Possible implications of animal models for the assessment of visceral pain.

Acute pain, provoked generally after the activation of peripheral nociceptors, is an adaptive sensory function that alerts the individual to avoid noxious stimuli. However, uncontrolled acute pain has a maladaptive role in sensory activity leading to development of a chronic pain state which persists even after the damage is resolved, or in some cases, in the absence of an initial local acute injury. Huge numbers of people suffer from visceral pain at least once during their life span, leading to substantial health care costs. Although studies reporting on the mechanism of visceral pain are accumulating, it is still not precisely understood. Therefore, this review aims to elucidate the mechanism of visceral pain through an evaluation of different animal models and their application to develop novel therapeutic approaches for treating visceral pain. To assess the nociceptive responses in viscera, several visceral pain models such as inflammatory, traction, stress and genetic models utilizing different methods of measurement have been devised. Among them, the inflammatory and traction models are widely used for studying the visceral pain mechanism of different disease conditions and post-operative surgery in humans and animals. A hapten, 2,4,6-trinitrobenzene sulfonic acid (TNBS), has been extensively used as an inflammatory agent to induce visceral pain. The traction model seems to cause a strong pain stimulation and autonomic reaction and could thus be the most appropriate model for studying the underlying visceral pain mechanism and for probing the therapeutic efficacies of various anesthetic and analgesics for the treatment of visceral pain and hyperalgesia.

Electroacupuncture intervention of visceral hypersensitivity is involved in PAR-2-activation and CGRP-release in the spinal cord.

Electroacupuncture (EA) relieves visceral hypersensitivity (VH) with underlying inflammatory bowel diseases. However, the mechanism by which EA treats ileitis-induced VH is not clearly known. To assess the effects of EA on ileitis-induced VH and confirm whether EA attenuates VH through spinal PAR-2 activation and CGRP release, goats received an injection of 2,4,6-trinitro-benzenesulfonic-acid (TNBS) solution into the ileal wall. TNBS-injected goats were allocated into VH, Sham acupuncture (Sham-A) and EA groups, while goats treated with saline instead of TNBS solution were used as the control. Goats in EA group received EA at bilateral Hou-San-Li acupoints for 0.5 h at 7 days and thereafter repeated every 3 days for 6 times. Goats in the Sham-A group were inserted with needles for 0.5 h at the aforementioned acupoints without any hand manipulation and electric stimulation. Visceromotor responses to colorectal distension, an indicator of VH, were recorded by electromyography. The terminal ileum and thoracic spinal cord (T11) were sampled for evaluating ileitis at days 7 and 22, and distribution and expression-levels of PAR-2, CGRP and c-Fos on day 22. TNBS-treated-goats exhibited apparent transmural-ileitis on day 7, microscopically low-grade ileitis on day 22 and VH at days 7-22. Goats of Sham-A, VH or EA group showed higher (P < 0.01) VH at days 7-22 than the Control-goats. EA-treated goats exhibited lower (P < 0.01) VH as compared with Sham-A or VH group. Immunoreactive-cells and expression-levels of spinal PAR-2, CGRP and c-Fos in the EA group were greater (P < 0.01) than those in the Control group, but less (P < 0.01) than those in Sham-A and VH groups on day 22. Downregulation of spinal PAR-2 and CGRP levels by EA attenuates the ileitis and resultant VH.

Survival and thermal resistance among four Salmonella serovars inoculated onto flaxseeds.

Thermal resistance among Salmonella serovars has been shown to vary, however, such data are minimal for Salmonella inoculated onto low moisture foods. We evaluated survival and subsequent thermal resistance for 32 strains of Salmonella from four serovars (Agona, Enteritidis, Montevideo, and Tennessee) on flaxseed over 24 weeks. After inoculation, flaxseeds were adjusted to aw = 0.5 and stored at 22 °C. After 24 weeks at 22 °C, strains of serovar Agona had a significantly slower rate of reduction compared to those of Enteritidis and Montevideo (adj. p < 0.05). Inoculated flaxseeds were processed at 71 °C with vacuum steam pasteurization at 4 time points during storage. Average initial D71°C values ranging from 1.0 to 1.5 min were similar across serovars. Over 24 weeks, D71°C varied in a serovar-dependent manner. D71°C at 8, 16, and 24 weeks did not change significantly for Enteritidis and Montevideo but did for Tennessee and Agona. While significant, the differences in D71°C over time were less than 1 min, indicating that storage time prior to heat treatment would have a minimal effect on the processing time required to inactivate Salmonella on flaxseed.

Salmonella enterica in Soils Amended with Heat-Treated Poultry Pellets Survived Longer than Bacteria in Unamended Soils and More Readily Transferred to and Persisted on Spinach.

