Development and validation of an improved method for the detection of Salmonella in cinnamon bark and oregano leaves using the adsorbent beta zeolite in the pre-enrichment media.

The detection of Salmonella in spices is challenging due to the presence of antibacterial components. In this study, we evaluated the use of an adsorbent beta zeolite in pre-enrichment media to improve the recovery of Salmonella from cinnamon bark and oregano leaves. Samples (25 g) were spiked with varying levels of S. Montevideo or S. Senftenberg. After 2 weeks of stabilization at RT, betazeolite was added to cinnamon and oregano samples prior to the addition of 225 mL or 475 mL of pre-enrichment media, respectively. Detection sensitivity and rate of the test method were compared to the FDA Bacteriological Analytical Manual (BAM) method which requires the use of 2.5 L pre-enrichment broth. While Salmonella could not be detected in the test method using the reduced volume of pre-enrichment media alone, the addition of beta zeolite resulted in a positivity rate of 62% and 72.6% for cinnamon bark and oregano leaves respectively (all spike levels and both serovars combined). Furthermore, while there were differences in the LOD50 compared to the BAM method, there was no significant difference in the minimum level of detection between the betazeolite and the BAM methods. Our results demonstrate that the use of betazeolite in the pre-enrichment media offers a method with reduced media volumes without compromising on the sensitivity or efficiency of Salmonella detection in cinnamon bark and oregano leaves.

A loop-mediated isothermal amplification (LAMP) assay for the consensus detection of human pathogenic Campylobacter species.

Most rapid identification methods for Campylobacter are designed to detect thermotolerant Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli). A growing number of thermosensitive Campylobacter species are now gaining recognition as emerging human pathogens. Methods are lacking for the rapid screening of these emerging species. Loop-mediated Isothermal Amplification (LAMP) is a nucleic acid amplification method that allows for the rapid and cost-effective detection of bacteria. Degenerate primers against the 16S rRNA sequences for C. jejuni, C. coli, C. lari, C. upsaliensis, C. ureolyticus, C. fetus, C. gracilis, C. rectus, and C. concisus were designed. Isothermal amplification was conducted using ATCC reference strains at 68 °C for 30 min using WarmStart® Colorimetric LAMP reagents. Positive reactions were indicated by a color change from pink to yellow; specificity to Campylobacter was confirmed using a restriction enzyme digest (RsaI). The developed LAMP reaction was specific for the reference strains, which was confirmed against an exclusivity panel that consisted of other enteric pathogens, including E. coli, Salmonella, Shigella, Helicobacter, and Arcobacter. This method was also evaluated for the detection of C. jejuni, C. coli, and C. lari in primary enrichment media from artificially contaminated fresh spinach samples. The LAMP method provides an option to rapidly screen for the presence of pathogenic Campylobacter spp. in field surveillance and trace-back analysis.

Genetically Engineered Human Kidney Cells for Real-Time Cytotoxicity Testing In Vitro.

Classic toxicology studies often utilize in vivo animal models. Newer approaches employing in vitro organ-specific cellular models have been developed in recent years to help accelerate the speed and reduce the cost of traditional toxicology testing. Toward the goal of supporting in vitro cellular model research with a regulatory application in mind, we have developed a 'designer' human kidney cell line called HK2-Vi that can fluorescently measure the cytotoxicity of potential toxins on proximal tubule cell viability in a direct exposure in vitro model. HK2-Vi was designed to be a reagent-less kinetic assay that can yield data on short- or long-term cell viability after toxin exposure. To generate HK2-Vi, we used monocistronic lentiviral transduction methods to genetically engineer a human kidney cell line called HK-2 to stably co-express two transgenes. The first is Perceval HR, which encodes a fluorescent biosensor of both cytosolic ATP and ADP and the second is pHRed, which encodes a biosensor of cytosolic pH. Relative levels of cellular ATP and ADP effectively serve as a reliable and robust indicator of cell viability. Because the fluorescence Perceval HR is pH-dependent, we co-expressed the pHRed genetic biosensor to correct for variations in pH if necessary. Heterogenous populations of transduced renal cells were enriched by flow cytometry before monoclonal cellular populations were isolated by cell culture methods. A single clonal population of co-transduced cells expressing both Perceval HR and pHRed was selected to be HK2-Vi. This established cell line can now serve as a tool for in vitro toxicology testing and the methods described herein serve as a model for developing designer cell lines derived from other organs.

Diet-induced obesity precipitates kidney dysfunction and alters inflammatory mediators in mice treated with Shiga Toxin 2.

