group 1 aborting ewes. In contrast, tissues and placentas of Group 1 live-born lambs were Neospora DNA-negative. Immunoreactive Neospora antigens were demonstrated in placentas associated with abortions, but not in tissues of aborted fetuses or those of the live-born lambs and their associated placentas. Anti-NcCyP and anti-NcPro titers were high in sera from Group 1 ewes and were further boosted by challenge infection, resulting in long-lasting (≥14.5 mos.) elevated titers. Lambs born to Group 1 ewes also had high NcCyP and NcPro titers in pre-colostrum sera. Immunofluorescence staining (IFA) of NcTZ with Group 1 post-immunization sera revealed both surface and internal TZ staining, a pattern consistent with that observed with rabbit sera to rNcCyP or rNcPro. Infection of NR-vaccinated ewes produced high but transient anti-NcCyP and anti-NcPro Ab titers. The results indicate that the NcCyP-NcPro vaccine elicited strong anti-N. caninum responses and conferred significant protection against abortion and transplacental transmission of N. caninum TZ in sheep.

Parasitism and Predation on Sentinel Egg Masses of Three Stink Bug Species (Hemiptera: Pentatomidae) in Native and Exotic Ornamental Landscapes.

This study evaluated parasitism and predation on sentinel egg masses of three stink bug species, the spined soldier bug, Podisus maculiventris (Say), the brown stink bug, Euschistus servus (Say), and the invasive brown marmorated stink bug (BMSB), Halyomorpha halys (Stål), in ornamental landscapes composed of either native or exotic plants. This study also compared the species composition of parasitoids attacking two native stink bug species (P. maculiventris and E. servus) with those attacking the invasive BMSB on the same tree species in the same habitat. Overall, egg parasitism and predation were much higher on the two native stink bug species compared with BMSB, with an average parasitism rate of 20.6% for E. servus, 12.7% for P. maculiventris, and only 4.2% for H. halys and an average predation rate of 8.2% for E. servus,17.7% for P. maculiventris, and 2.3% for H. halys. Egg predation was also significantly higher on P. maculiventris than on E. servus eggs. Eight parasitoid species attacked sentinel stink bug eggs in the ornamental landscaped plots. Trissolcus euschisti (Ashmead) (Hymenoptera: Scelionidae) was the predominant parasitoid for all three stink bug species. There were no significant differences in parasitism and predation rates on any of the stink bug species between native and exotic plots. Therefore, there is no evidence that ornamental landscapes composed of native plants increased parasitism or predation rates of sentinel egg masses of two native stink bug species or the invasive BMSB, compared with those composed entirely of exotic plants.

Temporal and Agricultural Factors Influence Escherichia coli Survival in Soil and Transfer to Cucumbers.

Biological soil amendments of animal origin (BSAAO) increase nutrient levels in soils to support the production of fruits and vegetables. BSAAOs may introduce or extend the survival of bacterial pathogens which can be transferred to fruits and vegetables to cause foodborne illness. Escherichia coli survival over 120 days in soil plots (3 m2) covered with (mulched) or without plastic mulch (not mulched), amended with either poultry litter, composted poultry litter, heat-treated poultry pellets, or chemical fertilizer, and transfer to cucumbers in 2 years (2018 and 2019) were evaluated. Plots were inoculated with E. coli (8.5 log CFU/m2) and planted with cucumber seedlings (Supremo). The number of days needed to reduce E. coli levels by 4 log CFU (dpi4log) was determined using a sigmoidal decline model. Random forest regression and one-way analysis of variance (ANOVA; P < 0.05) identified predictors (soil properties, nutrients, and weather factors) of dpi4log of E. coli and transfer to cucumbers. The combination of year, amendment, and mulch (25.0% increase in the mean square error [IncMSE]) and year (9.75% IncMSE) were the most prominent predictors of dpi4log and transfer to cucumbers, respectively. Nitrate levels at 30 days and soil moisture at 40 days were also impactful predictors of dpi4log. Differing rainfall amounts in 2018 (24.9 in.) and 2019 (12.6 in.) affected E. coli survival in soils and transfer to cucumbers. Salmonella spp. were recovered sporadically from various plots but were not recovered from cucumbers in either year. Greater transfer of E. coli to cucumbers was also shown to be partially dependent on dpi4log of E. coli in plots containing BSAAO.IMPORTANCE Poultry litter and other biological soil amendments are commonly used fertilizers in fruit and vegetable production and can introduce enteric pathogens such as Escherichia coli O157:H7 or Salmonella previously associated with outbreaks of illness linked to contaminated produce. E. coli survival duration in soils covered with plastic mulch or uncovered and containing poultry litter or heat-treated poultry litter pellets were evaluated. Nitrate levels on day 30 and moisture content in soils on day 40 on specific days were good predictors of E. coli survival in soils; however, the combination of year, amendment, and mulch type was a better predictor. Different cumulative rainfall totals from year to year most likely affected the transfer of E. coli from soils to cucumbers and survival durations in soil. E. coli survival in soils can be extended by the addition of several poultry litter-based soil amendments commonly used in organic production of fruits and vegetables and is highly dependent on temporal variation in rainfall.

