Transfer of Salmonella Enteritidis to four types of surfaces after cleaning procedures and cross-contamination to tomatoes

The objectives of the present study were to evaluate the spread of Salmonella Enteritidis to different cutting boards (wood, triclosan-treated plastic, glass, and stainless steel) from contaminated poultry skin (5 log CFU/g) and then to tomatoes and to analyze the effect of different protocols used to clean these surfaces to control contamination. The following procedures were simulated: (1) no cleaning after handling contaminated poultry skin; (2) rinsing in running water; (3) cleaning with dish soap and mechanical scrubbing; and (4) cleaning with dish soap and mechanical scrubbing, followed by disinfection with hypochlorite. The pathogen was recovered from all surfaces following procedure 1, with counts ranging from 1.90 to 2.80 log, as well as from the tomatoes handled on it. Reduced numbers of S. Enteritidis were recovered using the other procedures, both from the surfaces and from the tomatoes. Counts were undetectable after procedure 4. From all surfaces evaluated, wood was the most difficult to clean, and stainless steel was the easiest. The use of hypochlorite as a disinfecting agent helped to reduce cross-contamination.     

The evaluation of a PCR-based method for identification of Salmonella enterica serotypes from environmental samples and various food matrices

The most commonly used method for serotyping Salmonella spp. is based on the Kaufmann–White scheme, and is composed of serological reactions using antibodies to LPS agglutinins. The multiplex PCR used in this investigation was established by Kim et al. to serotype the 30 most common clinical Salmonella serotypes, as determined by CDC. The PCR assay consists of two five-plex reactions and a single two-plex PCR reaction, based on six genetic loci from Salmonella enterica serotype Typhimurium and four loci from S. enterica serotype Typhi. In this investigation, we further evaluated the method for serotyping Salmonella spp. using a reference collection, environmental samples collected from a Mid-Atlantic region tomato farm study, four food matrices spiked with different Salmonella serotypes and a proficiency test. The PCR assay was first evaluated using DNA isolated from pure cultures of isolates obtained from various clinical and environmental samples, and then DNA isolated from broth cultures of food matrices of “Red round” and Roma tomatoes, Romaine lettuce, green onions and Serrano peppers spiked with serotypes Newport, Typhimurium, Javiana and Saintpaul, respectively. The results showed that the PCR assay correctly serotyped Salmonella spp. from the clinical, environmental, spiked food matrices, and proficiency test samples. These findings are significant because the PCR assay was successful in the identification of Salmonella in the spiked samples in a broth culture containing other non-salmonella organism. This method may be a useful resource for the food safety community.     

Diversity and assessment of potential risk factors of Gram-negative isolates associated with French cheeses

The goal of this study was to identify at the species level a large collection of Gram-negative dairy bacteria isolated from milks or semi-hard and soft, smear-ripened cheeses (cheese core or surface samples) from different regions of France. The isolates were then assessed for two risk factors, antibiotic resistance and volatile and non-volatile biogenic amine production in vitro. In total, 173 Gram-negative isolates were identified by rrs and/or rpoB gene sequencing. A large biodiversity was observed with nearly half of all Gram-negative isolates belonging to the Enterobacteriaceae family. Overall, 26 different genera represented by 68 species including potential new species were identified among the studied Gram-negative isolates for both surface and milk or cheese core samples. The most frequently isolated genera corresponded to Pseudomonas, Proteus, Psychrobacter, Halomonas and Serratia and represented almost 54% of the dairy collection. After Pseudomonas, Chryseobacterium, Enterobacter and Stenotrophomonas were the most frequently isolated genera found in cheese core and milk samples while Proteus, Psychrobacter, Halomonas and Serratia were the most frequently isolated genera among surface samples. Antibiotic resistance profiles indicated that resistances to the aminosid, imipemen and quinolon were relatively low while more than half of all tested isolates were resistant to antibiotics belonging to the monobactam, cephem, fosfomycin, colistin, phenicol, sulfamid and some from the penam families. Thirty-six% of isolates were negative for in vitro biogenic amine production. Among biogenic amine-producers, cadaverine was the most frequently produced followed by isoamylamine, histamine and putrescine. Only low levels (<75 mg/l) of tyramine were detected in vitro.     

Interlaboratory comparison of real-time PCR protocols for quantification of general fecal indicator bacteria

The application of quantitative real-time PCR (qPCR) technologies for the rapid identification of fecal bacteria in environmental waters is being considered for use as a national water quality metric in the United States. The transition from research tool to a standardized protocol requires information on the reproducibility and sources of variation associated with qPCR methodology across laboratories. This study examines interlaboratory variability in the measurement of enterococci and Bacteroidales concentrations from standardized, spiked, and environmental sources of DNA using the Entero1a and GenBac3 qPCR methods, respectively. Comparisons are based on data generated from eight different research facilities. Special attention was placed on the influence of the DNA isolation step and effect of simplex and multiplex amplification approaches on interlaboratory variability. Results suggest that a crude lysate is sufficient for DNA isolation unless environmental samples contain substances that can inhibit qPCR amplification. No appreciable difference was observed between simplex and multiplex amplification approaches. Overall, interlaboratory variability levels remained low (<10% coefficient of variation) regardless of qPCR protocol.     

