Mihai C. Giurcanu

Factors That Affect Proliferation of Salmonella in Tomatoes Post-Harvest: The Roles of Seasonal Effects, Irrigation Regime, Crop and Pathogen Genotype

Main Objectives Fresh fruits and vegetables become increasingly recognized as vehicles of human salmonellosis. Physiological, ecological, and environmental factors are all thought to contribute to the ability of Salmonella to colonize fruits and vegetables pre- and post-harvest. The goal of this study was to test how irrigation levels, fruit water congestion, crop and pathogen genotypes affect the ability of Salmonella to multiply in tomatoes post-harvest. Experimental Design Fruits from three tomato varieties, grown over three production seasons in two Florida locations, were infected with seven strains of Salmonella and their ability to multiply post-harvest in field-grown tomatoes was tested. The field experiments were set up as a two-factor factorial split plot experiment, with the whole-plot treatments arranged in a randomized complete-block design. The irrigation treatment (at three levels) was the whole-plot factor, and the split-plot factor was tomato variety, with three levels. The significance of the main, two-way, and three-way interaction effects was tested using the (type III) F-tests for fixed effects. Mean separation for each significant fixed effect in the model was performed using Tukey’s multiple comparison testing procedure. Most Important Discoveries and Significance The irrigation regime per se did not affect susceptibility of the crop to post-harvest proliferation of Salmonella. However, Salmonella grew significantly better in water-congested tissues of green tomatoes. Tomato maturity and genotype, Salmonella genotype, and inter-seasonal differences were the strongest factors affecting proliferation. Red ripe tomatoes were significantly and consistently more conducive to proliferation of Salmonella. Tomatoes harvested in the driest, sunniest season were the most conducive to post-harvest proliferation of the pathogen. Statistically significant interactions between production conditions affected post-harvest susceptibility of the crop to the pathogen. UV irradiation of tomatoes post-harvest promoted Salmonella growth.

Distribution and Characterization of Salmonella enterica Isolates from Irrigation Ponds in the Southeastern United States.

ABSTRACT Irrigation water has been implicated as a likely source of produce contamination by Salmonella enterica. Therefore, the distribution of S. enterica was surveyed monthly in irrigation ponds (n = 10) located within a prime agricultural region in southern Georgia and northern Florida. All ponds and 28.2% of all samples (n = 635) were positive for Salmonella, with an overall geometric mean concentration (0.26 most probable number [MPN]/liter) that was relatively low compared to prior reports for rivers in this region. Salmonella peaks were seasonal; the levels correlated with increased temperature and rainfall (P < 0.05). The numbers and occurrence were significantly higher in water (0.32 MPN/liter and 37% of samples) than in sediment (0.22 MPN/liter and 17% of samples) but did not vary with depth. Representative isolates (n = 185) from different ponds, sample types, and seasons were examined for resistance to 15 different antibiotics; most strains were resistant to streptomycin (98.9%), while 20% were multidrug resistant (MDR) for 2 to 6 antibiotics. DiversiLab repetitive extragenic palindromic-element sequence-based PCR (rep-PCR) revealed genetic diversity and showed 43 genotypes among 191 isolates, as defined by >95% similarity. The genotypes did not partition by pond, season, or sample type. Genetic similarity to known serotypes indicated Hadar, Montevideo, and Newport as the most prevalent. All ponds achieved the current safety standards for generic Escherichia coli in agricultural water, and regression modeling showed that the E. coli level was a significant predictor for the probability of Salmonella occurrence. However, persistent populations of Salmonella were widely distributed in irrigation ponds, and the associated risks for produce contamination and subsequent human exposure are unknown, supporting continued surveillance of this pathogen in agricultural settings.

Residual Effects of New Insecticides on Egg and Nymph Densities of Bemisia tabaci (Hemiptera: Aleyrodidae)

ABSTRACT The residual effects of 4 new insecticides (cyazypyr, flupyradifurone, pyrifluquinazon, and sulfoxaflor) and 2 registered insecticides (pymetrozine and a combination of zeta-cypermethrin and bifenthrin) on egg and nymph densities of Bemisia tabaci biotype B were evaluated at 3, 7 or 14 days after treatment (DAT) of the tomato seedlings with insecticides. Whole plant egg and nymph counts were taken weekly for 3 wk after the introduction of whitefly adults. The study was repeated 3 times. Egg densities tended to be statistically higher at 14 DAT than 3 or 7 DAT in the zeta-cypermethrin/bifenthrin and pymetrozine treatments. The other materials demonstrated greater efficacy than zeta-cypermethrin/bifenthrin and pymetrozine on 14 DAT. Egg densities were very low in all insecticide treatments compared to untreated plants at 3 and 7 DAT. Egg densities on plants treated with cyazypyr, flupyradifurone, pyrifluquinazon, and sulfoxaflor were statistically similar within a given DAT interval for most trials. Treatment effects on nymph densities were similar to treatment effects on egg densities. Cyazypyr, flupyradifurone, pyrifluquinazon, and sulfoxaflor represent distinct modes of action, and should contribute to future integrated pest management and integrated resistance management plans for B. tabaci on tomato.

