A longitudinal study to examine the influence of farming practices and environmental factors on pathogen prevalence using structural equation modeling.

The contamination of fresh produce with foodborne pathogens has been an on-going concern with outbreaks linked to these commodities. Evaluation of farm practices, such as use of manure, irrigation water source, and other factors that could influence pathogen prevalence in the farming environment could lead to improved mitigation strategies to reduce the potential for contamination events. Soil, water, manure, and compost were sampled from farms in Ohio and Georgia to identify the prevalence of Salmonella, Listeria monocytogenes (Lm), Campylobacter, and Shiga-toxin-producing Escherichia coli (STEC), as well as Arcobacter, an emerging human pathogen. This study investigated agricultural practices to determine which influenced pathogen prevalence, i.e., the percent positive samples. These efforts identified a low prevalence of Salmonella, STEC, and Campylobacter in soil and water (< 10%), preventing statistical modeling of these pathogens. However, Lm and Arcobacter were found in soil (13 and 7%, respectively), manure (49 and 32%, respectively), and water samples (18 and 39%, respectively) at a comparatively higher prevalence, suggesting different dynamics are involved in their survival in the farm environment. Lm and Arcobacter prevalence data, soil chemical characteristics, as well as farm practices and weather, were analyzed using structural equation modeling to identify which factors play a role, directly or indirectly, on the prevalence of these pathogens. These analyses identified an association between pathogen prevalence and weather, as well as biological soil amendments of animal origin. Increasing air temperature increased Arcobacter and decreased Lm. Lm prevalence was found to be inversely correlated with the use of surface water for irrigation, despite a high Lm prevalence in surface water suggesting other factors may play a role. Furthermore, Lm prevalence increased when the microbiome's Simpson's Diversity Index decreased, which occurred as soil fertility increased, leading to an indirect positive effect for soil fertility on Lm prevalence. These results suggest that pathogen, environment, and farm management practices, in addition to produce commodities, all need to be considered when developing mitigation strategies. The prevalence of Arcobacter and Lm versus the other pathogens suggests that multiple mitigation strategies may need to be employed to control these pathogens.

Whole genome characterization of thermophilic Campylobacter species isolated from dairy manure in small specialty crop farms of Northeast Ohio.

Introduction: With more public interest in consuming locally grown produce, small specialty crop farms (SSCF) are a viable and growing segment of the food production chain in the United States. Methods: The goal of this study was to investigate the genomic diversity of Campylobacter isolated from dairy manure (n = 69) collected from 10 SSCF in Northeast Ohio between 2018 and 2020. Results: A total of 56 C. jejuni and 13 C. coli isolates were sequenced. Multi-locus sequence typing (MLST) identified 22 sequence types (STs), with ST-922 (18%) and ST-61 (13%) predominant in C. jejuni and ST-829 (62%) and ST-1068 (38%) predominant in C. coli. Interestingly, isolates with similar genomic and gene contents were detected within and between SSCF over time, suggesting that Campylobacter could be transmitted between farms and may persist in a given SSCF over time. Virulence-associated genes (n = 35) involved in the uptake and utilization of potassium and organic compounds (succinate, gluconate, oxoglutarate, and malate) were detected only in the C. jejuni isolates, while 45 genes associated with increased resistance to environmental stresses (capsule production, cell envelope integrity, and iron uptake) were detected only in the C. coli isolates. Campylobacter coli isolates were also sub-divided into two distinct clusters based on the presence of unique prophages (n = 21) or IncQ conjugative plasmid/type-IV secretion system genes (n = 15). Campylobacter coli isolates harbored genes associated with resistance to streptomycin (aadE-Cc; 54%) and quinolone (gyrA-T86I; 77%), while C. jejuni had resistance genes for kanamycin (aph3'-IIIa; 20%). Both species harbored resistance genes associated with ß-lactam (especially, blaOXA-193; up to 100%) and tetracycline (tetO; up to 59%). Discussion/Conclusion: Our study demonstrated that Campylobacter genome plasticity associated with conjugative transfer might provide resistance to certain antimicrobials and viral infections via the acquisition of protein-encoding genes involved in mechanisms such as ribosomal protection and capsule modification.

Current methodologies and future direction of Campylobacter isolation and detection from food matrices, clinical samples, and the agricultural environment.