Shiga Toxin (Stx)-producing E. coli (STEC) continue to be a prominent cause of foodborne outbreaks of hemorrhagic colitis worldwide, and can result in life-threatening diseases, including hemolytic uremic syndrome (HUS), in susceptible individuals. Obesity-associated immune dysfunction has been shown to be a risk factor for infectious diseases, although few studies have addressed the role of obesity in foodborne diseases. We hypothesized that obesity may affect the development of HUS through an alteration of immune responses and kidney function. We combined diet-induced obese (DIO) and HUS mouse models to look for differences in disease outcome between DIO and wild-type (WT) male and female C57 B l/6 mice. Following multiple intraperitoneal injections with endotoxin-free saline or sublethal doses of purified Stx2, we examined DIO and WT mice for signs of HUS development. DIO mice receiving Stx2 injections lost more body weight, and had significantly higher (p < 0.001) BUN, serum creatinine, and neutrophil counts compared to WT mice or DIO mice receiving saline injections. Lymphocyte counts were significantly (p < 0.05) lower in Stx2-treated obese mice compared to WT mice or saline-treated DIO mice. In addition to increased Stx2-induced kidney dysfunction, DIO mouse kidneys also had significantly increased expression of IL-1α, IL-1ß, IL-6, TNF-α, MCP-1, and KC RNA compared to saline controls (p < 0.05). Serum cytokine levels of IL-6 and KC were also significantly higher in Stx2-treated mice compared to saline controls, but there were no significant differences between the WT and DIO mice. WT and DIO mice treated with Stx2 exhibited significantly higher degrees of kidney tubular dilation and necrosis as well as some signs of tissue repair/regeneration, but did not appear to progress to the full pathology typically associated with human HUS. Although the combined obesity/HUS mouse model did not manifest into HUS symptoms and pathogenesis, these data demonstrate that obesity alters kidney function, inflammatory cells and cytokine production in response to Stx2, and may play a role in HUS severity in a susceptible model of infection.

Effects of maternal silver acetate exposure on immune biomarkers in a rodent model.

Male and female rats (26-day old) were exposed to 0.0, 0.4, 4 or 40 mg/kg body weight silver acetate (AgAc) in drinking water for 10 weeks prior to and during mating. Sperm positive females remained within their dose groups and were exposed to AgAc during gestation and lactation. Splenic and thymic lymphocyte subsets from F1 generation PD (postnatal day) 4 and 26 pups were assessed by flow cytometry for changes in phenotypic markers. Spleens from PD4 pups had lower percentages of CD8+ lymphocytes in 4 and 40 mg/kg AgAc exposed groups and reduced Concanavalin A (Con A) response at all AgAc exposure groups. Splenic maturation increased in PD26 pups compared to PD4 pups. Con A and lipopolysaccharide (LPS) mediated splenic responses were lower in PD26 pups exposed to 40 mg/kg AgAc. Changes in PD 26 pup splenocyte phenotypic markers included lower TCR + cells at 4 and 40 mg/kg AgAc exposure and higher B cell population in the 40 mg/kg AgAc. PD26 pup splenic natural killer cell (NK) activity was higher in the 0.4 AgAc group and unchanged in 4 and 40 mg/kg AgAc groups. In conclusion, maternal exposure to AgAc had a significant impact on rat splenic development during the early lactation period.

Tissue colonization and circulating T lymphocytes in laying hens upon oral challenge with Salmonella enterica serovars.

Evaluating the potential of Salmonella serovars for tissue colonization and egg contamination in laying hens is critical due to widespread consumption of poultry and egg-containing products. The 2009 FDA Egg Rule was implemented to target the eradication of Salmonella enterica Enteritidis (SE) from layers; however, other Salmonella serovars, such as Heidelberg (SH) and Typhimurium (ST), have also been associated with poultry-related outbreaks. We conducted this study to see if serovars other than SE could colonize in laying hens, cause egg contamination, and modulate circulating T-cell populations. Laying hens were orally gavaged with 107 colony forming units (CFU) of SE, SH, or ST and assessed for colonization in spleen, ovaries, and oviduct 10 d postchallenge. Splenic colonization was similar for all the serovars; however, colonization of ovaries and oviducts was significantly higher with SH compared to SE and ST. Furthermore, SH challenge resulted in egg contamination, while SE and ST did not result in contaminated eggs. Phenotypic evaluation of peripheral blood lymphocytes showed significant reduction in CD4 cells in SH-challenged birds and lower CD8α and CD8ß cells in SE-challenged birds compared to controls. Our data showed that non-SE serovars have equal or higher potential to colonize reproductive tissues of laying hens and may be accompanied by altered lymphocyte populations.

Immunophenotype and gene expression profile of mesenchymal stem cells derived from canine adipose tissue and bone marrow.

