text:
Necrotic enteritis (NE) caused by Clostridium perfringens infection has reemerged as a prevalent poultry disease worldwide due to reduced usage of prophylactic antibiotics under consumer preferences and regulatory pressures. The lack of alternative antimicrobial strategies to control this disease is mainly due to limited insight into the relationship between NE pathogenesis, microbiome, and host responses. Here we showed that the microbial metabolic byproduct of secondary bile acid deoxycholic acid (DCA), at as low as 50 µM, inhibited 82.8% of C. perfringens growth in Tryptic Soy Broth (P < 0.05). Sequential Eimeria maxima and C. perfringens challenges significantly induced NE, severe intestinal inflammation, and body weight (BW) loss in broiler chickens. These negative effects were diminished (P < 0.05) by 1.5 g/kg DCA diet. At the cellular level, DCA alleviated NE-associated ileal epithelial death and significantly reduced lamina propria cell apoptosis. Interestingly, DCA reduced C. perfringens invasion into ileum (P < 0.05) without altering the bacterial ileal luminal colonization. Molecular analysis showed that DCA significantly reduced inflammatory mediators of Infγ, Litaf, Il1β, and Mmp9 mRNA accumulation in ileal tissue. Mechanism studies revealed that C. perfringens induced (P < 0.05) elevated expression of inflammatory mediators of Infγ, Litaf, and Ptgs2 (Cyclooxygenases-2 (COX-2) gene) in chicken splenocytes. Inhibiting the COX signaling by aspirin significantly attenuated INFγ-induced inflammatory response in the splenocytes. Consistent with the in vitro assay, chickens fed 0.12 g/kg aspirin diet protected the birds against NE-induced BW loss, ileal inflammation, and intestinal cell apoptosis. In conclusion, microbial metabolic product DCA prevents NE-induced BW loss and ileal inflammation through attenuating inflammatory response. These novel findings of microbiome protecting birds against NE provide new options on developing next generation antimicrobial alternatives against NE.
Antimicrobial resistance is one of the emerging challenges requiring immediate and sustainable counter-actions from agriculture to healthcare1. Increasing antimicrobial resistance has caused the emergence of multiple drug-resistant microbes or “superbugs”. Recently, a “superbug” of an Escherichia coli strain resistant to the last resort antibiotic, Colistin, was reported in USA2. Overuse of antimicrobial agents in medical and agricultural practice is contributing to exacerbating the episodes of emerging antimicrobial resistant microbes1. Withdrawing antimicrobials in chicken production, however, has caused new problems for the chicken industry by reducing production efficiency and increasing diseases, such as Eimeria maxima- and Clostridium perfringens-induced necrotic enteritis (NE)3. NE is a multi-factorial disease and has a significant economic impact to the chicken industry with annual loss at billions of dollars4. Despite no clinical NE outbreak worldwide, subclinical NE (cholangiohepatitis) incidences were doubled from 2013 to 2014 in South-Eastern Norway and were associated with reduced in-feed antimicrobial usage5. Clinical signs of acute NE include watery to bloody (dark) diarrhea, severe depression, decreased appetite, closed eyes, and ruffled feathers. Dissection of dead or severely ill birds shows that the intestine is often distended with gas, very friable, and contains a foul-smelling brown fluid, with clearly visible necrotic lesions6. At the cellular level, NE birds display intestinal inflammation with diffuse and coagulative necrosis of villi and crypts, infiltration of immune cells into lamina propria, and crypt abscesses7,8. Although progress has been made toward understanding risk factors influencing the outcome of NE such as C. perfringens virulence, coccidiosis, and feed9, few effective non-antimicrobial strategies are available.
The human and animal intestine harbors up to trillions of microbes and this intestinal microbiota regulates various host functions such as the intestinal barrier, nutrition and immune homeostasis10,11,12. The enteric microbiota regulates granulocytosis and neonatal response to Escherichia coli K1 and Klebsiella pneumoniae sepsis13, suggesting the key role of the microbiota in protecting the host against systemic infection. At the gut level, fecal transplantation was reported decades ago to prevent Salmonella infantis chicken infection14. More recently, microbiota transplantation has shown tremendous success against recurrent human Clostridium difficile infection15 and Clostridium scindens metabolizing secondary bile acids have been shown to inhibit C. difficile infection16. Bile acids synthesized in the liver are released in the intestine and metabolized by gut microbiota into final forms of secondary bile acids17. Secondary bile acid of deoxycholic acid (DCA) is associated with a variety of human chronic diseases, such as obesity, diabetes, and colorectal cancer18,19. Recently, mouse anaerobes and their metabolic product DCA has been found to prevent and treat Campylobacter jejuni-induced intestinal inflammation in germ-free mice through attenuating host inflammatory signaling pathways20. However, whether bile acids play any role in NE pathogenesis is unknown.
NE is frequently concurrent with the episode of coccidiosis21. Coccidiosis is associated with strong immune response and intestinal inflammation in chickens22,23. Coccidia-induced intestinal inflammation may render a favorable environment for C. perfringens overgrowth, virulence expression, and invasion21. C. perfringens produces various toxins24 and induces hemolysis, epithelial barrier dysfunction, tissue necrosis and severe inflammation in non-chicken models25,26. Among inflammatory signaling pathways, cyclooxygenases (COX)-catalyzed prostanoids regulate various activities including cell proliferation, apoptosis and migration27, gastrointestinal secretion28, body temperature29, inflammation30, and pain sensation31. COX-1 and COX-3, translated from alternative splicing gene of Ptgs1, are constitutively expressed in intestinal cells and they maintain intestinal epithelial integrity. Inducible COX-2 (Ptgs2) activity is associated with various inflammatory diseases including inflammatory bowel disease32 and radiation-induced small bowel injury33. COX-2 increases gut barrier permeability and bacterial translocation across the intestinal barrier34,35. Paradoxically, COX-2 enhances inflammation resolution through prostaglandin D236. Although non-selective COX inhibitor, aspirin, is used to prevent various chronic diseases, it inflicts intestinal inflammation to the healthy intestine37. However, the role of COX signaling on NE remains elusive.
Currently, limited knowledge is available on the relationship among the NE pathogenesis, the microbiome, and the host inflammatory response. Because DCA attenuates C. jejuni-induced intestinal inflammation20 and prevents in vitro growth of C. difficile16, a same genus member of C. perfringens, we hypothesized that the microbiota metabolic product DCA attenuated NE through inhibiting intestinal inflammation. We found that DCA decreased NE-induced intestinal inflammation, C. perfringens invasion, intestinal cell death, and body weight loss. Blocking the inflammatory COX signaling pathways by aspirin reduced NE-induced intestinal inflammation. These novel findings of microbiome DCA and COX inhibitor against NE offer new strategies to prevent and treat C. perfringens-induced diseases.
Materials and Methods
Bile acid inhibition of C. perfringens assay
Similar to bile acid inhibition assay on Campylobacter jejuni growth38, 103 CFU C. perfringens was inoculated into 10 ml Tryptic Soy Broth (TSB) supplemented with 0.5% sodium thioglycollate, in the presence of taurocholic acid (TCA, 0.2 mM, final concentration), cholic acid (CA, 0.2 mM), or DCA (0, 0.01, 0.05, 0.1, or 0.2 mM), or 0, 0.2, or 1 mM of lithocholic acid (LCA) or ursodeoxycholic acid (UDCA). The different treatments of bacterial broth were cultured at 42 °C overnight (16–18 hours) under anaerobic conditions. The bacterial growth in tubes was observed for inhibition (clear broth) or no inhibition (cloudy broth). The bacterial growth was quantified by OD600 nm using a spectrophotometer (Nanodrop, Thermo Fisher).
Animal experiments performed were in accordance with the Animal Research: Reporting of In Vivo Experiments (https://www.nc3rs.org.uk/arrive-guidelines). The experiments were approved by the Care and Use Committee of the University of Arkansas. Cohorts of thirteen zero-day-old broiler chicks per group were obtained from Cobb-Vantress Hatchery (Siloam Springs, AR). Chicks were neck-tagged and randomly allocated to floor pens with new pine shavings as litter in an environmentally controlled isolated room suitable for up to biosafety level (BSL) III animal experiments. The birds were provided with their respective diet and water ad libitum, and temperature was maintained at 34 °C for the first 5 days of age and was then gradually reduced until a temperature of 23 °C was achieved at day 26 days of age. The birds were fed a corn-soybean meal-based starter diet during 0–10 days of age and a grower diet during 11–26 days of age. The basal diet was formulated as described before39. Treatment diets were supplemented with 1.5 g/kg CA or DCA (all from Alfa Aesar). No antibiotics, coccidiostats or enzymes were added to the feed. E. maxima kindly provided by Dr. John Barta was M6 strain, which was a single oocyst-derived isolate at Ontario Veterinary College in 1973. The E. maxima was propagated in chickens as describe before40. The recovered oocysts were sporulated as previously described41. C. perfringens kindly donated from USDA-ARS, College Park, TX42,43 was confirmed alpha-toxin positive using a multiplex PCR assay42,44. An aliquot of frozen C. perfringens was grown in TSB plus sodium thioglycollate overnight for the NE challenge study, and was serially diluted and plated on Tryptic Soy Agar plus sodium thioglycolate for enumerating CFU. In previous experiments, birds infected with this aliquot of C. perfringens alone didn’t show any signs of NE and had comparable body weight gain to noninfected birds (data not showed). In current experiments, birds were infected with 20,000 sporulated E. maxima oocytes/bird at 18 days of age and 109 CFU C. perfringens/bird at 23 and 24 days of age. Chicken body weight and feed intake were individually measured at 0, 18, 23, and 26 days of age. Bird health status was monitored daily after the pathogen infection. Four birds with average BW of the treatment group were sacrificed at 23 and 26 days of age. Because gross necrotic lesion was often observed in upper ileum in this NE model, the ileal tissue and digesta samples from all sacrificed birds were collected for RNA and DNA analysis. The ileal tissue of ~8 cm length was also Swiss-rolled for H&E staining and histopathology analysis. Images were acquired using a Nikon TS2 fluorescent microscope. Ileal inflammation was scored blindly using the H&E Swiss-roll slides (4 slides (birds)/treatment). Briefly, each Swiss-roll slide was divided into 4 areas and was scored. Total histopathological scores were then calculated by adding the four area scores. The following score scales used were based on previous ileitis45,46 and colitis scoring systems47: score 0: no inflammation, villi and crypt intact; score 1: small number infiltration cells in laminar propria of villi and crypts or villi minimally shortened; score 2: more extensive infiltration cells in laminar propria of villi and crypts, villi shortened >1/4 and edema, or crypt hyperplasia; score 3: pronounced infiltration cells in laminar propria of villi, crypts, submucosa, and muscularis, villi shortened >1/2 and edema, or crypt hyperplasia and regeneration; and score 4: necrosis, villus diffuse, ulcers, crypt abscesses, or transmural inflammation (may extend to serosa).
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
Cell apoptosis in intestinal tissue was visualized using TUNEL assay similar to described before48. Briefly, ileal tissue slides were deparaffinized with xylene bath for 3 times and then rehydrated with 100%, 95%, and 70% ethanol. The tissue was then incubated with TUNEL solution (5 µM Fluorescein-12-dUTP (Enzo Life Sciences), 10 µM dATP, 1 mM pH 7.6 Tris-HCl, 0.1 mM EDTA, 1U TdT enzyme (Promega)) at 37 °C for 90 min. The slides were counter-stained with DAPI for nucleus visualization. The fluorescent green apoptosis cells were evaluated and imaged using a Nikon TS2 fluorescent microscopy. The green dots in representative 3 areas per slide were counted as apoptosis cells and blue dots of nuclei were counted as total cells using ImageJ49 particle analysis and its plugin of Nikon ND2 reader. The results were showed as apoptosis cells per 1,000 total intestinal cells.