Untreated biological soil amendments of animal origin (BSAAO) are commonly used as biological fertilizers but can harbor foodborne pathogens like Salmonella enterica, leading to potential transfer from soils to fruits and vegetables intended for human consumption. Heat-treated poultry pellets (HTPP) can provide produce growers with a slow-release fertilizer with a minimized risk of pathogen contamination. Little is known about the impact of HTPP-amended soil on the survival of Salmonella enterica The contributions of RpoS and formation of viable but nonculturable cells to Salmonella survival in soils are also inadequately understood. We quantified the survival of Salmonella enterica subsp. enterica serovar Newport wild-type (WT) and rpoS-deficient (ΔrpoS mutant) strains in HTPP-amended and unamended soil with or without spinach plants over 91 days using culture and quantitative PCR methods with propidium monoazide (PMA-qPCR). Simulated "splash" transfer of S. Newport from soil to spinach was evaluated at 35 and 63 days postinoculation (dpi). The S. Newport WT and ΔrpoS mutant reached the limit of detection, 1.0 log CFU/g (dry weight), in unamended soil after 35 days, whereas 2 to 4 log CFU/g (dry weight) was observed for both WT and ΔrpoS mutant strains at 91 dpi in HTPP-amended soil. S. Newport levels in soils determined by PMA-qPCR and plate count methods were similar (P > 0.05). HTPP-amended soils supported higher levels of S. Newport transfer to and survival on spinach leaves for longer periods of time than did unamended soils (P < 0.05). Salmonella Newport introduced to HTPP-amended soils survived for longer periods and was more likely to transfer to and persist on spinach plants than was S. Newport introduced to unamended soils.IMPORTANCE Heat-treated poultry pellets (HTPP) often are used by fruit and vegetable growers as a slow-release fertilizer. However, contamination of soil on farms may occur through contaminated irrigation water or scat from wild animals. Here, we show that the presence of HTPP in soil led to increased S. Newport survival in soil and to greater likelihood of its transfer to and survival on spinach plants. There were no significant differences in survival durations of WT and ΔrpoS mutant isolates of S. Newport. The statistically similar populations recovered by plate count and estimated by PMA-qPCR for both strains in the amended and unamended soils in this study indicate that all viable populations of S. Newport in soils were culturable.

Survival and Growth of Wild-Type and rpoS-Deficient Salmonella Newport Strains in Soil Extracts Prepared with Heat-Treated Poultry Pellets.

Manure runoff can transfer pathogens to farmlands or to water sources, leading to subsequent contamination of produce. Untreated biological soil amendments, like manure, can be contaminated with foodborne pathogens, such as Salmonella Newport, which may lead to transfer of the pathogen to fruits or vegetables. Studies have reported the occurrence and survival of Salmonella in manure or manure slurries. However, data on the survival and growth of Salmonella Newport is lacking in matrices simulating runoff. We quantified the survival and growth of wild-type (WT) Salmonella Newport and rpoS-deficient (Δ rpoS) strains in sterile and nonsterile soil extracts prepared with (amended) or without (unamended) heat-treated poultry pellets at 25°C. Salmonella Newport WT and Δ rpoS populations reached a maximum cell density of 6 to 8 log CFU/mL in 24 to 30 h in amended and unamended soil extracts and remained in stationary phase for up to 4 days. Salmonella Newport in amended soil extracts exhibited a decreased lag phase (λ , 2.87 ± 1.01 h) and greater maximum cell densities ( Nmax, 6.84 ± 1.25 CFU/mL) compared with λ (20.10 ± 9.53 h) and Nmax (5.22 ± 0.82 CFU/mL) in unamended soil extracts. In amended soil extract, the Δ rpoS strain had no measurable λ , similar growth rates (µmax) compared with WT, and a lower Nmax compared with the WT strain. Unamended, nonsterile soil extracts did not support the growth of Salmonella Newport WT and led to a decline in populations for the Δ rpoS strain. Salmonella Newport had lower cell densities in nonsterile soil extracts (5.94 ± 0.95 CFU/mL) than it did in sterile soil extracts (6.66 ± 1.50 CFU/mL), potentially indicating competition for nutrients between indigenous microbes and Salmonella Newport. The most favorable growth conditions were provided by amended sterile and nonsterile soil extracts, followed by sterile, unamended soil extracts for both Salmonella Newport strains. Salmonella Newport may grow to greater densities in amended extracts, providing a route for increased Salmonella levels in the growing environments of produce.

Genomic and phenotypic diversity of Listeria monocytogenes clonal complexes associated with human listeriosis.

