Refining the evolutionary tree of the horse Y chromosome.

The Y chromosome carries information about the demography of paternal lineages, and thus, can prove invaluable for retracing both the evolutionary trajectory of wild animals and the breeding history of domesticates. In horses, the Y chromosome shows a limited, but highly informative, sequence diversity, supporting the increasing breeding influence of Oriental lineages during the last 1500 years. Here, we augment the primary horse Y-phylogeny, which is currently mainly based on modern horse breeds of economic interest, with haplotypes (HT) segregating in remote horse populations around the world. We analyze target enriched sequencing data of 5 Mb of the Y chromosome from 76 domestic males, together with 89 whole genome sequenced domestic males and five Przewalski's horses from previous studies. The resulting phylogeny comprises 153 HTs defined by 2966 variants and offers unprecedented resolution into the history of horse paternal lineages. It reveals the presence of a remarkable number of previously unknown haplogroups in Mongolian horses and insular populations. Phylogenetic placement of HTs retrieved from 163 archaeological specimens further indicates that most of the present-day Y-chromosomal variation evolved after the domestication process that started around 4200 years ago in the Western Eurasian steppes. Our comprehensive phylogeny significantly reduces ascertainment bias and constitutes a robust evolutionary framework for analyzing horse population dynamics and diversity.

Seroprevalence of canine distemper virus (CDV) in the free-roaming dog (Canis familiaris) population surrounding Chitwan National Park, Nepal.

Canine distemper virus (CDV) is a global multi-host pathogen that is capable of causing considerable mortality in a range of species and is important in the field of conservation medicine. Nepal's Chitwan National Park is a protected area providing habitat for 32% of the country's mammal species including endangered carnivores such as the Bengal tiger (Panthera tigris tigris) that are susceptible to CDV. The presence of free-roaming dogs around protected areas could represent a source of infectious disease for transmission to local wildlife. A cross-sectional demographic and canine distemper virus seroprevalence study of 100 free-roaming dogs from the Chitwan National Park buffer zone and surrounding area was conducted in November 2019. The overall seroprevalence indicating past exposure to canine distemper virus was 80.0% (95% CI: 70.8-87.3). Of the host variables assessed, sex and age were positively associated with seroprevalence at the univariable level, with male dogs demonstrating lower seroprevalence than females (OR = 0.32, 95% CI: 0.11-0.91) and adult dogs demonstrating higher seroprevalence than juveniles (OR = 13.94, 95% CI: 1.37-142.29). The effect of sex was no longer significant at the multivariable level, but the direction of the effect remained the same. The effect of age remained significant after multivariable analysis (OR = 9.00, 95% CI: 1.03-192.75). No spatial associations were demonstrated in relation to the buffer zone area or boundary of Chitwan National Park. Free-roaming dog neutering and vaccination programmes can provide a useful baseline for future CDV studies in the region, and a proxy to monitor disease threats to susceptible wildlife.

Geographical contrasts of Y-chromosomal haplogroups from wild and domestic goats reveal ancient migrations and recent introgressions.

By their paternal transmission, Y-chromosomal haplotypes are sensitive markers of population history and male-mediated introgression. Previous studies identified biallelic single-nucleotide variants in the SRY, ZFY and DDX3Y genes, which in domestic goats identified four major Y-chromosomal haplotypes, Y1A, Y1B, Y2A and Y2B, with a marked geographical partitioning. Here, we extracted goat Y-chromosomal variants from whole-genome sequences of 386 domestic goats (75 breeds) and seven wild goat species, which were generated by the VarGoats goat genome project. Phylogenetic analyses indicated domestic haplogroups corresponding to Y1B, Y2A and Y2B, respectively, whereas Y1A is split into Y1AA and Y1AB. All five haplogroups were detected in 26 ancient DNA samples from southeast Europe or Asia. Haplotypes from present-day bezoars are not shared with domestic goats and are attached to deep nodes of the trees and networks. Haplogroup distributions for 186 domestic breeds indicate ancient paternal population bottlenecks and expansions during migrations into northern Europe, eastern and southern Asia, and Africa south of the Sahara. In addition, sharing of haplogroups indicates male-mediated introgressions, most notably an early gene flow from Asian goats into Madagascar and the crossbreeding that in the 19th century resulted in the popular Boer and Anglo-Nubian breeds. More recent introgressions are those from European goats into the native Korean goat population and from Boer goat into Uganda, Kenya, Tanzania, Malawi and Zimbabwe. This study illustrates the power of the Y-chromosomal variants for reconstructing the history of domestic species with a wide geographical range.