Metagenomic Analysis of 16S Clostridium perfringens Amplicons Corroborates C. perfringens Counts on Select Agar and C. perfringens PCR Analyses of Bacteria in Broiler Farm Litter.

The purpose of this study was twofold-first, to determine whether analysis of bacterial 16S ribosomal RNA (rRNA) in poultry litter corroborated standard Clostridium perfringens counts and PCR assay, and second, to find whether a correlation between 16S rRNA analysis and netB or Tpel toxin PCR intensity with chick mortality existed. At three time points of growout (0, 2, and 4 wk) litter samples were collected from 23 broiler houses representing eight farms during a coccidiosis vaccine control program. DNA extracted from these samples was used for microbiota determination by sequencing the hypervariable V3-V4 region of bacterial 16s rRNA. Obtained sequences were analyzed by QIIME 2 and the Greengenes database for taxonomic composition and relative abundance of C. perfringens in the litter bacterial population. Clostridium perfringens counts on select agar and semiquantitative PCR for C. perfringens were compared with 16S analysis for equivalence testing. Relative abundance of C. perfringens estimated by 16S analysis and semiquantitative PCR for netB and Tpel toxin DNA were analyzed by Pearson linear correlation and statistical equivalence analyses with cumulative chick mortality at 4 and 9 wk growout. When data from all time points were combined, abundance estimates by C. perfringens 16S were statistically equivalent (α = 0.10) to both C. perfringens PCR and C. perfringens counts. Yet, no correlations were observed between any estimate of C. perfringens abundance and cumulative percent chick mortality at 4 or 9 wk growout. However, correlation analyses revealed a significant linear relationship between netB signal at 0 wk (r = 0.55) and 4 wk (r = 0.46) and cumulative mortality at 9 wk growout (P < 0.05). Similarly, abundance of Tpel at 0 and 2 wk showed a linear relationship with cumulative percent mortality at both 4 and 9 wk growout (0.44 ≤ r ≤ 0.54, P < 0.05). No correlations were observed between any other genera or species determined by 16S and cumulative percent chick mortality.

Ovipositional Behavior of the Egg Parasitoid Gryon pennsylvanicum (Hymenoptera: Scelionidae) on Two Squash Bug Species Anasa tristis (Hemiptera: Coreidae) and Anasa armigera: Effects of Parasitoid Density, Nutrition, and Host Egg Chorion on Parasitism Rates.