Campylobacter spp. are the leading cause of bacterial foodborne infections in both developed and developing countries. The food commodities primarily attributed to campylobacteriosis include raw milk, poultry, seafood, and fresh produce. Furthermore, insects, animal/bird fecal material, and agricultural water have been shown to be the sources of Campylobacter contamination in these commodities. Both established and emerging species of Campylobacter have been recovered from food and environmental sources. Therefore, optimal detection and isolation of Campylobacter spp., including the emerging species, is critical for improved surveillance, prevention, and traceback of Campylobacter outbreaks. This review focuses on the existing variability in Campylobacter enrichment and isolation procedures used by researchers and regulatory agencies worldwide, for various matrices. Additionally, the challenges associated with developing and validating new culture, molecular, and immunological methods for rapid and sensitive Campylobacter detection are discussed.

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.

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.

In vivo and in vitro evaluation of tissue colonization and survival capacity of Salmonella Oranienburg in laying hens.

Salmonella enterica serovar Oranienburg (SO) was linked to a human salmonellosis outbreak in the Midwest in 2015 and 2016 from consumption of eggs. However, unlike Salmonella enterica serovar Enteritidis (SE), little is known regarding the potential of SO to colonize in laying hens and contaminate eggs. We used in vivo and in vitro models to evaluate tissue colonization and survival capacity of SO. Twenty eight-week-old laying hens were each challenged with an oral dose of approximately 107 (n = 92) or 109 (n = 96) colony-forming units (CFU) in 1 mL saline and evaluated after 1, 2, and 4 wk. Standard microbiological methods with pre-enrichment and enrichment in selective media were used for detection of SO in tissues, egg shell wash, internal egg contents, and excreta. Peak colonization of spleen (86.9%), ovaries (31.6%), upper oviduct (15.8%), and lower oviduct (34.3%) was detected between 1 and 2 wk post-infection (pi), while at 4 wk SO was only recovered from spleens (25%). Salmonella enterica serovar Oranienburg was not recovered from internal egg contents. However, the presence of SO on egg shells was seen when there were traces of excreta. Shedding in excreta was found in 92 and 100% birds gavaged with 107 and 109 CFU at 2 wk pi, respectively. The invasion and proliferation of SO in ovarian granulosa cells (GC) was compared to that of SE, and while the invasion of SO into GC was comparable to SE, proliferation of SO was significantly lower (P < 0.05). The infective potential of SO was also assessed by enumerating survival in egg white over 4 wk under refrigerated conditions, resulting in 65% survival at 4 wk. Overall, our data suggested that SO infection in layers did not result in egg contamination via vertical transmission, and colonization of egg-forming tissues was limited to 2 wk pi. Survival within GC and egg white demonstrates the ability of SO to withstand antibacterial factors and the potential of SO to penetrate the yolk.

Genotypic and phenotypic characterization of multidrug resistant Salmonella Typhimurium and Salmonella Kentucky strains recovered from chicken carcasses.

Salmonella Typhimurium is the leading cause of human non-typhoidal gastroenteritis in the US. S. Kentucky is one the most commonly recovered serovars from commercially processed poultry carcasses. This study compared the genotypic and phenotypic properties of two Salmonella enterica strains Typhimurium (ST221_31B) and Kentucky (SK222_32B) recovered from commercially processed chicken carcasses using whole genome sequencing, phenotype characterizations and an intracellular killing assay. Illumina MiSeq platform was used for sequencing of two Salmonella genomes. Phylogenetic analysis employing homologous alignment of a 1,185 non-duplicated protein-coding gene in the Salmonella core genome demonstrated fully resolved bifurcating patterns with varying levels of diversity that separated ST221_31B and SK222_32B genomes into distinct monophyletic serovar clades. Single nucleotide polymorphism (SNP) analysis identified 2,432 (ST19) SNPs within 13 Typhimurium genomes including ST221_31B representing Sequence Type ST19 and 650 (ST152) SNPs were detected within 13 Kentucky genomes including SK222_32B representing Sequence Type ST152. In addition to serovar-specific conserved coding sequences, the genomes of ST221_31B and SK222_32B harbor several genomic regions with significant genetic differences. These included phage and phage-like elements, carbon utilization or transport operons, fimbriae operons, putative membrane associated protein-encoding genes, antibiotic resistance genes, siderophore operons, and numerous hypothetical protein-encoding genes. Phenotype microarray results demonstrated that ST221_31B is capable of utilizing certain carbon compounds more efficiently as compared to SK222_3B; namely, 1,2-propanediol, M-inositol, L-threonine, α-D-lactose, D-tagatose, adonitol, formic acid, acetoacetic acid, and L-tartaric acid. ST221_31B survived for 48 h in macrophages, while SK222_32B was mostly eliminated. Further, a 3-fold growth of ST221_31B was observed at 24 hours post-infection in chicken granulosa cells while SK222_32B was unable to replicate in these cells. These results suggest that Salmonella Typhimurium can survive host defenses better and could be more invasive than Salmonella Kentucky and provide some insights into the genomic determinants responsible for these differences.