Real time PCR of mRNA gene expression and C. perfringens quantification
Total RNA from ileal tissue or splenocytes was extracted using TRIzol as described before20,50. cDNA was prepared using M-MLV (NE Biolab). mRNA levels of proinflammatory genes were determined using SYBR Green PCR Master mix (Bio-Rad) on a Bio-Rad 384-well Real-Time PCR System and normalized to Gapdh. To quantify C. perfringens intestinal luminal colonization or tissue invasion, ileal digesta or tissue were weighed, bead-beaten, and extracted for DNA using phenol-chloroform method as described before20. C. perfringens in the digesta and tissue was quantified using specific C. perfringens 16S rDNA primers by the qPCR. The PCR reactions were performed according to the manufacturer’s recommendation. The following gene primers were used:
Cp16S_forward: 5′-CAACTTGGGTGCTGCATTCC-3′; Cp16S_reverse: 5′-GCCTCAGCGTCAGTTACAG-3′; Mmp9_forward: 5′-CCAAGATGTGCTCACCAAGA-3′; Mmp9_reverse: 5′-CCAATGCCCAACTTCTCAAT-3′; Litaf_forward: 5′-AGATGGGAAGGGAATGAACC; Litaf_reverse: 5′-GACGTGTCACGATCATCTGG-3′; Il1β_forward: 5′-GCATCAAGGGCTACAAGCTC-3′; Il1β_reverse: 5′-CAGGCGGTAGAAGATGAAGC-3′; Infγ_forward: 5′-AGCCGCACATCAAACACATA-3′; Infγ_reverse: 5′-TCCTTTTGAAACTCGGAGGA-3′; Ptgs2_forward: 5′-ACCAGCATTTCAACCTTTGC-3′; Ptgs2_reverse: 5′-CCAGGTTGCTGCTCTACTCC-3′; Gapdh_forward: 5′-GACGTGCAGCAGGAACACTA-3′; Gapdh_reverse: 5′-CTTGGACTTTGCCAGAGAGG-3′. Gene expression of fold change was calculated using ΔΔCt method51 and Gapdh as internal control.
Fluorescence in situ hybridization (FISH)
C. perfringens at ileal tissue was visualized using FISH assay similarly to previously described47. Briefly, tissue slides were deparaffinized, hybridized with the FISH probe, washed, stained with DAPI, and imaged using a Nikon TS2 fluorescent microscope system. The FISH probe sequence of Cp85aa18: 5′-/Cy3/TGGTTGAATGATGATGCC-3′52 was used to probe the presence of C. perfringens similar to a previous report50. Briefly, deparaffinized, formalin-fixed 5 μm thick sections were incubated for 15 min in lysozyme (300,000 Units/ml lysozyme; Sigma-Aldrich) buffer (25 mM Tris pH 7.5, 10 mM EDTA, 585 mM sucrose, and 0.3 mg/ml sodium taurocholate) at room temperature and hybridized overnight at 46 °C in hybridization chambers with the oligonucleotide probe (final concentration of 5 ng/μl in a solution of 30 percent formamide, 0.9 M sodium chloride, 20 mM Tris pH 7.5, and 0.01% sodium dodecyl sulfate). Tissue sections were washed for 20 min at 48 °C in washing buffer (0.9 M NaCl, 20 mM Tris pH 7.2, 0.1% SDS, 20% Formamide, and 10% Dextran Sulfate) and once in distilled water for 10 seconds. The slides were stained with DAPI for 2 min and dried at room temperature, mounted with 50% glycerol. C. perfringens in intestinal tissues were visualized using a Nikon TS2 fluorescent microscopy and representative images of each treatment were presented in the Results section.
C. perfringens-induced inflammatory response using primary splenocytes
Splenocytes were isolated similarly to described previously53. Briefly, chicken spleen was resected, homogenized into splenocytes using frosted glass slides, and pooled together in RPMI 1640 medium supplemented with 2% fetal bovine serum, 2 mM L-glutamine, 50 µM 2-mercaptoethanol. After lysed the red blood cells, the collected cells were plated at 2 × 106 cells/well in 6-well plates. The cells were pre-treated with 1.2 mM aspirin for 45 min. Cells were then challenged with murine INFγ (1 μg/ml, Pepro Tech), chicken INFγ (1 μg/ml, IBI Scientific), or C. perfringens (multiplicity of infection 100). The cells were lysed in TRIzol (Invitrogen) for RNA isolation after 2 or 4 hours of cytokines or C. perfringens treatment, respectively.
Dietary aspirin against NE
Birds fed diet supplemented with 0 and 0.12 g/kg aspirin were raised, infected, and sampled as abovementioned. Ileal histopathology images and scores were collected. To evaluate cell death, TUNEL assay was performed in the ileal slides of the birds and the apoptotic cells were quantified. The impact of aspirin on body weight gain was also measured.
For in vitro assay of bile acids against C. perfringens growth, data were first analyzed by One-way ANOVA for significant difference and then Bonferroni’s multiple comparison test using Prism 5.0 software. For other data, differences between treatments were analyzed pairwise using the nonparametric Mann–Whitney U test performed using Prism 5.0 software. The specific pairwise comparisons were showed in Results section and Figures. Sample (individual birds or cell wells) numbers of body weight, histology, and other assays were listed in respective subsections of Methods section. Values are shown as mean of samples in the treatment ± standard error of the mean as indicated. Experiments were considered statistically significant if P values were < 0.05.
Ethics approval and consent to participate
All animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Arkansas at Fayetteville.
DCA prevents C. perfringens in vitro growth
Based on previous findings and current state of knowledge, we reasoned that DCA would prevent C. perfringens growth. To test this hypothesis, in vitro inhibition experiments were conducted, in which C. perfringens was inoculated in TSB with sodium thioglycollate under anaerobic condition. The TSB was also supplemented with various concentrations of bile acids, including conjugated primary bile acid TCA, primary bile acid CA, and secondary bile acid DCA. The results showed that DCA inhibited C. perfringens growth at 0.01 (−33.8%) and 0.05 mM (−82.8%, clear broth), respectively, compared to control, while TCA (−16.4%) and CA (−8.2%) didn’t prevent the bacterial growth (cloudy broth) even at 0.2 mM (Fig. 1A). We then examined whether other secondary bile acids were also bacteriostatic in TSB. Interestingly, C. perfringens growth was inhibited by lithocholic acid (LCA; −22.6 and −23.8%) and ursodeoxycholic acid (UDCA; −10.0 and −25.3%) at 0.2 and 1 mM, respectively (Fig. 1B) and it was far less effective compared to DCA.
DCA prevents NE-induced intestinal inflammation in chicken ileum
To further address whether DCA reduced coccidia E. maxima- and C. perfringens-induced NE in birds, broiler chicks were fed CA or DCA diet. Because coccidiosis and NE induce severe intestinal inflammation7,8, the impact of DCA on chicken NE was investigated. Upper ileum tissue was collected as Swiss-roll, processed with H&E staining, and performed histopathology analysis. Notably, E. maxima (Em) infection induced severe intestinal inflammation (ileitis) as seen by immune cell infiltration (yellow arrows) into lamina propria, crypt hyperplasia, and mild villus height shortening compared to uninfected birds (Fig. 2A). NE birds displayed worse ileitis as seen by necrosis and fusion of villi and crypt (Green arrow), massive immune cell infiltration, and severe villus shortening. In contrast, DCA diet dramatically attenuated NE-induced ileitis and histopathology score (Fig. 2B), while CA diet also reduced NE-induced ileitis and histopathology score.
DCA attenuates NE-induced intestinal cell necrosis and apoptosis
Because inflammation often induces cell death54, it was then sought to examine whether cell death was relevant in DCA attenuating NE-induced ileitis. Since it was difficult to find reliable chicken antibodies to detect apoptosis or necrosis in chicken histology slides, we first resorted to classical histological analysis under high magnification. Consistently, the epithelial nuclei (dark blue) in heathy control bird villi were distributed close to the basal membrane (at the right side of the yellow dash line, Fig. 3A lower left panel). In contrast, the nuclei in inflamed villi epithelial cells of Em and NE birds were scattered from basal to the apical membranes, indicating epithelial cell death in villi of those birds. As a contrast, the DCA diet prevented epithelial cell nucleus translocation to apical side, suggesting cell death reduction. To further characterize the villus cell death, TUNEL assay was used, which detects later stage of cell apoptosis. Consistent with histopathology results, coccidiosis and NE induced scattered (Em birds) or concentrated (NE birds) apoptosis cells (green dots) in villus lamina propria, while cellular apoptosis was attenuated in the DCA treatment birds (Fig. 3B). To quantify the apoptosis, ImageJ was used to count apoptosis cells (TUNEL, green) and total cells (DAPI, blue). Consistent with histopathology results, DCA reduced NE-induced cell apoptosis (Fig. 3C).
DCA reduces C. perfringens invasion, NE-induced inflammatory response, and productivity loss
Given DCA inhibited C. perfringens growth in vitro, it was logic to reason that DCA might also reduce C. perfringens intestinal overgrowth in NE birds. To examine this possibility, C. perfringens colonization level was measured in the intestinal lumen using real-time PCR of C. perfringens 16S rDNA. Surprisingly, ileal luminal C. perfringens colonization in DCA birds was not significantly different from NE control birds (Fig. 4A), while bird histopathology was distinct between the two groups of birds. We then reasoned that the pathogen invasion into tissue was the main driving factors of NE pathogenesis, but not the pathogen luminal colonization level. To quantify the bacterial invasion, total DNA in ileal tissue was isolated and C. perfringens was measured using real time PCR. DCA attenuated more than 95% of C. perfringens invasion into ileal tissue (Fig. 4B). To have a better overview of the bacterial tissue invasion distribution, we used a fluorescence in situ hybridization (FISH) technique. We found that while C. perfringens was present deeply in the inflamed villus and crypt lamina propria of NE control birds, the bacterium was barely detectable in the ileal tissue of DCA birds (Fig. 4C). Because DCA reduced C. perfringens invasion and intestinal inflammation, the impact of DCA on various proinflammatory mediators was evaluated in ileal tissue using Real-Time PCR. C. perfringens induced inflammatory Infγ, Litaf (Tnfα), and Mmp9 mRNA accumulation in chicken ileal tissue, an effect attenuated by 51, 82, and 93%, respectively, in DCA fed chickens (Fig. 4D).
Body weight (BW) is one of important productivity parameters for meat chickens and reflects collective response during NE. The productivity results showed that DCA (solid black bar) but not CA (vertical-line bar) diet promoted bird daily BW gain during 0–18 days of age compared to birds fed control diets (open, tilted line, and dotted bars, Fig. 5). BW gain was reduced in birds infected with E. maxima (Em) (tilted line and dotted bars) during 18–23 days of age (coccidiosis phase) compared to noninfected birds (open bar). Subsequent C. perfringens infection reduced NE control birds (dotted bar) BW gain during 23–26 days of age (NE phase) compared to Em birds (tilted line bar). Consistent with histopathology analysis, DCA prevented productivity loss at coccidiosis and NE phases compared to the NE control birds. Interestingly, the primary bile acid CA diet attenuated body weight loss at NE phase but failed at coccidiosis phase compared to the NE control birds.
COX inhibitor aspirin alleviates C. perfringens-induced inflammatory response in splenocytes