Listeria monocytogenes is a pathogen of significant concern in many ready to eat foods due to its ability to survive and multiply even under significant environmental stresses. Listeriosis in humans is a concern, especially to high-risk populations such as those who are immunocompromised or pregnant, due to the high rates of morbidity and mortality. Whole genome sequencing has become a routine part of assessing L. monocytogenes isolated from patients, and the frequency of different genetic subtypes associated with listeriosis is now being reported. The recent abundance of genome sequences for L. monocytogenes has provided a wealth of information regarding the variation in core and accessory genomic elements. Newly described accessory genomic regions have been linked to greater virulence capabilities as well as greater resistance to environmental stressors such as sanitizers commonly used in food processing facilities. This review will provide a summary of our current understanding of stress response and virulence phenotypes of L. monocytogenes, within the context of the genetic diversity of the pathogen.

Electroacupuncture Attenuates Visceral Hypersensitivity by Inhibiting JAK2/STAT3 Signaling Pathway in the Descending Pain Modulation System.

Electroacupuncture (EA) has been used for treating visceral hypersensitivity (VH). However, the underlying molecular mechanism remains unclear. This study was aim to testify the effect of EA on ileitis-provoked VH, and to confirm whether EA attenuates VH through Janus kinase 2 (JAK2)/signal transducers and activators of transcription 3 (STAT3) signaling pathway in the periaqueductal gray (PAG)-the rostral ventromedial medulla (RVM)-the spinal cord dorsal horn (SCDH) axis. Methods: Goats were anesthetized and laparotomized for injecting 2,4,6-trinitro-benzene-sulfonic acid (TNBS)-ethanol solution (30mg TNBS dissolved in 40% ethanol) into the ileal wall to induce VH. EA was treated for 30min from day 7, then every 3 days for six times. VH was assessed by visceromotor response (VMR) and pain behavior response to 20, 40, 60, 80, and 100 mmHg colorectal distension pressures at day 7, 10, 13, 16, 19, and 22. The spinal cord in the eleventh thoracic vertebra and the brain were collected at day 22. The protein and mRNA levels of IL-6, JAK2, and STAT3 in the SCDH were detected with western blot and qPCR, respectively. The distribution of these substances was observed with immunohistochemistry in the ventrolateral PAG (vlPAG), RVM (mainly the nucleus raphe magnus, NRM), SCDH, the nucleus tractus solitaries (NTS) and the dorsal motor nucleus of vagi (DMV). Results: Goats administered with TNBS-ethanol solution showed diarrhea, enhanced VMR and pain behavior response, and increased IL-6, phosphorylated JAK2 and STAT3 (pJAK2 and pSTAT3) in the vlPAG, NRM, NTS and DMV, and their protein and mRNA levels in the SCDH. EA relieved diarrhea, VMR and pain behavior response, decreased IL-6, pJAK2 and pSTAT3 levels in the vlPAG, NRM, SCDH, NTS, and DMV except for pSTAT3 in the DMV, but did not affect mRNA level of these three substances in the SCDH. Conclusion: EA attenuates VH probably through inhibiting JAK2/STAT3 signaling pathway in the PAG-RVM-SCDH axis.

Efficacy of vacuum steam pasteurization for inactivation of Salmonella PT 30, Escherichia coli O157:H7 and Enterococcus faecium on low moisture foods.

Low moisture foods such as nuts, spices, and seeds have been implicated in several outbreaks due to Salmonella or E. coli O157:H7 contamination. Such foods may be consumed raw, and can be used as ingredients in other food products. While numerous thermal inactivation studies have been conducted for Salmonella on nuts, studies on other seeds and grains are minimal. Product water activity can influence the thermal resistance of pathogens, where thermal resistance increases as water activity decreases, leading to a requirement for higher temperatures and longer exposure times to achieve significant reduction of pathogen numbers. Vacuum steam pasteurization uses steam under vacuum, which can be operated at temperatures above and below 100°C. The objective of this study was to determine the efficacy of vacuum steam pasteurization for inactivation of pathogens on whole flaxseed, quinoa, sunflower kernels, milled flaxseed and whole black peppercorns. The use of E. faecium as a potential surrogate for Salmonella and E. coli O157:H7 in vacuum steam pasteurization was also evaluated. Pasteurization for 1min at 75°C yielded average log reductions of 5.48±1.22, 5.71±0.40 and 5.23±0.61 on flaxseed, 4.29±0.92, 5.89±0.26 and 2.39±0.83 on quinoa, and 4.01±0.74, 5.40±0.83 and 2.99±0.92 on sunflower kernels for Salmonella PT 30, E. coli O157:H7 and E. faecium, respectively. Similarly, on milled flaxseed and black peppercorns average log reductions of 3.02±0.79 and 6.10±0.64CFU/g were observed for Salmonella PT 30 after 1min of treatment at 75°C but, on average, >6.0 log reductions were observed after pasteurization at 85°C. Our data demonstrate that vacuum steam pasteurization can be effectively used to reduce pathogens on these low moisture foods at temperature as low as 75 and 85°C, and that E. faecium may be used as a potential surrogate for Salmonella PT 30 and E. coli O157:H7.