This study examined the ovipositional behavior of Gryon pennsylvanicum Ashmead (Hymenoptera: Scelionidae) on egg masses of two squash bug species Anasa tristis DeGeer and Anasa armigera Say (Hemiptera: Coreidae) by evaluating how parasitoid density and access to nutrition influenced percent parasitism on egg masses of different sizes in laboratory tests. When three parasitoids were exposed to A. tristis egg masses with only three to five eggs, 72.7% of parasitoids became trapped in the eggs and failed to emerge successfully. These results suggest that competition between larvae within the egg may have reduced the fitness of the surviving parasitoid. Continual access to honey water did not significantly influence parasitism rates on A. armigera egg masses and only increased parasitism on A. tristis egg masses with 20-25 eggs. Overall, parasitism rates were higher on A. armigera egg masses than on A. tristis egg masses, and parasitoids were more likely to emerge successfully from A. armigera eggs than from A. tristis eggs. Parasitoids spent the same amount of time probing eggs of the two species, but they spent significantly more time drilling into A. tristis eggs than A. armigera eggs. Measurements taken using transmission electron microscopy determined that the average combined width of the epicuticle and exocuticle of the egg chorion was significantly greater for A. tristis eggs than for A. armigera eggs. This difference may account for the lower rates of parasitism and parasitoid emergence and for the increased time spent drilling into A. tristis eggs compared with A. armigera eggs.

BMI Is Associated With Increased Plasma and Urine Appearance of Glucosinolate Metabolites After Consumption of Cooked Broccoli.

Introduction: Preclinical studies suggest that brassica vegetable diets decrease cancer risk, but epidemiological studies show varied effects, resulting in uncertainty about any health impact of brassicas. Factors controlling absorption of glucosinolate metabolites may relate to inconsistent results. We reported previously that subjects with BMI > 26 kg/m2 (HiBMI), given cooked broccoli plus raw daikon radish (as a source of plant myrosinase) daily for 17 days, had lower glucosinolate metabolite absorption than subjects given a single broccoli meal. This difference was not seen in subjects with BMI < 26 kg/m2 (LoBMI). Our objective in this current study was to determine whether a similar response occurred when cooked broccoli was consumed without a source of plant myrosinase. Methods: In a randomized crossover study (n = 18), subjects consumed no broccoli for 16 days or the same diet with 200 g of cooked broccoli daily for 15 days and 100 g of broccoli on day 16. On day 17, all subjects consumed 200 g of cooked broccoli. Plasma and urine were collected for 24 h and analyzed for glucosinolate metabolites by LC-MS. Results: There was no effect of diet alone or interaction of diet with BMI. However, absorption doubled in HiBMI subjects (AUC 219%, plasma mass of metabolites 202% compared to values for LoBMI subjects) and time to peak plasma metabolite values and 24-h urinary metabolites also increased, to 127 and 177% of LoBMI values, respectively. Conclusion: BMI impacts absorption and metabolism of glucosinolates from cooked broccoli, and this association must be further elucidated for more efficacious dietary recommendations. Clinical Trial Registration: This trial was registered at clinicaltrials.gov (NCT03013465).

Diurnal effects on sporangium and zoospore production by Phytophthora ramorum on Rhododendron 'Cunningham's White'.

We evaluated sporangium and zoospore production by three isolates of Phytophthora ramorum on Rhododendron 'Cunningham's White' leaves under light and dark conditions at both variable and constant (14 C) temperature. P. ramorum-infected leaves were detached and placed in funnels inside of a 62-L plastic storage container located in a growth chamber. Cool mist was introduced to the container to create a high-humidity environment. Sporangia and zoospores were collected over a 4-day period by misting leaves with 5 mL of distilled water, which was collected in conical test tubes that also contained runoff from the misting. Spores were collected daily just before a 13-h light period and again just before an 11-h dark period. Sporangia and zoospores in the collection tubes were counted using a dissecting microscope following staining with lactoglycerin/aniline blue. Large differences in sporangium and zoospore numbers observed for the dark versus light periods were observed on days 2, 3, and 4. A diurnal effect has been observed for production of propagules of other oomycetes, but such effects have not been previously reported for P. ramorum. This information will help provide a better understanding of patterns of inoculum production by P. ramorum and resulting fluctuations in inoculum density that will influence sudden oak death epidemics in forest ecosystems in the United States and other countries where it occurs.