Veterinary adult stem cell therapy is an emerging area of basic and clinical research. Like their human counterparts, veterinary mesenchymal stem cells (MSCs) offer many potential therapeutic benefits. The characterization of canine-derived MSCs, however, is poorly defined compared to human MSCs. Furthermore, little consensus exists regarding the expression of canine MSC cell surface markers. To address this issue, this study investigated characteristics of cultured canine MSCs derived from both adipose tissue and bone marrow. The canine MSCs were obtained from donors of various breeds and ages. A panel of cell surface markers for canine MSCs was selected based on current human and canine literature and the availability of canine-reactive antibodies. Using flow cytometry, canine MSCs were defined to be CD90(+)CD44(+)MHC I(+)CD14(-)CD29(-)CD34(-)MHC II(-). Canine MSCs were further characterized using real-time RT-PCR as CD105(+)CD73(+)CD14(+)CD29(+)MHC II(+)CD45(-) at the mRNA level. Among these markers, canine MSCs differed from canine peripheral blood mononuclear cells (PBMCs) by the absence of CD45 expression at the mRNA level. A novel high-throughput canine-specific PCR array was developed and used to identify changes in the gene expression profiles of canine MSCs. Genes including PTPRC, TNF, ß2M, TGFß1, and PDGFRß, were identified as unique to canine MSCs as compared to canine PBMCs. Our findings will facilitate characterization of canine MSCs for use in research and clinical trials. Moreover, the high-throughput PCR array is a novel tool for characterizing canine MSCs isolated from different tissues and potentially from different laboratories.

Maternal and postnatal dietary probiotic supplementation enhances splenic regulatory T helper cell population and reduces peanut allergen-induced hypersensitivity responses in mice.

Neonatal to early childhood is the critical period for establishing a balance of T helper 1 (Th1) versus T helper 2 (Th2) cellular immunity within the gut, which is strongly influenced by the source and establishment of gut microflora. Probiotic administration has been shown to attenuate Th2-biased cellular immunity and predisposition to food allergies. To test this hypothesis we provided ad libitum a probiotic-supplemented (Primalac 454 Feed Grade Microbials) or control diet to lactating dams with suckling pups and weaned pups until 10 weeks of age. Weaned mice were sensitized/challenged with peanut extract, saline or adjuvant at 6, 8 and 10 weeks of age. At 3, 6, 8 and 10 weeks, fecal samples were collected for microbial analysis, while blood samples were analyzed for total plasma IgE levels. At termination (10 weeks of age), splenic T lymphocyte population subtypes were determined using FACS analysis and Th1/Th2/Th17 gene expression by PCR array. Mice given the probiotic-supplemented diet had significantly enhanced probiotic fecal counts compared to controls at 3, 6, 8 and 10 weeks. Moreover, mice fed the probiotic-supplemented diet had enhanced splenic naturally occurring T regulatory cell populations, and reduced splenic gene expression of allergic mediator IL-13 compared to controls. These results provide evidence that early probiotic supplementation may provide host protection to hypersensitivity reactions to food allergens by attenuating food allergen inflammatory responses.

Ecto-5'-nucleotidase (CD73) regulates host inflammatory responses and exacerbates murine salmonellosis.

Food-borne Salmonella spp., are a major cause of hospitalization and death. Adenosine, an important immune regulator of inflammation, limits tissue damage during infection. CD39 (nucleoside triphosphate dephosphorylase) combined with ecto-5'-nucleotidase (CD73) metabolizes ATP to adenosine. We studied the expressions of CD39 and CD73 in tissues, and T helper cells in mice after Salmonella infection and evaluated the role of CD73 in regulating immune responses and bacterial clearance in wild-type and CD73-deficient (CD73(-/-)) mice. Both CD39 and CD73 transcript levels declined in the infected wild-type mice. Compared to wild-type mice, tissues from infected CD73(-/-) mice had significantly higher expression of pro-inflammatory cytokines and reduced anti-inflammatory responses. CD73(-/-) mice were more resistant to infection and had a greater inflammatory responses and a significantly lower bacterial load in the liver compared to wild-type mice. Thus, CD73 expression attenuates inflammation during murine Salmonellosis and impairs immunity, leading to increased bacterial colonization and prolonged infection.

Maternal and postnatal dietary probiotic supplementation enhances splenic regulatory T helper cell population and reduces ovalbumin allergen-induced hypersensitivity responses in mice.