Inflammatory events shape intestinal diseases and targeting the inflammatory response attenuates disease progress such as in Inflammatory Bowel Disease55 and campylobacteriosis53. To dissect how the host inflammatory response is involved in NE-induced ileitis, a primary chicken splenocyte cell culture system was then used similarly to previous report47. After isolation from chickens, the splenocytes were infected with C. perfringens (MOI 100) for 4 hours. The results showed that C. perfringens increased inflammatory mediators of Infγ, Litaf (Tnfα), Mmp9 and Ptgs2 (protein COX-2) mRNA accumulation by 1.54, 1.69, 1.72 and 8.65 folds, respectively, compared to uninfected splenocytes (Fig. 6A). Because COX-2 is an important mediator in the inflammatory response32, COX inhibitor aspirin was then used in C. perfringens-infected chicken splenocytes. Interestingly, aspirin failed to reduce C. perfringens-induced inflammatory gene expression (data not shown). We then reasoned that COX signaling acted on C. perfringens-induced inflammatory cytokines. Inflammatory cytokine of recombinant chicken INFγ (ch-INFγ) was then used to challenge splenocytes in the presence of aspirin. Aspirin reduced ch-INFγ-induced inflammatory gene expression of Infγ, Litaf (Tnfα), and Mmp9 by 44, 45, and 65%, respectively (Fig. 6B). Because no chicken TNFα was available, murine INFγ (mINFγ) and mTNFα were used. Consistently, aspirin also reduced murine INFγ (mINFγ)-induced inflammatory gene expression of Infγ, Litaf, and Mmp9 by 41, 27, and 45%, respectively (Fig. 7A). Similarly, aspirin reduced mTNFα-induced inflammatory gene expression of Infγ, Litaf, and Mmp9 by 49, 53, and 27%, respectively (Fig. 7B). These data indicate that C. perfringens induces inflammatory cytokines and COX-2 while blocking COX signaling by aspirin reduces inflammatory cytokine-induced responses, suggesting that aspirin poses protection potential against NE detrimental inflammatory response.
Aspirin attenuates NE-induced ileitis, intestinal cell apoptosis, and productivity loss
To functionally assess the protective effect of aspirin against NE-induced ileitis, broiler chickens fed with aspirin diet (ASP) were infected with E. maxima and C. perfringens as describe before. Ileal tissues were collected and histopathology analysis was performed to assess NE. Consistently, NE birds had severe ileal necrosis with immune cell infiltration and villus shortening (Fig. 8A). Notably, ASP attenuated NE-induced intestinal inflammation and histopathological score (Fig. 8A,B). At cellular level, ASP reduced NE-induced immune cell apoptosis in villus lamina propria (Fig. 8C,D). On growth performance, ASP birds grew slower compared to control diet birds during 0–18 days of age (Fig. 9). This is because aspirin inhibits all COX isoforms and COX-1 and -3 are important for intestinal homeostasis and growth. Notably, ASP attenuated NE-induced BW loss by 60% during NE phase of 23–26 days of age, while no difference between ASP and NE birds during coccidiosis phase of 18–23 days of age.
Although NE has reemerged as a prevalent chicken disease worldwide in the antimicrobial free era5, the lack of comprehensive molecular mechanism insight into NE severely hinders the development of antimicrobial alternatives to control this disease56. Many virulence factors of Eimeria and C. perfringens are identified but few findings are effective to control NE in chicken production9,57, suggesting that important players/factors in NE pathogenesis were overlooked, such as microbiome and host response. We reported here that microbiota metabolic product DCA attenuated chicken NE by reducing chicken inflammatory COX signaling pathways. These new findings offer approaches for exploring novel antimicrobial alternatives to control C. perfringens-induced diseases.
It is a relatively new concept to manipulate microbiota and its metabolic products against infectious diseases. Fecal transplantation was used in chickens decades ago to prevent S. infantis infection14. Microbiome plays an important role in susceptibility to human C. difficile infection58. Anaerobe C. scindens-transformed secondary bile acids prevent C. difficile germination and growth16. LCA and DCA but not primary bile acid CA inhibit C. difficile vegetable growth and toxin production16,59. However, whether secondary bile acids prevent or treat C. difficile infection in human or animal models is still unknown. It has been recently found that orally gavaging DCA attenuated C. jejuni-induced intestinal inflammation in germ-free mice20. Based on the knowledge, we then reasoned that DCA might prevent E. maxima- and C. perfringens-induced chicken NE. Indeed, dietary DCA prevented NE and its associated productivity loss. The reduction of ileitis was coupled with reduced C. perfringens intestinal tissue invasion and intestinal inflammation and cell death. Intriguingly, DCA failed to reduce C. perfringens ileal luminal colonization, suggesting that the mechanism of DCA action is independent of intestinal luminal colonization exclusion and is possibly through modulating other factors such as inflammation.
At the cellular level, the intestinal tract of NE-inflicted birds displays severe small intestinal inflammation, showing massive immune cells infiltration into lamina propria, villus breakdown, and crypt hyperplasia60,61. Inflammation often induces cell death54. Normal intestinal epithelial cells have polarity62 and their nuclei are located toward the basal membrane63, while stressed dying (apoptosis or necrosis) cells lose polarity64 and their nuclei disperse from basal to apical membranes65. Intestinal inflammation is also critical to clear invaded microbes and to resolve inflammation, while overzealous inflammation causes more bacterial invasion and further collateral damage and inflammation66. Infectious bacteria often hijack the inflammatory pathways to gain survival and invasion advantage. For example, Salmonella Typhimurium induces extensive inflammation in mouse intestine and thrives on the inflammation67. Interestingly, S. Typhimurium infection causes immunosuppressive effect in neonatal chickens because of lymphocyte depletion68. Unlike S. Typhimurium infection in neonatal birds, coccidia infection induces strong immune response and intestinal inflammation in chickens22,23. Furthermore, NE birds suffered more intestinal inflammation compared to Em birds in current study. Consistent with the “over-inflammation” model, NE birds with severe intestinal inflammation showed extensive immune cell infiltration, increased inflammatory cytokine gene expression, and epithelial cell hyperplasia and death (dispersed nuclei and apoptosis). Conversely, DCA attenuated the intestinal inflammation and inflammatory cytokines and improved the growth performance and reduced the NE pathology. Consistent with the reasoning of anti-inflammation reducing NE, blocking inflammatory COX-2 signaling pathway by aspirin alleviated intestinal inflammation, villus apoptosis and NE-induced BW loss. These findings indicate that DCA attenuates NE through decreasing inflammatory signaling pathways.
Different strains of C. perfringens produce a variety of toxins including alpha (CPA), beta (CPB), epsilon (ETX), iota (ITX), enterotoxin (CPE), necrotic enteritis B-like toxin (NetB), and others69. Among them, researchers have reported that NetB but not CPA induced NE in chickens70. However, NE doesn’t have strong association with NetB positive C. perfringens in US71. In our NE model, no difference of NetB gene expression in ileal digesta between healthy control and NE birds (data not shown) suggests limited role of the toxin. Generally, C. perfringens toxins induce cell death of apoptosis, necrosis, necroptosis, and pyroptosis in the presence of extracellular calcium influx69. Higher dietary calcium increases chicken mortality and reduces growth performance in coccidiosis-induced NE model72, suggesting the important role of toxin-induced and calcium-mediated cell death in NE. The reduction of intestinal cell death by DCA and aspirin suggests possible toxin involvement in our NE model. Future work on C. perfringens toxins, DCA, and inflammatory response will be conducted.
In contrast to the accumulating evidence of bile acids against infectious enteritis in recent studies16,20, the microbial-derived metabolites have long been implicated in chronic diseases18. Plasm bile acids is correlated with increased body mass index of obese patients73. Plasma chenodeoxycholic acid, CA and DCA concentrations are higher in type 2 diabetes patients compared to healthy subjects74. Bile acid promoting fatty acid digestion and absorption may play important role in the diseases. Furthermore, DCA and LCA in the colonic contents are increased in humans consuming a high fat diet75. The increase of the two bile acids in feces is associated with elevated incidence of colorectal cancer76. DCA damages genomic DNA by oxidation77 and deficiency in base excision repair of oxidative DNA damage is linked to increased risk of intestinal tumors in mice78. Future work is needed to investigate how DCA induces the chronic diseases but attenuates the infectious enteritis.
Altogether, these data reveal that the microbial metabolic product secondary bile acid DCA attenuates NE, through reducing NE-induced host inflammatory response. These findings highlight the importance of elucidating the molecular relationship between infectious pathogen, microbiome, and host response. These discoveries could be applied to control NE and other intestinal diseases targeting microbiome and host inflammatory response.
Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.
Neu, H. C. The crisis in antibiotic resistance. Science 257, 1064–1073 (1992).
McGann, P. et al. Escherichia coli Harboring mcr-1 and blaCTX-M on a Novel IncF Plasmid: First report of mcr-1 in the USA. Antimicrob Agents Chemother. https://doi.org/10.1128/AAC.01103-16 (2016).
Casewell, M., Friis, C., Marco, E., McMullin, P. & Phillips, I. The European ban on growth-promoting antibiotics and emerging consequences for human and animal health. The Journal of antimicrobial chemotherapy 52, 159–161, https://doi.org/10.1093/jac/dkg313 (2003).
Wade, B. & Keyburn, A. The true cost of necrotic enteritis. Poultry World 31, 16–17 (2015).
Kaldhusdal, M., Benestad, S. L. & Lovland, A. Epidemiologic aspects of necrotic enteritis in broiler chickens - disease occurrence and production performance. Avian pathology: journal of the W.V.P.A 45, 271–274, https://doi.org/10.1080/03079457.2016.1163521 (2016).
Timbermont, L., Haesebrouck, F., Ducatelle, R. & Van Immerseel, F. Necrotic enteritis in broilers: an updated review on the pathogenesis. Avian Pathol 40, 341–347, https://doi.org/10.1080/03079457.2011.590967 (2011).
Olkowski, A. A., Wojnarowicz, C., Chirino-Trejo, M. & Drew, M. D. Responses of broiler chickens orally challenged with Clostridium perfringens isolated from field cases of necrotic enteritis. Research in veterinary science 81, 99–108, https://doi.org/10.1016/j.rvsc.2005.10.006 (2006).
Olkowski, A. A., Wojnarowicz, C., Chirino-Trejo, M., Laarveld, B. & Sawicki, G. Sub-clinical necrotic enteritis in broiler chickens: novel etiological consideration based on ultra-structural and molecular changes in the intestinal tissue. Research in veterinary science 85, 543–553, https://doi.org/10.1016/j.rvsc.2008.02.007 (2008).
Prescott, J. F., Parreira, V. R., Mehdizadeh Gohari, I., Lepp, D. & Gong, J. The pathogenesis of necrotic enteritis in chickens: what we know and what we need to know: a review. Avian pathology: journal of the W.V.P.A 45, 288–294, https://doi.org/10.1080/03079457.2016.1139688 (2016).
Caricilli, A. M., Castoldi, A. & Camara, N. O. Intestinal barrier: A gentlemen’s agreement between microbiota and immunity. World J Gastrointest Pathophysiol 5, 18–32, https://doi.org/10.4291/wjgp.v5.i1.18 (2014).
Subramanian, S. et al. Persistent gut microbiota immaturity in malnourished Bangladeshi children. Nature. https://doi.org/10.1038/nature13421 (2014).
Belkaid, Y. & Hand, T. W. Role of the microbiota in immunity and inflammation. Cell 157, 121–141, https://doi.org/10.1016/j.cell.2014.03.011 (2014).
Deshmukh, H. S. et al. The microbiota regulates neutrophil homeostasis and host resistance to Escherichia coli K1 sepsis in neonatal mice. Nat Med. https://doi.org/10.1038/nm.3542 (2014).
Rantala, M. & Nurmi, E. Prevention of the growth of Salmonella infantis in chicks by the flora of the alimentary tract of chickens. British poultry science 14, 627–630, https://doi.org/10.1080/00071667308416073 (1973).
Silverman, M. S., Davis, I. & Pillai, D. R. Success of self-administered home fecal transplantation for chronic Clostridium difficile infection. Clin Gastroenterol Hepatol 8, 471–473, https://doi.org/10.1016/j.cgh.2010.01.007 (2010).
Buffie, C. G. et al. Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile. Nature 517, 205–208, https://doi.org/10.1038/nature13828 (2015).
Ridlon, J. M., Kang, D. J. & Hylemon, P. B. Bile salt biotransformations by human intestinal bacteria. Journal of lipid research 47, 241–259, https://doi.org/10.1194/jlr.R500013-JLR200 (2006).
Ma, H. & Patti, M. E. Bile acids, obesity, and the metabolic syndrome. Best practice & research. Clinical gastroenterology 28, 573–583, https://doi.org/10.1016/j.bpg.2014.07.004 (2014).
Bernstein, C. et al. Carcinogenicity of deoxycholate, a secondary bile acid. Archives of toxicology 85, 863–871, https://doi.org/10.1007/s00204-011-0648-7 (2011).