Development of a novel cross-streaking method for isolation, confirmation, and enumeration of Salmonella from irrigation ponds.

The 2013 Produce Safety Rules in Food Safety Modernization Act (FSMA) require regular testing for generic Escherichia coli in agricultural water intended for pre-harvest contact with the edible portion of fresh produce. However, the use of fecal contamination indicators frequently does not correctly reflect distribution of foodborne pathogens such as Salmonella enterica, and ensuring food safety may require direct detection and enumeration of pathogens in agricultural settings. Herein we report the evaluation of different cost-effective methods for quantification, isolation, and confirmation of Salmonella in irrigation pond water and sediment samples. A most probably number (MPN) dual enrichment culture method was used in combination with differential and selective agars, XLT4 and CHROMagar™ Salmonella plus (CSP). The necessity for PCR confirmation was evaluated, and methods were compared by cost and performance measures (i.e., sensitivity, specificity, positive predictive value, and negative predictive value). Statistical analyses showed that using XLT4 as the initial selective agar to isolate Salmonella colonies improved recovery compared to CSP agar; however, PCR confirmation was required to avoid false positive results on either agar. Therefore, a novel cross-streaking method utilizing CHROMagar™ agar for individual colony confirmation of Salmonella presence/absence on XLT4 was developed. This method classifies the colony as positive if typical Salmonella appearance is observed on both agars. Statistical analysis showed that this method was as effective as PCR for species confirmation of pure individual strains isolated from enrichment cultures (sensitivity=0.99, specificity=1.00, relative to PCR). This method offers a cost-effective alternative to PCR that would increase the capacity and sensitivity of Salmonella evaluation.

Bootstrapping in non-regular smooth function models

We study the large sample behavior of the standard bootstrap, the m-out-of-n bootstrap, and the oracle bootstrap (Giurcanu and Presnell, 2009) [14] percentile confidence intervals in non-regular smooth function models. We show that the oracle bootstrap percentile confidence intervals are consistent while the standard bootstrap and the m-out-of-n bootstrap confidence intervals are inconsistent. Further analysis of coverage probabilities reveals that, for large samples, the iterated oracle bootstrap percentile confidence intervals are more accurate than their non-iterated versions. We also describe the large sample local behavior of the bootstrap confidence intervals for parameter values near the points of inconsistency of the standard bootstrap. In a simulation study, we describe the finite sample local behavior of various bootstrap confidence intervals.

A meta-analysis on the effect of dietary application of exogenous fibrolytic enzymes on the performance of dairy cows

The aim of this study was to use meta-analytical methods to estimate effects of adding exogenous fibrolytic enzymes (EFE) to dairy cow diets on their performance and to determine which factors affect the response. Fifteen studies with 17 experiments and 36 observations met the study selection criteria for inclusion in the meta-analysis. The effects were compared by using random-effect models to examine the raw mean difference (RMD) and standardized mean difference between EFE and control treatments after both were weighted with the inverse of the study variances. Heterogeneity sources evaluated by meta-regression included experimental duration, EFE type and application rate, form (liquid or solid), and method (application to the forage, concentrate, or total mixed ration). Only the cellulase-xylanase (C-X) enzymes had a substantial number of observations (n = 13 studies). Application of EFE, overall, did not affect dry matter intake, feed efficiency but tended to increase total-tract dry matter digestibility and neutral detergent fiber digestibility (NDFD) by relatively small amounts (1.36 and 2.30%, respectively, or <0.31 standard deviation units). Application of EFE increased yields of milk (0.83 kg/d), 3.5% fat-corrected milk (0.55 kg/d), milk protein (0.03 kg/d), and milk lactose (0.05 kg/d) by moderate to small amounts (<0.30 standard deviation units). Low heterogeneity (I 2 statistic <25%) was present for yields and concentrations of milk fat and protein and lactose yield. Moderate heterogeneity (I 2 = 25 to 50%) was detected for dry matter intake, milk yield, 3.5% fat-corrected milk, and feed efficiency (kg of milk/kg of dry matter intake), whereas high heterogeneity (I 2 > 50%) was detected for total-tract dry matter digestibility and NDFD. Milk production responses were higher for the C-X enzymes (RMD = 1.04 kg/d; 95% confidence interval: 0.33 to 1.74), but were still only moderate, about 0.35 standardized mean difference. A 24% numerical increase in the RMD resulting from examining only C-X enzymes instead of all enzymes (RMD = 1.04 vs. 0.83 kg/d) suggests that had more studies met the inclusion criteria, the C-X enzymes would have statistically increased the milk response relative to that for all enzymes. Increasing the EFE application rate had no effect on performance measures. Application of EFE to the total mixed ration improved only milk protein concentration, and application to the forage or concentrate had no effect. Applying EFE tended to increase dry matter digestibility and NDFD and increased milk yield by relatively small amounts, reflecting the variable response among EFE types.