Comparative responses of chicken macrophages to infection with Salmonella enterica serovars.

Poultry products such as meat and eggs are known reservoirs for Salmonella serovars. Macrophages play an important role by limiting bacterial replication using several defense mechanisms including immune and inflammatory mediators, antibacterial proteins, reactive nitrogen and oxygen species. In this study, we evaluate transcriptional changes in Toll-like receptors, immune/inflammatory cytokines, chemokines, antibacterial factors, and nitric oxide (NO) production in HD11 chicken macrophages in response to intracellular persistence of poultry-derived Salmonella enterica Enteritidis (SE), Typhimurium (ST), and Heidelberg (SH) that were associated with human salmonellosis. Invasion of ST was higher than SE or SH; however, SH persistence in HD11 cells at 18 h post infection (hpi) was more pronounced than the other 2 serovars. In comparison to the uninfected control HD11 cells, expression of TLR5 was >2 fold higher for SE and SH which was followed by up-regulation of downstream signal transduction molecules. Significant up-regulation of antibacterial peptides, proinflammatory chemokines, cytokines, and NO production were observed in response to SE, SH, and ST at 18 hpi. These results indicate that although antibacterial factors contribute to the clearance of invading Salmonella, some of the differences in response could also be due to the different virulence properties of these serovars.

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.

Differential antibacterial response of chicken granulosa cells to invasion by Salmonella serovars.

In the United States, Salmonella enterica ser. Enteritidis (SE) is among the leading bacterial cause of foodborne illness via consumption of raw or undercooked eggs. The top Salmonella serovars implicated in U.S. foodborne outbreaks associated with chicken consumption include SE, Typhimurium (ST), Heidelberg (SH), Montevideo, Mbandka, Braenderup, and Newport. While enforcement actions target the eradication of SE from layer hens, there is a growing concern that other serovars could occupy this niche and be a cause of egg-transmitted human salmonellosis. Therefore, we tested the invasion and survival of SE, SH, ST, and Salmonella enterica ser. Hadar (S. Hadar) at 4 and 20 h post infection (hpi) in chicken ovarian granulosa cells (cGC); a cellular layer which surrounds the previtelline layer and central yolk in egg-forming follicles. We also evaluated cGC transcriptional changes, using an antibacterial response PCR array, to assess host response to intracellular SalmonellaWe observed that invasion of cGC by SE, SH, and ST was significantly higher than invasion by S. Hadar, with ST showing the highest level of invasion. The Bacterial Survival Index, defined as the ratio of intracellular bacteria at 20 and 4 h, were 18.94, 7.35, and 15.27 for SE, SH, and ST, respectively, with no significant difference in survival between SE or ST compared to SH. Evaluation of cGC anti-Salmonella gene responses indicated that at 4 hpi there was a significant decrease in Toll-like receptor (TLR)-4 mRNA in cGC infected with SE, whereas TLR5 and myeloid differentiation primary response gene 88 were significantly down regulated across all serovars. At 4 hpi, invasion by Salmonella serovars resulted in significant upregulation of several antimicrobial genes, and proinflammatory cytokines and chemokines (PICs). At 20 hpi, all the serovars induced PICs with SH being the strongest inducer. Additionally, SE, SH and ST differentially induced signal transduction pathways. Although only a single strain from each serovar was tested, cGC presents a useful ex vivo cell culture model to assess the virulence potential of Salmonella serovars.

Vitamin D2 from UVB light exposed mushrooms modulates immune response to LPS in rats.