Sun, X. et al. Microbiota-Derived Metabolic Factors Reduce Campylobacteriosis in Mice. Gastroenterology 154, 1751–1763 e1752, https://doi.org/10.1053/j.gastro.2018.01.042 (2018).
Williams, R. B. Intercurrent coccidiosis and necrotic enteritis of chickens: rational, integrated disease management by maintenance of gut integrity. Avian pathology: journal of the W.V.P.A 34, 159–180, https://doi.org/10.1080/03079450500112195 (2005).
Rose, M. E., Long, P. L. & Bradley, J. W. Immune responses to infections with coccidia in chickens: gut hypersensitivity. Parasitology 71, 357–368 (1975).
Dalloul, R. A. & Lillehoj, H. S. Poultry coccidiosis: recent advancements in control measures and vaccine development. Expert Rev Vaccines 5, 143–163, https://doi.org/10.1586/147605220.127.116.11 (2006).
Paiva, D. & McElroy, A. Necrotic enteritis: Applications for the poultry industry. The Journal of Applied Poultry Research 23, 557–566, https://doi.org/10.3382/japr.2013-00925 (2014).
Uzal, F. A. et al. Towards an understanding of the role of Clostridium perfringens toxins in human and animal disease. Future Microbiol 9, 361–377, https://doi.org/10.2217/fmb.13.168 (2014).
Freedman, J. C., Shrestha, A. & McClane, B. A. Clostridium perfringens Enterotoxin: Action, Genetics, and Translational Applications. Toxins 8, https://doi.org/10.3390/toxins8030073 (2016).
Biondi, C. et al. Prostaglandin E2 inhibits proliferation and migration of HTR-8/SVneo cells, a human trophoblast-derived cell line. Placenta 27, 592–601, https://doi.org/10.1016/j.placenta.2005.07.009 (2006).
Dey, I., Lejeune, M. & Chadee, K. Prostaglandin E2 receptor distribution and function in the gastrointestinal tract. Br J Pharmacol 149, 611–623, https://doi.org/10.1038/sj.bjp.0706923 (2006).
Lazarus, M. The differential role of prostaglandin E2 receptors EP3 and EP4 in regulation of fever. Mol Nutr Food Res 50, 451–455, https://doi.org/10.1002/mnfr.200500207 (2006).
Bos, C. L., Richel, D. J., Ritsema, T., Peppelenbosch, M. P. & Versteeg, H. H. Prostanoids and prostanoid receptors in signal transduction. Int J Biochem Cell Biol 36, 1187–1205, https://doi.org/10.1016/j.biocel.2003.08.006 (2004).
Lee, Y., Rodriguez, C. & Dionne, R. A. The role of COX-2 in acute pain and the use of selective COX-2 inhibitors for acute pain relief. Curr Pharm Des 11, 1737–1755 (2005).
Wallace, J. L. Prostaglandin biology in inflammatory bowel disease. Gastroenterol Clin North Am 30, 971–980 (2001).
Tessner, T. G., Muhale, F., Riehl, T. E., Anant, S. & Stenson, W. F. Prostaglandin E2 reduces radiation-induced epithelial apoptosis through a mechanism involving AKT activation and bax translocation. J Clin Invest 114, 1676–1685, https://doi.org/10.1172/JCI22218 (2004).
Martin-Venegas, R., Roig-Perez, S., Ferrer, R. & Moreno, J. J. Arachidonic acid cascade and epithelial barrier function during Caco-2 cell differentiation. Journal of lipid research 47, 1416–1423, https://doi.org/10.1194/jlr.M500564-JLR200 (2006).
Zamuner, S. R., Warrier, N., Buret, A. G., MacNaughton, W. K. & Wallace, J. L. Cyclooxygenase 2 mediates post-inflammatory colonic secretory and barrier dysfunction. Gut 52, 1714–1720 (2003).
Hatazawa, R., Ohno, R., Tanigami, M., Tanaka, A. & Takeuchi, K. Roles of endogenous prostaglandins and cyclooxygenase isozymes in healing of indomethacin-induced small intestinal lesions in rats. J Pharmacol Exp Ther 318, 691–699, https://doi.org/10.1124/jpet.106.103994 (2006).
Pavlidis, P. & Bjarnason, I. Aspirin Induced Adverse Effects on the Small and Large Intestine. Curr Pharm Des 21, 5089–5093 (2015).
Alrubaye, B. et al. Microbial metabolite deoxycholic acid shapes microbiota against Campylobacter jejuni chicken colonization. PloS one 14, e0214705, https://doi.org/10.1371/journal.pone.0214705 (2019).
Galarza-Seeber, R. et al. Leaky Gut and Mycotoxins: Aflatoxin B1 Does Not Increase Gut Permeability in Broiler Chickens. Frontiers in veterinary science 3, 10, https://doi.org/10.3389/fvets.2016.00010 (2016).
Basak, S. C., Lee, S., Barta, J. R. & Fernando, M. A. Differential display analysis of gene expression in two immunologically distinct strains of Eimeria maxima. Parasitol Res 99, 28–36, https://doi.org/10.1007/s00436-005-0087-6 (2006).
Long, P. L., Millard, B. J., Joyner, L. P. & Norton, C. C. A guide to laboratory techniques used in the study and diagnosis of avian coccidiosis. Folia Vet Lat 6, 201–217 (1976).
Shivaramaiah, S. et al. The role of an early Salmonella Typhimurium infection as a predisposing factor for necrotic enteritis in a laboratory challenge model. Avian diseases 55, 319–323, https://doi.org/10.1637/9604-112910-ResNote.1 (2011).
McReynolds, J. L. et al. Evaluation of immunosuppressants and dietary mechanisms in an experimental disease model for necrotic enteritis. Poult Sci 83, 1948–1952 (2004).
Heikinheimo, A. & Korkeala, H. Multiplex PCR assay for toxinotyping Clostridium perfringens isolates obtained from Finnish broiler chickens. Letters in applied microbiology 40, 407–411, https://doi.org/10.1111/j.1472-765X.2005.01702.x (2005).
Tanaka, M. & Riddell, R. H. The pathological diagnosis and differential diagnosis of Crohn’s disease. Hepato-gastroenterology 37, 18–31 (1990).
Odze, R. Diagnostic problems and advances in inflammatory bowel disease. Modern pathology: an official journal of the United States and Canadian Academy of Pathology, Inc 16, 347–358, https://doi.org/10.1097/01.MP.0000064746.82024.D1 (2003).
Sun, X., Threadgill, D. & Jobin, C. Campylobacter jejuni induces colitis through activation of mammalian target of rapamycin signaling. Gastroenterology 142, 86–95 e85, https://doi.org/10.1053/j.gastro.2011.09.042 (2012).
Joo, Y. E. et al. Tomato lycopene extract prevents lipopolysaccharide-induced NF-kappaB signaling but worsens dextran sulfate sodium-induced colitis in NF-kappaBEGFP mice. PloS one 4, e4562, https://doi.org/10.1371/journal.pone.0004562 (2009).
Rueden, C. T. et al. ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinformatics 18, 529, https://doi.org/10.1186/s12859-017-1934-z (2017).
Sun, X. & Jobin, C. Nucleotide-binding oligomerization domain-containing protein 2 controls host response to Campylobacter jejuni in Il10−/− mice. J Infect Dis 210, 1145–1154, https://doi.org/10.1093/infdis/jiu148 (2014).
Yuan, J. S., Reed, A., Chen, F. & Stewart, C. N. Jr. Statistical analysis of real-time PCR data. BMC Bioinformatics 7, 85, https://doi.org/10.1186/1471-2105-7-85 (2006).
Sangild, P. T. et al. Diet- and colonization-dependent intestinal dysfunction predisposes to necrotizing enterocolitis in preterm pigs. Gastroenterology 130, 1776–1792, https://doi.org/10.1053/j.gastro.2006.02.026 (2006).
Sun, X., Liu, B., Sartor, R. B. & Jobin, C. Phosphatidylinositol 3-kinase-gamma signaling promotes Campylobacter jejuni-induced colitis through neutrophil recruitment in mice. J Immunol 190, 357–365, https://doi.org/10.4049/jimmunol.1201825 (2013).
Yang, Y., Jiang, G., Zhang, P. & Fan, J. Programmed cell death and its role in inflammation. Military Medical Research 2, 12, https://doi.org/10.1186/s40779-015-0039-0 (2015).
Nielsen, O. H. & Rask-Madsen, J. Mediators of inflammation in chronic inflammatory bowel disease. Scandinavian journal of gastroenterology. Supplement 216, 149–159 (1996).
Caly, D. L., D’Inca, R., Auclair, E. & Drider, D. Alternatives to Antibiotics to Prevent Necrotic Enteritis in Broiler Chickens: A Microbiologist’s Perspective. Frontiers in microbiology 6, 1336, https://doi.org/10.3389/fmicb.2015.01336 (2015).
Chapman, H. D. et al. A selective review of advances in coccidiosis research. Advances in parasitology 83, 93–171, https://doi.org/10.1016/B978-0-12-407705-8.00002-1 (2013).
Abt, M. C., McKenney, P. T. & Pamer, E. G. Clostridium difficile colitis: pathogenesis and host defence. Nature reviews. Microbiology 14, 609–620, https://doi.org/10.1038/nrmicro.2016.108 (2016).
Sorg, J. A. & Sonenshein, A. L. Bile salts and glycine as cogerminants for Clostridium difficile spores. J Bacteriol 190, 2505–2512, https://doi.org/10.1128/JB.01765-07 (2008).
Long, J. R., Pettit, J. R. & Barnum, D. A. Necrotic enteritis in broiler chickens. II. Pathology and proposed pathogenesis. Canadian journal of comparative medicine: Revue canadienne de medecine comparee 38, 467–474 (1974).
Al-Sheikhly, F. & Truscott, R. B. The pathology of necrotic enteritis of chickens following infusion of broth cultures of Clostridium perfringens into the duodenum. Avian diseases 21, 230–240 (1977).
Royer, C. & Lu, X. Epithelial cell polarity: a major gatekeeper against cancer? Cell death and differentiation 18, 1470–1477, https://doi.org/10.1038/cdd.2011.60 (2011).
Xin, Z. S. 188–270 (Springer, 1998).
Delgado, M. E., Grabinger, T. & Brunner, T. Cell death at the intestinal epithelial front line. The FEBS journal 283, 2701–2719, https://doi.org/10.1111/febs.13575 (2016).
Grant, T. D. & Specian, R. D. Epithelial cell dynamics in rabbit cecum and proximal colon P1. The Anatomical record 264, 427–437 (2001).
Sherman, M. A. & Kalman, D. Initiation and resolution of mucosal inflammation. Immunologic Research 29, 241, https://doi.org/10.1385/ir:29:1-3:241 (2004).
Winter, S. E. et al. Gut inflammation provides a respiratory electron acceptor for Salmonella. Nature 467, 426–429, https://doi.org/10.1038/nature09415 (2010).
Hassan, J. O. & Curtiss, R. 3rd Virulent Salmonella typhimurium-induced lymphocyte depletion and immunosuppression in chickens. Infect Immun 62, 2027–2036 (1994).
Navarro, M. A., McClane, B. A. & Uzal, F. A. Mechanisms of Action and Cell Death Associated with Clostridium perfringens Toxins. Toxins (Basel) 10, https://doi.org/10.3390/toxins10050212 (2018).
Keyburn, A. L. et al. NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens. PLoS Pathog 4, e26, https://doi.org/10.1371/journal.ppat.0040026 (2008).
Martin, T. G. & Smyth, J. A. Prevalence of netB among some clinical isolates of Clostridium perfringens from animals in the United States. Vet Microbiol 136, 202–205, https://doi.org/10.1016/j.vetmic.2008.10.026 (2009).
Paiva, D. M., Walk, C. L. & McElroy, A. P. Influence of dietary calcium level, calcium source, and phytase on bird performance and mineral digestibility during a natural necrotic enteritis episode. Poult Sci 92, 3125–3133, https://doi.org/10.3382/ps.2013-03298 (2013).
Prinz, P. et al. Plasma bile acids show a positive correlation with body mass index and are negatively associated with cognitive restraint of eating in obese patients. Frontiers in neuroscience 9, 199, https://doi.org/10.3389/fnins.2015.00199 (2015).
Cariou, B. et al. Fasting plasma chenodeoxycholic acid and cholic acid concentrations are inversely correlated with insulin sensitivity in adults. Nutrition & metabolism 8, 48, https://doi.org/10.1186/1743-7075-8-48 (2011).
Reddy, B. S. et al. Effect of high-fat, high-beef diet and of mode of cooking of beef in the diet on fecal bacterial enzymes and fecal bile acids and neutral sterols. The Journal of nutrition 110, 1880–1887, https://doi.org/10.1093/jn/110.9.1880 (1980).
Hill, M. J. Bile flow and colon cancer. Mutation research 238, 313–320, https://doi.org/10.1016/0165-1110(90)90023-5 (1990).
Rosignoli, P. et al. Genotoxic effect of bile acids on human normal and tumour colon cells and protection by dietary antioxidants and butyrate. European journal of nutrition 47, 301–309, https://doi.org/10.1007/s00394-008-0725-8 (2008).
Sakamoto, K. et al. MUTYH-null mice are susceptible to spontaneous and oxidative stress induced intestinal tumorigenesis. Cancer research 67, 6599–6604, https://doi.org/10.1158/0008-5472.CAN-06-4802 (2007).
The authors would like to thank the support from staff at Poultry Health Laboratory and Feed Mill in Department of Poultry Science at University of Arkansas, Fayetteville. We also thank P. L. Matsler on helping our histology slides. This research was supported grants of Arkansas Biosciences Institute, USDA National Institute of Food and Agriculture (NIFA) Hatch project 1012366, USDA NIFA Hatch/Multi State project 1018699, USDA NIFA project 2018-06686 to X. Sun. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Wang, H., Latorre, J.D., Bansal, M. et al. Microbial metabolite deoxycholic acid controls Clostridium perfringens-induced chicken necrotic enteritis through attenuating inflammatory cyclooxygenase signaling. Sci Rep 9, 14541 (2019). https://doi.org/10.1038/s41598-019-51104-0
A secondary bile acid from microbiota metabolism attenuates ileitis and bile acid reduction in subclinical necrotic enteritis in chickens
Journal of Animal Science and Biotechnology (2020)
Scientific Reports (2020)