Low progesterone concentration during the development of the first follicular wave reduces pregnancy per insemination of lactating dairy cows

Objectives were to determine the effect of progesterone (P4) concentration on fertility of lactating dairy cows induced to ovulate follicles of the first follicular wave. Lactating dairy cows (n=989) at 38±3d postpartum were balanced by parity and body condition score and randomly assigned to 3 treatments: first follicular wave (FFW), first follicular wave with exogenous P4 (FFWP), or second follicular wave (SFW). All cows had their estrous cycle presynchronized with 2 injections of prostaglandin (PG) F(2α) given 14 d apart. Cows in the FFW and FFWP treatments started the ovulation synchronization protocol 3 d after the last PGF(2α) of the presynchronization protocol, whereas SFW cows received a GnRH injection (100 μg of gonadorelin diacetate; Cystorelin, Merial Ltd., Duluth, GA) 3 d after the last PGF(2α) of the presynchronization protocol and started the synchronization protocol 7 d later. The synchronization protocol consisted of GnRH on d -10, PGF(2α) on d -3, and GnRH concurrent with timed artificial insemination (AI) on d 0. Cows in the FFWP treatment received 2 controlled internal drug release inserts containing 1.38 g of P4 from d -8 to -3. Progesterone concentration was determined on d -10, -8, -6, -3, and 0 from all cows and at 7, 14, and 21 d after AI from a subsample of cows (n=170). Cows (n=715) had their ovaries scanned by ultrasound on d -10, -3, and 7 d. Pregnancy was diagnosed at 38 and 66 d after AI. Concentration of P4 from study d -8 to -3 was lowest for FFW cows (1.4±0.1 ng/mL) and similar between SFW (3.7±0.2 ng/mL) and FFWP (3.7±0.1 ng/mL) cows. Diameter of the dominant follicle on study d -3 was greater for FFW cows (16.5±0.3 mm) than for SFW cows (15.4±0.3 mm), but diameter of the dominant follicle of FFWP cows was not different (15.9±0.3 mm) compared with that of SFW and FFW cows. The incidence of multiple ovulation was largest for FFW cows (SFW=19.5, FFW=33.6, FFWP=19.0%), but pregnancy per AI (P/AI) at 66 d was smallest for FFW cows (SFW=38.9, FFW=22.3, FFWP=32.0%). Anovular cows in the SFW (19.4 vs. 42.8%) and FFWP (22.1 vs. 37.2%) treatments had reduced P/AI compared with cyclic cows, despite having similar or greater P4 concentration from study d -8 to -3, respectively. Estrus and ovulation synchronization protocols for lactating dairy cows must result in growth of ovulatory follicle under P4 concentration >2 ng/mL to ensure high P/AI.     

Effect of amino acids on red pigments and citrinin production in Monascus ruber

Amino acids were used as sole nitrogen sources to examine their effects on the production of water-soluble red pigments and citrinin by Monascus ruber ATCC 96218 cultivated on chemically defined media. In general, when glycine, tyrosine, arginine, serine, or histidine were used as sole nitrogen sources, they favored the production of red pigments, and restricted the synthesis of the mycotoxin. In contrast, the production of citrinin was enhanced in media supplemented with either glutamate, alanine, or proline. Histidine was found to be the most valuable amino acid as it resulted in the highest production of red pigments and almost completely eliminated the formation of mycotoxin.     

Fatty acid profile and sensory characteristics of table eggs from laying hens fed hempseed and hempseed oil

Hempseed (HS) is rich in omega-3 polyunsaturated fatty acids, with approximately 17% of total fatty acids as alpha-linolenic acid. As such, HS and its oil may be used in hen diet formulations to produce eggs enriched in essential fatty acids. Because omega-3 eggs have the potential for unpleasant aromas and flavors, the current study was designed to assess the fatty acid profile and sensory attributes of eggs procured from hens consuming diets containing hempseed oil (HO) or HS. A total of 48 individually caged White Bovan hens received 1 of 6 diets containing 4%, 8%, 12% HO, 10%, 20% HS or 0% hemp (w/w) for 12 wk. Total omega-3 polyunsaturated fatty acid content was highest in the 12% HO group (15.3 mg/g of yolk) compared to the control (2.4 mg/g of yolk). Trained panellists (n= 8) found no significant differences (P≥ 0.05) in aroma or flavor between cooked eggs from different dietary treatments, with the exception of sweet flavor. The 4% HO group yielded the least sweet eggs compared to the 20% HS group, which was highest. For yolk color, L*, a*, and b* values (Mean ± SEM) for control eggs were 61.2 ± 0.10, 1.1 ± 0.05, and 43.0 ± 0.22, respectively. Addition of hemp led to significant (P < 0.001) reductions in L*, and significant increases in a* and b*, with the largest changes observed in the 20% HS treatment (L*= 58.7 ± 0.10; a*= 5.8 ± 0.05; b*= 60.5 ± 0.22). The results show that hemp use in hen diets leads to increased omega-3 polyunsaturated fatty acid content and color intensity of egg yolks, but does not have adverse effects on the sensory profiles of the cooked eggs. PRACTICAL APPLICATION: This study provides evidence that HS and hempseed oil (HO) can safely be utilized as feed ingredients for laying hens to produce table eggs that are enriched in essential fatty acids. Additionally, the eggs procured from these hens had similar aroma and flavor compared to eggs from hens not fed any hemp. The greater the dietary hemp inclusion, the more pigmented the resulting yolks became in terms of darkness, redness, and yellowness.     