SCOPE: Poor vitamin D (vitD) status is linked to increased risk of infectious diseases, thus there is need for vitD-rich foods. UVB-exposed mushrooms synthesize vitD2 but knowledge of bioavailability and function in immune response is lacking. METHODS AND RESULTS: One hundred rats were fed one of five diets--control, 20 IU vitD3/day; no vitD3/day; 5% unexposed mushroom, 2.4 IU vitD2/day; 2.5% UVB mushroom, 300 IU vitD2/day; and 5% UVB mushroom, 600 IU vitD2/day--for 10 wk and challenged with either saline or the endotoxin LPS. Blood and tissues were collected at 3 h postchallenge. Plasma 25-hydroxyvitamin D (25OHD) levels from UVB-exposed mushroom fed rats were significantly elevated and associated with higher natural killer cell activity and reduced plasma inflammatory response to LPS compared to control diet fed rats. Microarray evaluation of rat spleens for changes in inflammatory gene expression showed significant upregulation of proinflammatory genes after LPS compared to saline controls in all groups. However, compared to control rats, upregulation of the proinflammatory genes was markedly reduced in the groups fed vitD2-enriched mushrooms. CONCLUSION: Rats fed UVB-exposed mushrooms had significantly higher plasma total 25OHD levels that were associated with increased innate immune response and anti-inflammatory effects.

Dietary fatty acids and immune response to food-borne bacterial infections.

Functional innate and acquired immune responses are required to protect the host from pathogenic bacterial infections. Modulation of host immune functions may have beneficial or deleterious effects on disease outcome. Different types of dietary fatty acids have been shown to have variable effects on bacterial clearance and disease outcome through suppression or activation of immune responses. Therefore, we have chosen to review research across experimental models and food sources on the effects of commonly consumed fatty acids on the most common food-borne pathogens, including Salmonella sp., Campylobacter sp., Shiga toxin-producing Escherichia coli, Shigella sp., Listeria monocytogenes, and Staphylococcus aureus. Altogether, the compilation of literature suggests that no single fatty acid is an answer for protection from all food-borne pathogens, and further research is necessary to determine the best approach to improve disease outcomes.

Effects of fructooligosaccharide-inulin on Salmonella-killing and inflammatory gene expression in chicken macrophages.

Salmonella Enteritidis (SE) is one of the leading causes of food-borne salmonellosis, and macrophages play an essential role in eliminating this pathogen. Among the interventions to improve Salmonella clearance in chickens are the use of prebiotics and direct fed microbials (DFM) in animal feed as they have immunomodulatory effects. Therefore, we tested the influence of a prebiotic fructooligosaccharide (FOS)-inulin on the ability of the chicken macrophage HD11 cell line to phagocytose and kill SE, and express selected inflammatory cytokines and chemokines in an in vitro model. There were significantly fewer viable intracellular SE in HD11 cells treated with FOS-inulin than the untreated cells. However, SE phagocytosis, nitric oxide expression or production were not influenced by the prebiotic treatment. Among the inflammatory markers tested, IL-1ß expression was significantly lower in HD11 cells treated with FOS-inulin. These results suggest that FOS-inulin has the ability to modulate the innate immune system as shown by the enhanced killing of SE and decreased inflammasome activation.

Bacillus subtilis-based direct-fed microbials augment macrophage function in broiler chickens.

The present study was conducted to evaluate the function of Bacillus subtilis-based direct-fed microbials (DFMs) on macrophage functions, i.e., nitric oxide (NO) production and phagocytosis in broiler chickens. DFMs used in this study were eight single strains designated as Bs2084, LSSAO1, 3AP4, Bs18, 15AP4, 22CP1, Bs27, and Bs278, and one multiple strain DFM product (Avicorr™) containing equal amount of Bs2084, LSSAO1 and 15AP4. NO concentrations were monitored in plasma and in the supernatants from the peripheral blood-derived monocytic cells (PBMC)-derived macrophages stimulated by either chicken recombinant interferon gamma (IFNγ) or lipopolysaccharide (LPS) from Escherichia coli or Salmonella typhi. In addition, phagocytosis of fluorescent beads and green fluorescent protein (GFP)-labeled Salmonella by PBMC-derived macrophage was assayed. Plasma NO levels were significantly higher in groups given 3AP4 or Bs27 diets compared with the control group at days 7 and 14. NO production by PBMC-derived macrophages stimulated with IFNγ or LPS was apparent, although the effect was strain-dependent. Phagocytosis of fluorescent beads or GFP-labeled Salmonella by macrophages was augmented in groups on DFM-supplemented diets compared with those fed the control diet. This study describes the immunomodulatory effects of Bacillus-based DFMs on innate immunity in broiler chickens.

Modern India and the vitamin D dilemma: evidence for the need of a national food fortification program.