The ergonomic design of this sleek performance hat pairs well with sunglasses and shows off the subtle laser-cut Half Dome logo on the brim.
- This hat has an comfortable fit, vents for breathability and a low-profile Velcro® adjustment
- Lightweight and quick drying
- Venting mesh panels
- Velcro® adjustment at back
- Ultraviolet Protection Factor (UPF) 50
- Fabric: 100% Polyester plain weave
- Lining: 97% Recycled polyester / 3% Polyester FlashDry™ mesh


text:
Would you like to sell books at your event on Bainbridge Island?
Many local organizations are realizing the community service and fund-raising possibilities of bringing featured authors and experts to meetings, conferences, and off-site book fairs. Eagle Harbor Books has years of experience serving schools, non-profits, business groups, churches, and associations for these types of events.
We’ll provide books and sales kits for your events, and even staffing depending on the size and scope of the event. We’ll help to publicize your event and a portion of the proceeds from the sales will be returned to your group. These programs are each unique, so please give us a call, send us an email, or drop by the store for help in planning your event.

domain: literature


text:
The Cutest Two Little Peas In A Pod! This delightful Limoges box features TWO PEAS IN A POD with bright eyes looking at you! Inside the upper lid is the inscription "Two Peas In A Pod". This magnificent Limoges box is artfully and colorfully hand-painted with great detailing and has been fired numerous times to achieve its brilliant colors. The collectible Limoges box is adorned with intricate metalwork and an ornate clasp in the shape of a BEE. This Limoges porcelain box is meticulously crafted entirely in Limoges, France and bears the insignia of the artist.
DIMENSIONS: 3.25" W, 1" D, 1" H
You might also like . . .
Product No.: 6621
Product No.: 7699
Product No.: 2980

domain: artistic


Exhibition provides view ‘Through the Eyes of the Artist’
Bringing binational unity to the San Diego art scene, Studio Door in North Park presents the second annual exhibition of “Through the Eyes of the Artist.” Organized by Studio C artists, Laura Lehman and Carlos Castrejon, the show displays artists from both sides of the border of the United States and Mexico and their personal artistic messages.
Featured artists include Luis Alderete, Debra Babylon, Laurie Bezer, Carlos Castejon, Kenda Francis, Therese Cipiti Herron, Kris Daniels, Frank Higley, Nancy Jacobsen, Laura Lehman, Judy McClain, Judy Pike, Patric Stillman and Jose Strimling.
A reception, being held at 6 p.m. Saturday, April 8, will usher in the opening of HYPE at Studio Door, situated next door to the main gallery. In perfect artistic alignment, the popular North Park event Ray at Night runs the same night, along with additional artist receptions at OBR Architecture and Karras Chiropractor. In other words, art lovers, get your peepers ready, this isn’t an evening you will want to miss.
(Editor’s note: Ray at Night is the longest running monthly art walk in San Diego. It is held the second Saturday of every month, from 6-10 p.m. Art lovers, music fans, and foodies come out to peruse 15 galleries and business for their art fix, while swaying to live music, and munching on goodies from local food trucks.)
“By bringing together artists from across the region and the border, we create a stronger creative bond.” - Carlos Castrejon
Look for Kendra Francis’ work Juma, which portrays a leopard surrounded by a halo of machine guns. Its bright colors of blue, pink, and teal oscillate somewhere between happy and peaceful while the guns, spikes, and stars are reminiscent of Cold War Russian propaganda posters. Its blending of multiple imagery has us pondering ideas of peace, current politics and social issues, animal rights and poaching, and our own views of gun control.
Judy Pike’s Texting is a starkly rendered image of our obsession with texting. A mixture of drawing and painting, with a subdued palette of tans, blacks, and greys, the image is not joyful, but withdrawn and a bit sad. An isolated, poor-postured gentleman looks downward at his phone while letters, numbers, and punctuation float in midair near his head. It directs us to think about the 1s and 0s of data and our digital world, and our allegiance to the computer. The background of this man’s life isn’t green and alive, but bleak and absent, giving us pause about our own world that exists beyond the digital.
The exhibition runs through April 30 and is open noon to 7 p.m. Tuesday through Sunday.
3750 30th St., North Park, 619.255.4920, thestudiodoor.com
Sign up for the Pacific Insider newsletter
PACIFIC magazine delivers the latest restaurant and bar openings, festivals and top concerts, every Tuesday.
You may occasionally receive promotional content from the Pacific San Diego.