Effect of the irrigation regime on the susceptibility of pepper and tomato to post-harvest proliferation of Salmonella enterica.

Raw produce is increasingly recognized as a vehicle of human gastroenteritis. Non-typhoidal Salmonella, pathogenic Escherichia coli, and other human pathogens have been isolated from fruits and vegetables in the field and in the marketplace, which led to the hypothesis that these microbes can use plants as alternate hosts. However, environmental and physiological factors that facilitate persistence of these bacteria in the crop production environment and make produce more vulnerable to post-harvest contamination have not been fully delineated. This study tested the effect of irrigation regimes on the susceptibility of peppers and tomatoes to post-harvest proliferation of Salmonella. The experiments were carried out over three experimental seasons in two locations using seven strains of Salmonella. The irrigation regime per se did not affect susceptibility of tomatoes and peppers to post-harvest proliferation of Salmonella; however, in some of the seasons, irrigation regime-dependent differences were observed. Red peppers and tomatoes were more conducive to proliferation of Salmonella than green fruit in all seasons. Inter-seasonal differences were the strongest factors affecting proliferation of Salmonella in peppers.

High relative humidity pre-harvest reduces post-harvest proliferation of Salmonella in tomatoes

Outbreaks of human illness caused by enteric pathogens such as Salmonella are increasingly linked to the consumption of fruits and vegetables. Knowledge on the factors affecting Salmonella proliferation on fresh produce therefore becomes increasingly important to safeguard public health. Previous experiments showed a limited impact of pre-harvest production practices on Salmonella proliferation on tomatoes, but suggested a significant effect of harvest time. We explored the data from two previously published and one unpublished experiment using regression trees, which allowed overcoming the interpretational difficulties of classical statistical models with higher order interactions. We assessed the effect of harvest time by explicitly modeling the climatic conditions at harvest time and by performing confirmatory laboratory experiments. Across all datasets, regression trees confirmed the dominant effect of harvest time on Salmonella proliferation, with humidity-related factors emerging as the most important underlying climatic factors. High relative humidity the week prior to harvest was consistently associated with lower Salmonella proliferation. A controlled lab experiment confirmed that tomatoes containing their native epimicrobiota supported significantly lower Salmonella proliferation when incubated at higher humidity prior to inoculation. The complex interactions between environmental conditions and the native microbiota of the tomato crop remain to be fully understood.

Abstract 668: Electron paramagnetic resonance (EPR) pO2 image-guided tumor biopsies to analyze hypoxia-induced proteins

Stereotactic MCa4 and FSa tumor tissue biopsies registered with electron paramagnetic resonance (EPR) O2 imaging were used to identify sampling sites suitable to measure protein levels of three hypoxic cell biomarkers, hypoxia-induced factor 1-alpha (HIF-1α), carbonic anhydrase IX (CA9), and vascular endothelial growth factor (VEGF). EPR imaging provided quantitative localization of pO2 levels in tumors grown in the legs of C3H mice. Since hypoxic cells (pO2 Grant Support: This work was supported by grants from the NIH, including R01 CA98575 and P41 EB002034. Citation Format: Richard Miller, Boris Epel, Martyna Elas, Martyna Krzykawska-Serda, Matthew Maggio, Eugene Barth, Mihai Giurcanu, Howard Halpern. Electron paramagnetic resonance (EPR) pO2 image-guided tumor biopsies to analyze hypoxia-induced proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 668.

Oracle M‐Estimation for Time Series Models

We propose a thresholding M-estimator for multivariate time series. Our proposed estimator has the oracle property that its large-sample properties are the same as of the classical M-estimator obtained under the a priori information that the zero parameters were known. We study the consistency of the standard block bootstrap, the centred block bootstrap and the empirical likelihood block bootstrap distributions of the proposed M-estimator. We develop automatic selection procedures for the thresholding parameter and for the block length of the bootstrap methods. We present the results of a simulation study of the proposed methods for a sparse vector autoregressive VAR(2) time series model. The analysis of two real-world data sets illustrate applications of the methods in practice.