Split Versus Non-Split Morning Dosing Regimen for Assessment of Quality of Bowel Preparation for Colonoscopy.

BACKGROUND: Different bowel preparation regimens are available. Currently we are giving the entire preparation on the day of colonoscopy. Multiple studies have shown splitting the regimen might improve the quality of bowel preparation with lesser side effects and better compliance. The study was done to compare the efficacy and tolerability of split bowel preparation regimen with non-split dosing regimen. METHODS: Single centered observational comparative study was done in a tertiary care hospital. One hundred ninety eight patients requiring elective colonoscopy were assigned to receive one of the two preparations (split versus morning) prior to colonoscopy. Main outcomes were bowel preparation quality and patient compliance and tolerability. RESULTS: There was no significant difference between the two regimen for the mean total Boston Bowel Preparation Scale (6.79VS 6.74,P value -0.777).Patient compliance was better for split dosing compared to single dosing (99 vs 5 p value-<0.001).There were more side effects in the single dosage compared to split dosing except for sleep disturbance which was more in split dosing. CONCLUSIONS: The study found that split-dose and single dose polyethylene glycol solution for bowel preparation before colonoscopy had similar efficacy in the quality of bowel preparation. Split-dose polyethylene glycol appears to be superior to single-dose PEG for patient compliance and side effects.

FGF5 and EPAS1 gene polymorphisms are associated with high-altitude adaptation in Nepalese goat breeds.

Several studies have reported the gene polymorphisms associated with high-altitude adaptation in goats. The FGF5 gene is a regulator in the hair-growth and a SNP c.-253G>A located within 5'UTR has been reported to cause long-haired phenotype. The EPAS1 gene is a transcription factor for various genes that have hypoxia-adaptive functions and a nonsynonymous SNP (Q579L) located in exon 5 has been reported to be associated with the mean corpuscular hemoglobin concentration. Nepal has large difference in altitudes in the north-south direction and four indigenous goat breeds are bred depending on the altitude. We used a total of 130 animals in Nepal, Chyangra (n = 37), Sinhal (n = 24), Khari (n = 33), and Terai (n = 36), and genotyped these two gene polymorphisms to compare the gene frequencies among the breeds and investigate the associations between breeding altitudes and allele frequencies. The genotyping results revealed that the mutant allele frequency in both polymorphisms tended to increase, as the breeding altitude of each population increased. In addition, correlation coefficients showed a relatively strong positive correlation between the breeding altitude and the mutant allele frequencies (r = 0.87 in FGF5 and r = 0.68 in EPAS1). These results suggested that both polymorphisms would significantly contribute to the high-altitude adaptation in Nepalese goat breeds.

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.

Microbial and Chemical Shelf-Life of Vacuum Steam-Pasteurized Whole Flaxseed and Milled Flaxseed.

Flaxseed is an oilseed with many health benefits. Flaxseed may be consumed raw or in processed form. In the raw form, there is a potential for microbial contamination. Several pasteurization methods have been used to reduce microbial contamination. However, such treatments may affect chemical properties of foods. In this study, vacuum steam-pasteurization was conducted on whole flaxseed and milled flaxseed using 4 different conditions (3 min at 75 °C, 3 min at 90 °C, 9 min at 90 °C, and 3 min at 105 °C). Microbial and chemical shelf-life was monitored for 28 wk (36 wk for aerobic plate counts). Significant reduction (P < 0.05) in microbial counts (total aerobic plate counts, and yeast and mold counts) occurred after pasteurization and during storage of both whole flaxseed and milled flaxseed. Although both the moisture content and aw increased after pasteurization, they were similar to the unpasteurized samples during storage. Peroxide value, free fatty acid, headspace volatiles, fatty acid profiles, oil content, and secoisolariciresinol diglucoside (SDG) content were chemical indices measured. Only small changes were observed in the chemical indices after vacuum steam-pasteurization for both pasteurized whole flaxseed and milled flaxseed as compared to the unpasteurized flaxseed at most instances. Vacuum steam-pasteurization can be used as a safe alternative for the microbial reduction of low-moisture products, such as flaxseed, without significantly affecting chemical stability. PRACTICAL APPLICATION: Vacuum steam-pasteurization can be effectively used for the treatment of whole flaxseed and milled flaxseed to reduce spoilage microorganisms, such as total aerobes and yeasts and molds. In addition, this pasteurization method had minimal effects on several chemical shelf-life parameters with positive impact on SDG of the processed flaxseed.