text:
Around 38% of the respondents have by no means had sexual intercourse, and majority don’t have a couple of sexual partner all through their lifetime. There are 5,891 (37.0) respondents who wouldn’t have children, and around 4,480 (28.3%) having only one or two youngsters. Most of the respondents have a excessive (≥5/7) HIV data rating, have a high ladies empowerment rating (≥6/10), and a low tolerance to home violence. The distributions of the number of lifetime sexual companions and HIV knowledge score were found to differ between those that were tested for HIV and these who have been by no means examined for HIV. Despite these, none of the quantitative publicity variables had proven a robust proof of association with HIV testing .
2.3 million more are in vulnerable employment, with insufficient earnings and social safety. In the search for first rate work, many young Filipinos transfer from rural to city areas, with some opting to go abroad.
Larger negative results of extra bodily demanding jobs had been characteristics of the older age cohorts. At entry into the CLHNS, they ranged in age from 15–45 y, imply parity was 2.23 ± 2.20, 19% were pregnant for the primary time, 29.2% had less than a sixth grade schooling, and 12.7% had completed some schooling beyond highschool. SES, food plan, reproductive variables, and work-related activity varied considerably over time . Changes over time mirrored rising urbanization and family income, improved household hygiene, and acquisition of extra belongings.
The incidence and magnitude of reproductive stresses various with age, but their effect on weight was similar throughout age groups. As women aged, more and more experienced menopause, which was related to decrease weight.
The Filipino Girls Trap
Full follow-up surveys have been performed in 1991, 1994–1995, 1998–1999, 2002, and 2005. From the primary 2 y of bimonthly information, we chosen 3 time points for inclusion in longitudinal fashions. To maximize sample dimension, if a woman was lacking or pregnant at considered one of these early surveys, we substituted data from the prior survey . The CLHNS has been reviewed and accredited by the University of North Carolina Institutional Review Board, Office of Human Research Ethics. Filipino girls asiawomen.org are unique in the way they convey with others and respect other peoples’ choices. They additionally look after all kith and kin belonging to their tribe, clan, and people. Unlike American women, Filipino girls sincerely and unconditionally respect males and admit their power and essential role in everything.
It has additionally resisted efforts to promote sexuality training and safer intercourse in colleges. Many of the efforts to advance LGBT rights have met with resistance from the Catholic Church, which has been an influential political drive on matters of sex and sexuality. While the CBCP rejects discrimination towards LGBT individuals in principle, it has frequently opposed efforts to prohibit that discrimination in practice. In 2017, for instance, the Church sought amendments to pending anti-discrimination legislation that may prohibit same-sex marriage and allow non secular objectors to choose out of recognizing LGBT rights. President Rodrigo Duterte has generally been supportive of LGBT rights as nicely. During his time as mayor, Davao City handed an LGBT-inclusive anti-discrimination ordinance, and on the campaign trail, he vocally condemned bullying and discrimination in opposition to LGBT folks. A sexual orientation in which a lady is primarily sexually or romantically drawn to different women.
In that case, an Asian woman was ready in a subway station in Manhattan and observed that somebody had set her backpack on hearth. On Tuesday, nevertheless, a law enforcement official mentioned that police now imagine the victim was Hispanic and the violence may have began when he referred to as his attacker, who was Black, a racial slur. A torrent of hate and violence against individuals of Asian descent across the United States began final spring, in the early days of the coronavirus pandemic. Last week, the New York Police Department announced that it might begin deploying undercover officers to neighborhoods with giant Asian populations in response to the rising attacks. All unprovoked assaults on individuals of Asian descent will now be referred for investigation as potential hate crimes, police officials mentioned. Anti-Asian hate crimes have lengthy been underreported due to components that embody language barriers and distrust of the police, in accordance with neighborhood advocates. The police released a photo of a person they said was wished within the attack on a 65-year-old girl in Manhattan.
The U.S.A. introduced a model new public schooling system which retained opportunity to every baby regardless of gender. President Duterte is not a person who troubles himself with women’s protection and safety.
Successful Strategies For Filipino Girls That You Could Use Starting Today
The man should courtroom the girl and prove his love for her earlier than he can win her heart. In the bigger more urbanized cities, this conservative courtship concept is not so emphasised as a lot. Parents favor their daughter to be courted in their home, so they can have an opportunity to know the person.
As quickly as she arrived at her new employer’s home, she knew she was in trouble. Her passport and phone had been taken away and she wasn’t allowed to contact her household.
Whether or not it was in response to the petition, the brand new decree has provided a small sense of aid and recognition. I was therefore not stunned to hear to from them that, within the face of the Italian government’s lack of support in the Covid-19 pandemic, Filipina in northern Italian cities had been taking excellent care of one another. Diaz has paved the method, placing Filipino weightlifting on the map, now others are following and simply think about what a gold medal in Tokyo would do for women and for weightlifting throughout the country. Her husband is now with another lady but, from time to time, he goes to her home demanding to see their kids. The Philippines took a step towards making divorce authorized in March with the lower home of Congress passing a regulation allowing individuals to dissolve marriages, within the face of opposition from the president and bishops in the primarily Roman Catholic nation. “With ladies being away, their proper to take part in neighborhood life and decisionmaking is challenged. They are additionally weak to abuse and trafficking,” he said. But the movement of women can also be having an unhappier influence on households in a rustic where males have long been the primary earners, and often had an even bigger say in the household.
“As an individual of both Asian and Pacific Islander descent, I truly have been watching with shock and dismay at the persevering with acts of violence and hate being directed in path of people who look like me,” said Police Chief Peter Decena. The program is on the market to all members of the Filipino group in Calgary, no matter their immigration status. Three novellas–including Obsession, Platinum, and Cadena de Amor–examine the Philippine experience by way of the lives of three female characters, a prostitute, a pupil activist, and a politician. Being a pretentious prick that can’t get sufficient of his muscular tissues, money or car is not any proper way tips on how to get a philippines lady to like you.

domain: sociology


text:
Rates Payment Management
A4 Office Products occupy 2 adjacent warehouses on the Rotherwas Industrial Estate, Hereford which were reconfigured resulting in savings of over 15% on past rates bills since 2010.
BWCS occupied a small office in Ledbury, Herefordshire just off the high street. After auditing their accounts past overpayments were found resulting in a refund of over £8000.
© SMB Surveyors
SMB is a trademark of Survey My Building Limited
Registered Office: Atelier B1, The Old Brewery, Lodway, Pill, Bristol, BS20 0DH

domain: economics


text:
These carved arrowheads make a unique gift and are wonderful for adding to crystal grids.
The amethyst, clear quartz and rose quartz are offered in parcels of 5 arrowheads in assorted sizes measuring between 1 1/4" to 1 3/4" long.
The black obsidian are offered individually and measure between 2 1/4" long
Each parcel of 5 comes with a crystal description card and a drawstring pouch. The black obsidian also come with a drawstring pouch and a crystal description card.


This Brooklyn based duo will have you swooning over lovers you never knew you had. Their eclectic sounds embrace the best parts of electronic R&B and indie pop twisting together heavy bass and sultry lyrics that sing to the soul. Their newest single, Rob You has us anxiously awaiting their newest album that we'll be sure to have on repeat.
What does BLKKATHY have to say about their latest single? They shared the inspiration behind "Rob You" with us.
"Rob You" is basically about how crazy absurd it is to date a stranger and think you'll be safe (emotionally). It's based on another song by Curtis Cook and Elise Silver.
We are certainly not cynics about love, sometimes it's awesome, but it's rough too. Every time I break up, I'm like "Damn! It's like that?! I thought you cared about me." Then I remember that dating for a few weeks or months doesn't really make someone any less of stranger to me or me to them.
It's a total miracle every time we trust someone new. I'm so grateful for the capacity to do that. But I still feel like a dumbass for doing it sometimes."


[Author Prev][Author Next][Thread Prev][Thread Next][Author Index][Thread Index]
Re: Therostat & Fan Switc
Mark Eissler <email@example.com> replies:
ME> Replacing the thermostat will go a lot easier if you remove the power
ME> steering pump (well, don't remove it completely, just release the belt
ME> tension, unbolt the pump, and move it to the side, with hoses
ME> attached, so you can get better access). I suppose you could change
ME> the thermo. without removing the p/s pump. But you might sprain your
ME> resist, break a nail, or something....
Agree totally, the pump is one of the easiest components
to remove on the engine. (how'd that one slip by the
engineers). It would also be an excellent time to
pull out the Bentley and a multimeter and check
those senders/sensors that also become accessible.
Checking the hoses, including that "invisible"
3 inch hose connecting the steel coolant piping
could also save alot of future agrevation.
... Why do Scots wear kilts? Because sheep can hear zippers.
* Blue Wave/QWK v2.20 [NR] *


Submitting patches for Gpredict
This page provides some basic guidelines for submitting patches to the Gpredict code.
- Gpredict uses git for source code management and patches should always be submitted against the latest code in the master branch.
- Clean your code for whitespace errors (hint:
git diff --check)
- Commit logically separate changesets, i.e. one commit per topic. Don't combine several topics into one patch and don't split one topic over many commits.
- Use meaningful commit messages. As a general rule, your messages should start with a single line that’s no more than about 50 characters and that describes the changeset concisely, followed by a blank line, followed by a more detailed explanation.
- If the changes fix a known bug or implement a requested feature, the ID of the bug/feature should appear in the first line.
- Work in a separate branch and create the patches using
git format-patch -M origin/master. If not possible
git diff origin/masterwill also be acceptable for small changes.
- Submit the patches to one of the following:
Note that submitting a patch is no guarantee that it will be applied.
This section shows an example workflow for creating a patch according the the requirements listed above.
First, get the latest code from the git repository:
$ git clone git://gpredict.git.sourceforge.net/gitroot/gpredict/gpredict gpredict.git $ cd gpredict.git
If you already have a copy pull the latest changes:
$ git pull
Ensure that your name and email address is properly configured. If it is configured globally you're fine, if not you can configure it locally for gpredict:
$ git config --global user.name "Firstname Lastname" $ git config --global user.email "email@example.com"
Next, create a branch where you will be doing your work:
$ git checkout -b mywork
When finished, check your code for whitespace errors:
$ git diff --check
and commit your changes:
$ git add file1 file2 file3 $ git commit
Format the commit message according to the requirements. In case of small changes a 50 char or less one liner may be sufficient. In such cases you can use
git commit -m 'My commit message'.
Before you create the patch, it is important to update your local copy to the latest code and rebase your changes accordingly:
$ git checkout master $ git pull $ git checkout mywork $ git rebase master
This will replay your changes on top of the latest changes from master. See
git help rebase for more information. If a conflict occurs during the rebase, it must be resolved before you can proceed.
When ready, create the patch(es):
$ git format-patch -M master
This will create some nicely formatted patches, one patch per commit. Author information will be included in each patch.
Submit the patches at the appropriate place, see the last bullet of the requirements.


text:
This tee is inspired by Saint Therese of Lisieux and one of her famous quotes, "The world is thy ship, not thy home." Reminding us our true home is awaiting us in Heaven. This Boxy tee is perfect to wear with your high-waisted skirts and jeans...super cute and fashionable!
•Premium Comfort Colors™ Boxy Tee (NOT a crop tee)
•Soft ring-spun cotton fabric with 100% cotton threads

domain: design


text:
CAPITALISM DIED many deaths. Its resurrections from the ashes of socialist fatalism had already lost their novelty. Still, the proverbial crisis in capitalism keeps the Cassandras in business; it continues to make convenient socialists out of suddenly deflated free marketeers; and it alone sustains the moral absolutism of anti-globalists. There are still people out there who remind us that the argument capitalism claims to have finally won in the last century remains unresolved. They tell us that the wealth gap is so wide that it’s impossible to romanticise the creativity of the capitalist. They despise the vulgarity and inequalities of the McWorld, its inherent inhumanity. It invalidates the idea of a just world. So capitalism must die. It does, once in a while.
The one in 2008 was momentous. It crashed when it looked invincible, and that too in a world which had already abandoned socialism for good. It crashed when only archival socialists wept for the phony theologies of equality, and when the last masters of the universe lived on Wall Street. And every fall, every rupture, in history is personalised by one morality tale, one totemic legend. In 2008, it was the Lehman Brothers. Its sudden fall made American capitalism almost bankrupt; it made all of us poorer, and some of us wiser. The eternal had become ephemeral, bringing to an end the mythology of the American Dream. When the curtain fell on a stage littered with shattered lives, the crash of capitalism was as emotionally exhausting as a Greek tragedy.
In The Lehman Trilogy by the Italian playwright Stefano Massini, first staged in Paris six years ago, American capitalism is dramatised as a generational saga, beginning with a dream and the magic of distance, ending with a fatal ringing of the telephone. The history of capitalism here is the history of migration, of the movement of man and his desire, his fantasies and follies, his greater ability to build a collapsible monument for himself. I saw the version adapted for the English stage by Ben Power, and directed by Sam Mendes, at London’s Piccadilly Theatre. The epic never looked so intensely spartan, and in more than three hours of riveting theatre, the humanisation of wealth was stretched between the solitude of the migrant salesman to the temptation of gods.
The eternal had become ephemeral, bringing to an end the mythology of the American Dream
Share this on
It begins with the arrival of Henry Lehman, a Jew from Bavaria, in New York, on September 11th, 1844. He starts his picaresque from the cotton capital of Alabama, and soon he is joined by his younger brothers Emanuel and Mayer, setting the stage for one of capitalism’s most resourceful dynasties. As salesmen, bankers, builders, buyers, and lenders, they made money not to spend, but to make more money; and there was nothing untouched by the ever-expanding dreams of the Lehman clan, be it railways, automobile, airlines, entertainment, or politics. They were survivors and adventurers; they assumed they had the wherewithal to cross the boundaries of desire wealth could create in man. The thrill was the pursuit; the fall was a tragic error, an existential sham.
The grandeur of the original European production of The Lehman Trilogy was provided by the psychedelic passage of history in capitalism’s holy land. Spanning 150 years of man’s ingenuity in wealth creation, it was an overpopulated saga. The English adaptation is a triumph of re-imagination by two originals, Power and Mendes. The entire saga is played by three male actors, mostly speaking in third person, who cross generations as well as gender. Call it an amazing piece of theatrical reductionism, but what it achieves is sensational. It’s as if the Homeric has been downsized with a sense of humour. In their virtuoso performance, Simon Russell Beale, perhaps the last of the classical actors on stage, Adam Godley and Ben Miles, become narrators and enactors of a long stretch of history, the remains of which still make us cautious. On a glassy stage filled with cardboard boxes, and the shifting backdrop made spectacular by laser technology, the three mimic the passions and pathologies of capitalism to the music of time.
In its moral extremism, The Lehman Trilogy is very Old Testament. In its political resonance, it is here and now: no wall is unbreachable for the most enterprising migrant. And at another level, it is the story of history’s loneliest traveller, the Jew, struggling to reduce the distance between promise and land.
In the biography of capitalism, we have come to the narrative point where poignancy has been replaced by pornography
Share this on
BARNEY FEIN TOO carries within him the legacy of the Jewish traveller, lonelier and bathetic in the end. Fein is a fat, Viagra-propelled, Harvey Weinstein-like movie mogul in Bitter Wheat, the new play by David Mamet, who is also the director. In the biography of capitalism, we have come to the narrative point where poignancy has been replaced by pornography. The words are sharp, visceral, and savage, and they are always so in a Mamet script or play, and the man who utters them here epitomises the priapic vulgarity of power, played to poisonous perfection by John Malkovich. He is the play. He is its rotten soul. He is a fat god steeped in self-pity, a victim of glandular punishment, and another misread Jewish story in America. Dialogues are arrows in Bitter Wheat; seductions and repudiations are abrupt and in bad taste. The individual aesthetics of Fein is far removed from the award-winning movies he makes.
When I watched it at its premiere in London’s Garrick Theatre, the world it portrayed looked like one resisted by a Lehman, whose celluloid venture didn’t go beyond a King Kong. In spite of Malkovich’s scene-stealing flamboyance, Bitter Wheat is an old fashioned production, maybe deliberately so. Mamet is more interested in the message than the method. In the end, you can’t miss the message from another shattered American dream: even the vulgarian is worthy of tragic isolation. The worst movie Fein has never made is his own life.
POST SCRIPT: And Brexit politics provides the theatre of the absurd, in which Britain’s most popular politician and the frontrunner for Tory leadership is not allowed to have a quarrel with his girlfriend in the privacy of her flat. Across the wall, Guardian variety vigilantes are listening with tape recorders.