Neonatal to early childhood is the critical period for establishing a balance of T helper 1 (Th1) versus T helper 2 (Th2) cellular immunity within the gut, which is strongly influenced by the source and establishment of gut microflora. Probiotic administration has been shown to attenuate Th2-biased cellular immunity and predisposition to food allergies. To test this hypothesis we provided ad libitum a probiotic-supplemented (Primalac 454 Feed Grade Microbials) or control diet to lactating dams with suckling pups and weaned pups until 10 weeks of age. Weaned mice were sensitized/challenged with egg allergen ovalbumin, saline or adjuvant at 6, 8 and 10 weeks of age. At 3, 6, 8 and 10 weeks, fecal samples were collected for microbial analysis, while blood samples were analyzed for ovalbumin-IgE and total plasma IgE levels. At termination, splenic T helper cell lymphocyte population subtypes were determined using FACS analysis and Th1/Th2/Th17 gene expression by PCR array. At 21 days of age, pups suckled by lactating dams fed the probiotic supplemented diet had significantly enhanced Lactobacillus acidophilus fecal counts compared to controls. Moreover, mice fed the probiotic supplemented diet had enhanced splenic naturally occurring and induced regulatory T cell populations, enhanced TGFß gene expression and reduced expression of allergic mediator IL13 compared to controls. These results provide evidence that early probiotic supplementation may provide host protection from hypersensitivity reactions to food allergens by attenuating food allergen inflammatory responses.

Effect of processing on recovery and variability associated with immunochemical analytical methods for multiple allergens in a single matrix: sugar cookies.

Among the major food allergies, peanut, egg, and milk are the most common. The immunochemical detection of food allergens depends on various factors, such as the food matrix and processing method, which can affect allergen conformation and extractability. This study aimed to (1) develop matrix-specific incurred reference materials for allergen testing, (2) determine whether multiple allergens in the same model food can be simultaneously detected, and (3) establish the effect of processing on reference material stability and allergen detection. Defatted peanut flour, whole egg powder, and spray-dried milk were added to cookie dough at seven incurred levels before baking. Allergens were measured using five commercial enzyme-linked immunosorbent assay (ELISA) kits. All kits showed decreased recovery of all allergens after baking. Analytical coefficients of variation for most kits increased with baking time, but decreased with incurred allergen level. Thus, food processing negatively affects the recovery and variability of peanut, egg, and milk detection in a sugar cookie matrix when using immunochemical methods.

Effect of processing on recovery and variability associated with immunochemical analytical methods for multiple allergens in a single matrix: dark chocolate.

Immunodetection of allergens in dark chocolate is complicated by interference from the chocolate components. The objectives of this study were to establish reference materials for detecting multiple allergens in dark chocolate and to determine the accuracy and precision of allergen detection by enzyme-linked immunosorbent assay (ELISA) before and after chocolate processing. Defatted peanut flour, whole egg powder, and spray-dried milk were added to melted chocolate at seven incurred levels and tempered for 4 h. Allergen concentrations were measured using commercial ELISA kits. Tempering decreased the detection of casein and ß-lactoglobulin (BLG), but had no significant effect on the detection of peanut and egg. Total coefficients of variation were higher in tempered than untempered chocolate for casein and BLG, but total and analytical CVs were comparable for peanut and egg. These findings indicate that processing has a greater effect on recovery and variability of casein and BLG than peanut and egg detection in a dark chocolate matrix.

Identification of an I-Ed-restricted T-cell epitope of Escherichia coli outer membrane protein F.

A predominant T-cell epitope of Escherichia coli outer membrane protein F (OmpF) that encompasses amino acids 295 to 314 was identified in H-2(d) mice. BALB/c-derived T-cell hybridomas generated against this region were CD3(+), CD4(+), CD8(-), and T-cell receptor alphabeta(+) and secreted TH-1-associated cytokines (interleukin-2 [IL-2] and gamma interferon), but not a TH-2-associated cytokine (IL-4), when restimulated with peptide 295-314. Class II(+) mouse lymphoma (A20) cells, but not class II(-) mouse mastocytoma (P815) cells, supported IL-2 secretion of hybridomas when substituted for syngeneic splenocytes as antigen-presenting cells (APCs). Antibodies specific for I-E(d) blocked IL-2 secretion by hybridomas, but I-A(d)-specific antiserum did not. When transfected L cells expressing I-A(d) (AalphaAbeta(d)), I-E(d) (EalphaEbeta(d)), or the hybrid molecule I-EalphaAbeta(d) were used as APCs, hybridomas recognized peptide only when presented by the I-E(d)-transfected cells. When peptide 295-314 truncated at either the C or the N terminus of the sequence was used, the minimal epitope was determined. Critical residues were determined by using alanine-substituted peptide analogues. T-cell hybridomas were only stimulated by peptides that encompassed amino acids 295 to 303 (9-mer), and the core sequence required a minimum of three additional amino acids at either the amino or the carboxy terminus to induce IL-2 secretion. Critical residues were determined to be phenylalanine at position 295, threonine at position 300, and tyrosines at positions 301 and 302. This study is the first to identify a minimal T-cell epitope and major histocompatibility complex restriction element of the OmpF protein and confirms previous observations that there is considerable degeneracy in the length of peptides that can bind I-E(d) and variability in the amino acid composition of the C and N termini of these peptides.