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.

Plasma Cytochrome c Detection Using a Highly Sensitive Electrochemiluminescence Enzyme-Linked Immunosorbent Assay.

BACKGROUND: Cytochrome c is an intermembrane mitochondrial protein that is released to the bloodstream following mitochondrial injury. METHODS AND RESULTS: We developed an electrochemiluminescence immunoassay to measure cytochrome c in human and rat plasma, which showed high sensitivity with broad dynamic range (2-1200 ng/mL in humans and 5-500 ng/mL in rat) and high assay reproducibility (inter-assay coefficient <6% in humans and <10% in rat). In patients after blunt trauma, plasma cytochrome c directly correlated with injury severity. In rats after cardiac resuscitation, plasma cytochrome c inversely correlated with survival and responsiveness to mitochondrial protective interventions. CONCLUSIONS: The cytochrome c assays herein presented have high sensitivity, wide dynamic range, and high reproducibility well suited for biomarker of mitochondrial injury.

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.

Health and mineral nutrition status of yaks in southern Mustang, Nepal.

Biochemical values and mineral concentrations in blood plasma were investigated to evaluate the statuses of health and mineral nutrition among yaks in Mustang District, Nepal. In total, 118 plasma samples of female yaks collected in April and September/October of 2013-2015 were offered. Seventy-four percent of yaks showed lower plasma total-cholesterol concentrations than the lowest limit of reference range (100 mg/dL) and the values in spring (83.41 mg/dL) were lower (P < 0.05) than those in autumn (95.05 mg/dL). All the yaks had lower plasma albumin concentrations than the lowest limit of reference range (3.0 g/dL) and 66% of yaks showed lower plasma inorganic phosphorus concentrations than the critical level of phosphorus deficiency (4.5 mg/dL). Thirty-five percent of yaks showed lower plasma calcium concentrations than the lowest limit of normal range (8 mg/dL) and the concentrations were lower in spring than in autumn (P < 0.01). Seventy-five percent of yaks presented lower copper concentrations than the critical level (0.65 mg/L) and the concentrations were lower in spring than in autumn (P < 0.01). Since the low plasma total-cholesterol might have indicated shortage of dry matter and energy intake, attention should be paid to the nutritional statuses of energy, phosphorus, calcium and copper in winter and early spring.

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.

A novel model for studying ileitis-induced visceral hypersensitivity in goats.

BACKGROUND: Visceral hypersensitivity (VH) is a common condition in many gastrointestinal disorders such as inflammatory bowel diseases (IBDs) in human and animals. Most studies often induce Crohn's disease/colitis to investigate VH in small experimental animals. Although farm animals commonly suffer from IBDs, their VH has not been investigated so far. Because goats can suffer from Johne's disease, a naturally occurring Crohn's-like disease, they may be suitable to be used for studying the mechanism underlying VH in common intestinal disorders of large animals. In the present study, 60 healthy goats of either sex were equally divided into a 2, 4, 6-trinitrobenzenesulfonic acid (TNBS) group and saline group. A volume of 1.2 ml of TNBS-ethanol solution (30 mg TNBS in 40 % ethanol) or an equal volume of isotonic saline was injected into the wall of the terminal ileum through laparotomy. The severity of the developing ileitis was determined according to macro- and microscopic pathologic scores and the levels of myeloperoxidase, interleukin-1ß, interleukin-6 and tumor necrosis factor-α, and VH was evaluated with visceromotor responses (VMR) to colorectal distension on days 3, 7, 14, 21 and 28. VMRs were assessed with a continuous ramp distention mode with 6 s for each pressure (20, 40, 60, 80 and 100 mmHg). RESULTS: Compared to the saline group, the TNBS-treated goats showed apparent transmural pathological changes and a significant increase (P < 0.05) in macroscopic and microscopic change scores, and levels of myeloperoxidase, interleukin-1ß, interleukin-6 and tumor necrosis factor-α in the ileum, and VMR to colorectal distension. The goats exhibited apparent ileitis at days 3 to 21, and VH at days 7 to 28 following TNBS treatment. CONCLUSION: This experiment successfully established a reproducible ileitis and VH with administration of TNBS-ethanol solution in the ileal wall of goats. This model is useful for studying the pathogenesis of the IBD and the mechanism underlying VH, and for evaluating the efficacy of new therapeutic regimens.

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.