Since much of the outlandish frock that hits the runways for Fashion Week is meant more for show than real life, it’s not too hard to fathom a growing new trend: designer digital outfits.
That’s right, virtual garments that you can digitally slap on yourself for Instagram and on any online avatars you may have.
A story in Agence France-Presse recounts the work of U.K.-based influencer Daniela Loftus, who quit her job in the traditional fashion industry to devote all of her time to her new venture, This Outfit Does Not Exist.
“When I first started talking about this, my friends were like, ‘What are you talking about?’ But my 14-year-old cousins understood it immediately,” Loftus tells AFP.
For gamers, spending real money on virtual goodies — like overpowered weapons, vehicles, or a new look for their characters, known as a skin — is old hat. But Loftus is taking the concept from the gaming world to the real one — or, arguably, the fake-real one of social media.
The idea being, you buy one of Loftus’ outfits, and with some Photoshop, her dress becomes yours — all without the need to actually get dressed up or to shell out Met Gala-level fashion cash.
Loftus isn’t alone: DressX is another company from which you can buy a fresh — albeit fake — look for yourself, from dresses to hats and other accessories.
In DressX’s case, users can skip the Photoshop. Submit a picture, and a look they’ve paid for is custom “tailored” to their image, which is sent back dressed to the nines — or, more accurately, to the ones and zeroes.


text:
June 20, 2021 12:55:48 am
A clash broke out between police and workers of the Rashtriya Lok Dal (RLD) and Samajwadi Party in Baghpat’s Barut town in Uttar Pradesg on Saturday after a SP-backed Zila Panchayat member’s brother and two sons were arrested.
The protesters also blocked the Meerut-Baraut link road for over three hours, demanding the release of newly elected Panchayat member Mehboob Ali’s brother, Matloob, and two sons, Adil and Raju.
RLD’s Baghpat chief Jagpal Tevatia accused the local police of acting like agents of the ruling BJP.
“Police are arresting innocent people to ensure the victory of BJP-backed candidate for the upcoming Zila Panchayat chief’s post. We will not tolerate this. We will continue our agitation till all those arrested on fabricated charges are freed,” he said.
However, police denied the allegations made by the protesters.
Baraut DSP Alok Kumar Singh said, “The three persons were arrested in connection with an FIR lodged against them under the SC/ST Act. These arrests have no link to the Zila Panchayat elections. Local police were doing their duty and a few political workers tried to interrupt them. We will take action against those who deterred police of discharging their duty.”
According to police, trouble erupted when officers from Binaluli police station arrested Matloob, Adil and Raju on Saturday afternoon and were on the way to the police station. The SP and RLD workers forcibly tried to get them released, they added.
Mehboob Alvi said, “Our party workers staged a demonstration at the Binauli police station to protest against the hooliganism of the local police at the behest of the BJP government, which cannot tolerate victory of Opposition-backed members at any election. .”
📣 The Indian Express is now on Telegram. Click here to join our channel (@indianexpress) and stay updated with the latest headlines
- The Indian Express website has been rated GREEN for its credibility and trustworthiness by Newsguard, a global service that rates news sources for their journalistic standards.

domain: law


text:
MINEOLA, N.Y. (CBSNewYork) - The Nassau County district attorney is calling a case of animal cruelty "unthinkable and horrific" as a young married couple are accused in the deaths of two puppies and near death of a third.
Police say 29-year-old Ellie Knoller and his wife, 30-year-old Jessica Kuncman Knoller, surrendered to the Nassau County authorities on Wednesday.
Ellie is accused of killing two puppies and nearly killing a third in less than three week, just days after adopting or buying the dogs.
District attorney Madeline Singas says the healthy puppies suffered catastrophic injuries.
Blunt force trauma caused the first puppy to die of internal bleeding.
Then very next day, the couple adopted a second puppy that shortly later also died from cardiac and respiratory arrest. The cause of death was again blunt force trauma causing internal bleeding
The very next week, a third puppy they adopted, a little Golden Doodle named Bella, was brought to a Westbury veterinarian with fractured ribs, bruising on her lungs, bleeding in her eyes and a broken leg.
She survived, but the couple is under arrest. He is charged with being a serial puppy killer. She is charged with doing nothing to help Bella, causing unjustifiable suffering.
Nassau County News Conference On Animal Cruelty Charges
"In my 28 years as a prosecutor, I've never seen a case where someone committed this kind of serial violence against different puppies," said Singas.
Defense attorneys say that the couple are normal dog lovers and would not have brought their sick dogs to a veterinarian if they wanted them to die.
"This has been an on-going investigation, they've known about this for the last two months," said Greg Madey, the defense attorney for Ellie Knoller. "They're horrible accusations, but just as bad to be charged with something like this.
"The way this started is they brought the dogs to the vet, this was something they were caring for," he said. "They brought them to the vet when there was a problem or they detected the animal was injured or sick, and once that happens, there's an investigation that's started. They're upset."
The couple was released without bail. After pleading not guilty, the judge forbade them from buying, keeping or caring for any animals while the case proceeds.
Due to the cost of Bella's medical care, the NCSPCA is seeking help from the public through their website, LuckyToBeAlive.NCSPCA.US.
for more features.