Tailoring the chemo-resistive response of self-assembled polysaccharide-CNT sensors by chain conformation at tunnel junctions

The performances of the e-noses used for the detection of toxic volatiles or anticipated diagnosis of diseases are closely related to the nanostructure of their conductive polymer composite (CPC) vapour sensors. It is attractive to be able to adjust a nanometric parameter such as the tunnel junctions’ gap between nanofillers in the percolated network of CPC sensors, to tune their macroscopic chemo-resistive properties, i.e., sensitivity and selectivity to organic vapours. Therefore, three types of transducers (multiwalled carbon nanotubes (CNTs), CNT functionalized by amylose, and CNT functionalized by amylopectine) were hierarchically assembled by spray layer by layer and exposed to water, methanol and toluene vapours. Amylose and amylopectine macromolecules, which have the same chemical nature but a different structure, linear and highly branched, are found (by atomic force microscopy) to adsorb in different conformations on CNT surface, helical and random, respectively. Interestingly, amylose is enhancing the sensitivity of CNT transducers to water by a factor 5, whereas amylopectine increases the sensitivity to toluene by a factor 1.5. These results suggest that changing the conformation of the polysaccharides adsorbed on CNT not only allows to tailor CPC chemo-resistive sensors sensitivity but also their selectivity by a specific orientation of chemical groups.     

A Low-Protein,High-Carbohydrate Diet Increases Fatty Acid Uptake and Reduces Norepinephrine-Induced Lipolysis in Rat Retroperitoneal White Adipose Tissue

A low-protein, high-carbohydrate (LPHC) diet for 15 days increased the lipid content in the carcass and adipose tissues of rats. The aim of this work was to investigate the mechanisms of this lipid increase in the retroperitoneal white adipose tissue (RWAT) of these animals. The LPHC diet induced an approximately two- and tenfold increase in serum corticosterone and TNF-α, respectively. The rate of de novo fatty acid (FA) synthesis in vivo was reduced (50%) in LPHC rats, and the lipoprotein lipase activity increased (100%). In addition, glycerokinase activity increased (60%), and the phosphoenolpyruvate carboxykinase content decreased (27%). Basal [U-¹⁴C]-glucose incorporation into glycerol-triacylglycerol did not differ between the groups; however, in the presence of insulin, [U-¹⁴C]-glucose incorporation increased by 124% in adipocytes from only control rats. The reductions in IRS1 and AKT content as well as AKT phosphorylation in the RWAT from LPHC rats and the absence of an insulin response suggest that these adipocytes have reduced insulin sensitivity. The increase in NE turnover by 45% and the lack of a lipolytic response to NE in adipocytes from LPHC rats imply catecholamine resistance. The data reveal that the increase in fat storage in the RWAT of LPHC rats results from an increase in FA uptake from circulating lipoproteins and glycerol phosphorylation, which is accompanied by an impaired lipolysis that is activated by NE.     

A multi-residue method for the determination of 90 pesticides in matrices with a high water content by LC-MS/MS without clean-up

A method using QuEChERS extraction and LC-MS/MS in electrospray positive ionisation mode was developed and validated for the analysis of 90 pesticides in a high water content matrix (tomato) in a single chromatographic run. To assess the intra-laboratory reproducibility of the method, validation was conducted on four different days by two different analysts. The validation data was treated using a spreadsheet developed in-house, which sets the most appropriate model for linear fit by determining whether the residuals of the calibration curves are homocedastic or heterocedastic. A statistical test for the significance of regression was also carried out. Calibration was always matrix-matched and the curves were obtained over the range 0.0075-0.10 or 0.020-0.125 mg kg(-1). Identification of analytes was based on retention times and MRM ratios. Recoveries were assessed at four different levels for each analyte and were between 73 and 106%, with relative standard deviations under reproducibility conditions of <20%. The measurement uncertainties of the method for each pesticide analysed were below 50%. Previous validation of the same method, applied to papaya samples and satisfactory results obtained in various proficiency tests with different high water content matrices, demonstrated the applicability of the method to these classes of commodities, without clean-up. The validated method will be applied routinely in the pesticide residues monitoring programme that constitutes the National Residue and Contaminant Control Plan of Brazil.