India is located between 8.4 and 37.6 degrees N latitude with the majority of its population living in regions experiencing ample sunlight throughout the year. Historically, Indians obtained most of their vitamin D through adequate sun exposure; however, darker skin pigmentation and the changes which have accompanied India's modernization, including increased hours spent working indoors and pollution, limit sun exposure for many. Inadequate sun exposure results in reduced vitamin D synthesis and ultimately poor vitamin D status if not compensated by dietary intake. Dietary vitamin D intake is very low in India because of low consumption of vitamin D rich foods, absence of fortification and low use of supplements. All these factors contribute to poor vitamin D status as measured by low circulating levels of 25-hydroxy vitamin D. Our review searches the published literature specific to India for evidence that would confirm the need to fortify food staples with vitamin D or stimulate public health policies for vitamin D supplementation and dietary guidelines tailored to the Indian diet. This review documents findings of widespread vitamin D deficiency in Indian populations in higher and lower socioeconomic strata, in all age groups, in both genders and people in various professions. Moreover, poor vitamin D status in India is accompanied by increased bone disorders including osteoporosis, osteomalacia in adults and rickets and other bone deformities in children. Without a concerted national effort to screen for vitamin D status, to implement policies or guidelines for vitamin D fortification and/or supplementation and to re-assess recommended dietary intake guidelines, dramatic increase in the number of bone disorders and other diseases may lie ahead.

Use of flow cytometry in an apoptosis assay to determine pH and temperature stability of shiga-like toxin 1.

Shiga toxins and Shiga-like toxins (Stx) are a relatively large group of cytotoxins produced by certain serotypes of Shigella and E. coli (STEC). These toxins are responsible for diarrhea, hemorrhagic colitis and may induce hemolytic uremic syndrome (HUS) with serious consequences in young children. The toxins are proteins made up of 5 small B subunits responsible for binding to an outer membrane ligand on host cells and surround the larger, biologically active A subunit. For Shiga-like toxin 1 (Stx1), the cellular receptor is the carbohydrate globotriose. Stx1was purified from STEC. We utilized induction of apoptosis in the human monocyte cell line THP-1, as a biological endpoint to test the stability of Stx1 activity added to fruit punch at different pH (2-9) and temperatures (4 and 20 degrees C). A flow cytometric method was used to test for early and late apoptotic events based on binding of R-phycoerytherin-labeled annexin V to exposed membrane phosphatidyl serine. Membrane permeability to 7-Amino-actinomycin corresponds with late apoptosis or necrosis. The combination of acid pH and higher storage temperature resulted in greatest degree of toxin inactivation. This approach provides a rapid and high throughput method to determine the functional activity of Stx1, and related toxins in a food matrix.

Impact of dietary components on chicken immune system and Salmonella infection.

Salmonella enterica serovars are facultative intracellular pathogens that may cause serious illness in poultry and humans. Human infection by two common serovars, Salmonella enteritidis (SE) and Salmonella typhimurium (ST) usually occurs via food-borne transmission. Consumption of raw or undercooked contaminated eggs usually causes SE infection, while ST is transmitted by contaminated chicken meat. There are several reports on dietary interventions, including fatty acid modifications, probiotic or prebiotic treatment on the immune system and/or Salmonella clearance in chickens. The aim of this review is to compile the information on the role of major dietary components on chicken immune system and Salmonella clearance. This may help design better poultry nutrition to lower Salmonella infection in chickens and, therefore, reduce human salmonellosis.

Live attenuated Listeria monocytogenes expressing HIV Gag: immunogenicity in rhesus monkeys.

Induction of strong cellular immunity will be important for AIDS vaccine candidates. Natural infection with wild-type Listeria monocytogenes (Lm), an orally transmitted organism, is known to generate strong cellular immunity, thus raising the possibility that live attenuated Lm could serve as a vaccine vector. We sought to examine the potential of live attenuated Lm to induce cellular immune responses to HIV Gag. Rhesus macaques were immunized with Lmdd-gag that expresses HIV gag and lacks two genes in the D-alanine (D-ala) synthesis pathway. Without this key component of the bacterial cell wall, vaccine vector replication critically depends on exogenous D-ala. Lmdd-gag was given to animals either solely orally or by oral priming followed by intramuscular (i.m.) boosting; D-ala was co-administered with all vaccinations. Lmdd-gag and D-ala were well tolerated. Oral priming/oral boosting induced Gag-specific cellular immune responses, whereas oral priming/i.m. boosting induced systemic as well as mucosal anti-Gag antibodies. These results suggest that the route of vaccination may bias anti-Gag immune responses either towards T-helper type 1 (Th1) or Th2 responses; overall, our data show that live attenuated, recombinant Lmdd-gag is safe and immunogenic in primates.