domain: pet


text:
Last Modified on Apr 17, 2014<< Continued from page 1Next page >>
[YEA] 02/01/2009: Jodie from Eau Claire, WI: "The pumpkin really works! Wow. Thanks so much for your suggestions on using pure pumpkin for clearing up pet constipation issues. My dog was spayed a few days ago, and hasn't been able to go to the bathroom since I brought her home. I fed her 1 tsp. of pumpkin with her dinner, and a few hours later, she was finally able to start going to the bathroom again. Thanks!"
[YEA] 01/26/2009: Sara from Albertville, Alabama: "Cat Constipation: Hi, I found this wonderful site searching for natural cure/treatment for Sjogrens disease. In cruising the site I found this section on pet treatments. I would like to tell everyone who has cats and feeds them dry food to give them a tablespoon of canned pumpkin 3/4 times a week to prevent feline constipation.NOT pumpkin pie filling-just plain old pumpkin. I lost a cat to this after surgery to remove the blockage. One cannot also feed them milk as this is not digestable."
[YEA] 11/29/2008: Jesse from Los Angeles, CA: "My dog loves to eat. He suffered constipation the day after our Thanksgiving party. i suspect someone tossed him a bone or beef BBQ. Last night was really hard for him. After reading your web site, i went to the local store and bought a canned pumpkin. This did the trick for him. I gave him 5-7 spoons every 4 hours. The next morning he was jumping and relieved.
Thanks for the information. You help save my wonderful dog and help me a trip to the vet."
[YEA] 10/16/2008: Pam from Smithsburg, MD, USA: "The pumpkin method also worked for my golden retriever, Lucy. I think at times she tends to overeat grass. The vet is the one who told me about the pumpkin so I felt it was a safe method. Now I have to really hide it in her food as she will not eat anything remotely related to fruits or vegetables concerning people food. Over her 2-1/2 years she has become a very picky eater for both dog and people food."
[YEA] 09/30/2008: TERESA from CLINTON, MS: "MY DOG WAS CONSTIPATED AFTER BEING OUTSIDE FOR AN OUT OF TOWN STAY, WHEN HE IS USE TO STAYING INSIDE. I THINK HE OVERATE THE GRASS. THE PUMPKIN WORKED GREAT, I GAVE HIM A SMALL AMOUNT IN A BOWL AND HE ATE IT FREELY AND THEN WE WENT ON A WALK. DURING THE WALK THE PROBLEM WAS TAKEN CARE OF AFTER TWO DAYS OF NO GOING TO THE BATHROOM. THANKS FOR THE ADVICE..."
[YEA] 09/03/2008: Silvia from Toronto, On, Canada: "My kitten was constipated but what worried me most was the gas. My kitten eats raw but I have no clue how to give her pumpkin so this is what I did: I shred the pumpkin and then I put it in the blender with melted butter and obtained a paste. She loves it, I just give her 1 teaspoon a day and after 2 days the pup is too soft so I have to stop it. Anyway, great remedy. Thanks"
[YEA] 07/05/2008: Shaun M. from Cottage Grove, MN: "Wow!! My dog was constipated due to recently being spayed. Gave her a tablespoon of pumpkin when we went to bed, woke up to lots of "treats"! Thanks"
[YEA] 02/25/2008: Estelle from Taylors, SC: "Canned pumpkin for dog's constipation. Tried the canned pumpkin and it worked like a charm on my little Chihuahua. Thanks for the information."
[YEA] 01/09/2008: Kris from Benton City, Washington: "I have used Pumpkin for constipation for my dog who suffers from chronic problems daily. Contrary to others beliefs. I give him a tablespoon every day. It works great! I also have another dog that has problems with diarrhea I also give him the same amount as it has fiber and works for both problems. This is a marvelous cure all. I am thirlled that I found this information on this site."
[YEA] 07/28/2007: Mary from Costa Mesa, CA: "My dog needed to see a Vet for constipation a few years back. He had to stay there for an enema, and it wasn't cheap. Today I recognized the signs and symptoms, Googled it, and found your Pumpkin remedy. He ate it willingly and it did the trick! I'm amazed and thankful."
[YEA] 05/14/2007: Cassie from North Vancouver, BC: "The most bland pumpkin filling really works to cure both constipation AND diarrhea in a dog any age. If I known that my 10 month old Berner puppy has gotten into something, I will actually give him a heaping tablespoon to push out the junk him faster and the end result being less "mess".
BEWARE, if you feed it to a dog too often it stops being effective."
[YEA] 04/25/2007: Lisa from Wichita Falls, Texas: "I have used the pumpkin for constipation in my dog.It really works!I just put the pumpkin in my dogs mouth and keep it closed until he swallows.Because he is not going to eat it if I put it in some other food.But it does work great!! I relly love the Earth Clinic site by the way!!"
[YEA] 04/19/2007: Ginny from Ashland, OR: "Pumpkin puree really works to cure constitpation. Thanks! Some antibiotics seem to create constipation and I used a couple teaspoons of pumpkin puree with piecies of cooked chicken to flavor it with each meal during the period of time they were on anitbiotics and my two mini dachsunds ate it with no problem and in a day were back to normal. I have shared this with others and appreciate you and your contributions greatly."Replies
[YEA] 02/23/2012: Happy from Somerset, Nj replies: "I never reply to these, but wanted to say thanks! This worked great for my dog."
11/06/2011: Sebie from Friendswood, Texas, Usa: "My dog seems to be constipated she is not eating, but she is drinking and urinating. She is breathing heavy at times, walking slow, and her stomach tightens up with quivering knots. Can someone please help."Replies
11/08/2011: Linda from Toronto, Ontario, Canada replies: "did she swallow something that is stuck in her stomach? I'd get her to a homeopathic vet a.s.a.p."
11/16/2011: Beth from Plainfield, Il replies: "TRY CANNED PUMPKIN MIXED WITH FOOD."
10/31/2011: Adriana from Brooklyn, Ny, Ny: ""My cat stopped urinating. We took him to the vet to have a catheter put in, plus a 3 day hospital stay and antibiotics. After that we switched him to special canned food with a very low ash. After 2 weeks he started showing signs of it again and stopped urinating. Luckily I found this website and went out and bought some ACV. I use 50/50 and I put some diluted on his back of neck. Also I got cranberry tabs diluted with water too. The effect was miraculous! He started urinating a little within 6 hours (or less) and is almost back to his normal active self.
BUT now I notice He is straining to go poo. He's in raw diet now, I dont know if this happen because the ACV. I'm still feeding him ACV/water (once a day) through a plastic syringe, the cranberry/water (once a day) and mineral oil once a day. He's not drinking water for himself. Please do let me know if you have any information on this. Thanks a lot for this website and all of your posts. I am so grateful to have found this great site."Replies
11/01/2011: Timh from Louisville, Usa replies: "Adriana, sure sounds like your kitty is quite ill. The mineral oil, if doesn't work first day or two, should be discontinued as it will worsen the condition. You may try calling the vet for followup advice but be careful with the pharmaceuticals as your kitty seems already dehydrated. Ask Vet about an enema, or consider giving one yourself. Filtered water with about 2cc's over-the-counter sodium diphosphate (Fleet) should help. If not, try a few grains of milled flax seed in food should elicit a responce within an hr. Be careful to not use too much, as I have used only a pinch for as many as 4 kitties in one dose as sufficient. Hope this helps and good luck."
11/01/2011: Valerie from Fort Worth, Texas, United States replies: "We had a kittie with end stage renal disease and diabetes. It came on so suddenly we were shocked as he was well one day and very ill the next. The vet had sent us home to basically keep him comfortable. I researched and found that sweet potatoes are almost a perfect food and very well digested for renal problems. At first he was too weak to eat and we gave him pureed sweet potatoes through a small syringe without the needle. By the third feeding he was lifting his head and licking it off my fingers. The next day he was up and walking around. We gave him sweet potatoes every day, we also gave iv bolus' of fluids every day and checked his ear with a diabetic blood testing kit and gave him insulin as he needed it or glucose if too low.
Have you checked your kitties blood sugar? You can buy the kits at any pharmacy and test the ear. Youtube has wonderful videos on how to perform the procedure. I also gave him a small amount of goldenseal for inflammation and fever mixed with water through a needless syringe when he was not feeling well. When he was not well we put him in a child's playpen to keep track of him and so he would not exert energy. We put a sound machine with the heart beat under the playpen, a positive ion lamp near him and used a tuning fork over his body three times a day. He lived 8 more months after the vet said there was nothing to do. Moral of the story, go to the vet BUT also research, research, research, and look to add alternative therapies when able. Good luck with your precious baby.. my thoughts are with you."


text: Ok, go easy as this is my first post. Spotted this whilst looking through Asda's site. Sorry if it's been posted before. But £17.93 delivered for this game seemed pretty cheap to me. I paid nearly £40 at the end of August. I think it's a great game, however if racing games, especially motorbikes, aren't your thing then fair enough.

domain: entertainment


text:
I really just could not think of a more befitting name for this post. I found this flatware box at the thrift store for just under $5.
Here it is as I found it:
Ooh good find, right? There are so many possible uses for a little chest like this…
Ok a LOT of TLC. So gross. It smelled so musty and yet I brought it home anyway for $5 more dollars than I should have. I would never have had the nerve to donate this!
But luckily for me, someone else did. 🙂
And for the inside:
No more smelly, crusty mess. I used a scraper to scrape away as much of the crusty paper as possible. Then I shellacked the interior with Zinsser spray shellac twice to get rid of the musty odor. There’s not a trace of it left!
Then I lined it with pretty paper and fabric. The paper is Mod Podged in place and the bottom is lined with a piece of foam core cut-to-fit and wrapped in fabric.
The outside got a new paint job including a grey grain sack stripe…
Then I used this stencil to add this bold PARIS graphic to the top. I used spray adhesive on the back of the stencil to hold it in place. It makes it just tacky enough to stay put while painting.
The thing about stencils is that they usually look like stencils. The breaks in the letters are a dead giveaway…
So I paint them in with a small paint brush! Here I also smoothed any areas where the paint bled a little.
And voila! Much better. I also sanded the surface with a little 220 grit sand paper to make it smooth. I sealed it with Annie Sloan’s clear wax.
There really are so many uses for a chest like this.
It could hold jewelry of course, but how about a place to hide the remotes? A quick place to stash the daily mail that needs sorting? A pretty new sewing box or craft stash box?
So many possibilities!
I try not to pass these up when I find them.
You might also like this one I did a couple of years ago:
Or this one I shared as part of my March Madness, 31 Thrifty Makeovers in 31 Days series:
These all sold quickly, so I’m always on the lookout for more.
In my next post I’ll be sharing how I made over this pretty yard sale find that I scored for $40:
She turned out really pretty!
UPDATE: See how she looks HERE!
See ya then!
Don’t Miss a Thing!
If you like what you see, there are several ways to follow along!


text:
We asked our friend Brad at Pacific Pickle Works and his family to make a pickle recipe with our Persimmon Vinegar. They share our love for supporting local farmers and food that's good for you.
Try this delicious and easy recipe. Let us know what you think email@example.com
2-3 lbs fresh Sweet Nante carrots
12oz Persimmon Vinegar
12oz Filtered or distilled water
1oz Kosher or Sea salt (2 Tbl + 2 tsp if using Diamond Crystal Kosher Salt, 1-1/2 Tbl if using a denser sea salt)
1 Tbsp sugar (completely optional, but helps to balance acidity of the pickle brine)
Spices per jar (only a suggestion, but feel free to improvise)
1 sprig fresh tarragon
1 sprig fresh thyme
1/4 tsp whole black pepper
1/8 tsp mustard seed
1/8 tsp crushed red pepper
1/8 tsp celery seed
2 whole allspice
Makes 4-5 16oz jars
Directions (and notes from Brad):
Trim and wash (scrub really) your carrots, but do not peel them. Some people like to peel them, but I feel that they look better, taste better and have a more natural look if you leave them with the skin in tact. You can chose what shape to cut them in, but I like quartered sticks for snacking. Cut to size so that they fit nicely in the jar you have chosen.
Distribute your chosen spices in the jars. You can increase or the quantities depending on how flavorful you want them. Be careful with the allspice and celery seed if you use them as a little goes a long way. For allspice, more than just 2 or 3 peppercorns will be very distinct in your final product. Add the fresh tarragon and thyme. If you want these fresh ingredients to be visually distinct in your jars, tilt the jar in your hand while you lay the fresh herbs along the side of the jar and then layer some carrots on top of them to hold the herbs against the side of the jar. This will make for a nice presentation if you are giving these as gifts. Continue to fill the remainder of the jar with your carrots until tightly packed. If you have some small carrot pieces left over, use these to top off the remainder of the jar without going above the fill line. Most canning style jars have an apparent ring near the top of the jar about 1/2” or so from them top for this purpose.
To prepare the brine, combine all of the brine ingredients in a pot and heat until the salt and sugar are dissolved. The sugar in this recipe is optional as there is already a touch of sweetness that comes off of the vinegar and from the carrots if you have some good ones. I like the little bit of sugar to help balance out the acidity of the vinegar, but it’s completely optional.
There are two methods of quick pickle preparation that you can choose depending on how you like your pickles. You can either heat the brine to boiling and then pour over your pickles and place in the fridge after they cool down a bit (an hour or so). Or, you can pour your brine (heat enough to dissolve the solids, but not necessary boiling) over your pickles leaving 1/4” headspace, then place the lids on securely and put in a canner (a pot of boiling water) with the jars completely immersed for 8 minutes.
The refrigerator method will leave you with a very fresh and crisp pickle, but it will take a few days for the flavors to develop and the jars must remain in the fridge until you eat them.
For refrigerator pickles, I would suggest giving them 3-4 days at a minimum in order for the flavors to penetrate the carrots.
For this recipe I actually would suggest using the hot water bath method for a couple of reasons. Doing the hot water bath will not only make them shelf stable so that you can put them in the pantry and give them as gifts, but the extra bit of heat will allow the flavors to more fully penetrate the carrots. Since carrots are so crunchy by nature, that little bit of heat is good to soften them up a touch, but still leave them quite crunchy. If using the hot water bath process, let cool overnight and enjoy as early as the next day.

domain: food


In an effort to prevent more insurers from abandoning the Obamacare exchange in Tennessee, the state’s insurance regulator is allowing health insurers refile 2017 rate requests by Aug. 12 after Cigna and Humana said their previously requested premium hikes were too low.
Cigna and Humana filed to increase last year’s premiums an average of 23 and 29 percent, respectively, on June 10. But in the interim, both insurance companies have told state regulators that the requests would not cover the expected claims, said Kevin Walters, spokesman for the Tennessee Department of Commerce and Insurance.
. . .


text:
Want to join the conversation?
Premier energy company $SO's Board of Directors yesterday announced a regular quarterly cash dividend of $0.5425 per share or $2.17 annualized dividend on its common stock, payable on Dec. 4, 2015, to shareholders of record as of Nov. 16, 2015.
I have a feeling "CLOUDy" days are ahead, with $AMZN leading the pack.
$AAL is giving pay hikes to its employees but investors are sulking.
$APC stock tumbled more than 5.6% after last week's fatal home explosion in Firestone, Colorado prompted the company to close more than 3,000 wells for weeks.
Unicorn Frappuccino. The latest hit from $SBUX. Can't wait to see how this will affect its results.
At what point should $AMZN consider a stock split? $1000 per share seems pretty high.

domain: finance