Visceral pain triggered by traction on the ileocecal ligament with ileitis.

BACKGROUND: Visceral pain is a common symptom of several gastrointestinal disorders. Despite tremendous progress in understanding its basic mechanisms, it remains a significant health challenge for clinicians. The present study quantified the intensity of visceral pain using ileocecal ligament traction on an inflamed ileum in goats. MATERIALS AND METHODS: A total of 36 male goats weighing 20.05±2.1 kg were randomly allocated equally into a 2,4,6-trinitrobenzenesulfonic acid (TNBS) group (n=18) and a saline group (n=18). Ileitis was induced via the injection of 30 mg TNBS dissolved in 30% ethanol into the ileal wall through a laparotomy. An equal volume of normal saline was injected into the ileal wall of the saline goats. Behavioral responses to traction (2, 4, and 6 N) on the ileocecal ligament were observed on days 3, 7, and 14. Six goats from each group received a laparotomy and partial intestinal resection for ileal sample collection immediately after behavioral testing on days 3, 7, and 14. Ileal histopathological changes were assessed and concentrations of myeloperoxidase, IL-1ß, IL-6, and TNFα investigated using enzyme-linked immunosorbent assay. RESULTS: The TNBS-treated goats exhibited remarkably increased macroscopic scores, mast-cell counts, myeloperoxidase, and TNFα concentrations on days 3 and 7 compared to the saline group, and increased microscopic scores and IL-1ß and IL-6 concentrations on days 3-14. The TNBS-treated goats exhibited behavioral changes in response to traction in the same pattern as their microscopic changes and cytokine levels. The traction force correlated positively with pain-behavior responses. CONCLUSION: Traction on the ileocecal ligament of goats with ileitis provoked an apparent, stable, and reproducible ileum-derived pain. The current model may be helpful in evaluating the efficacy of new drugs for the management of visceral pain and in investigating its underlying mechanisms.

Visceral Hypersensitivity Is Provoked by 2,4,6-Trinitrobenzene Sulfonic Acid-Induced Ileitis in Rats.

BACKGROUND AND AIMS: Crohn's Disease (CD), a chronic Inflammatory Bowel Disease, can occur in any part of the gastrointestinal tract, but most frequently in the ileum. Visceral hypersensitivity contributes for development of chronic abdominal pain in this disease. Currently, the understanding of the mechanism underlying hypersensitivity of Crohn's ileitis has been hindered by a lack of specific animal model. The present study is undertaken to investigate the visceral hypersensitivity provoked by 2,4,6-trinitrobenzene sulfonic (TNBS)-induced ileitis rats. METHODS: Male Sprague-Dawley rats were anaesthetized and laparotomized for intraileal injection of TNBS (0.6 ml, 80 mg/kg body weight in 30% ethanol, n = 48), an equal volume of 30% Ethanol (n = 24), and Saline (n = 24), respectively. Visceral hypersensitivity was assessed by visceromotor responses (VMR) to 20, 40, 60, 80, and 100 mmHg colorectal distension pressure (CRD) at day 1, 3, 7, 14, 21, and 28. Immediately after CRD test, the rats were euthanized for collecting the terminal ileal segment for histopathological examinations and ELISA of myleoperoxidase and cytokines (TNF-α, IL-1ß, IL-6), and dorsal root ganglia (T11) for determination of calcitonin gene-related peptide by immunohistochemistry, respectively. RESULTS: Among all groups, TNBS-treatment showed transmural inflammation initially at 3 days, reached maximum at 7 days and persisted up to 21 days. The rats with ileitis exhibited (P < 0.05) VMR to CRD at day 7 to day 21. The calcitonin gene-related peptide-immunoreactive positive cells increased (P < 0.05) in dorsal root ganglia at day 7 to 21, which was persistently consistent with visceral hypersensitivity in TNBS-treated rats. CONCLUSION: TNBS injection into the ileum induced transmural ileitis including granuloma and visceral hypersensitivity. As this model mimics clinical manifestations of CD, it may provide a road map to probe the pathogenesis of gut inflammation and visceral hypersensitivity, as well as for establishing the therapeutic protocol for Crohn's ileitis.