Cecal Samples Research Articles

Methylmercury Chronic Exposure and a High-Fat Diet Induce Gut Microbiome Alterations and Intestinal Barrier Disruption in Mice.

Methylmercury (MeHg) is markedly toxic to humans. Our study explores whether MeHg and high-fat diet (HFD) can impair the intestinal barrier with microbiota dysbiosis in mice. Weanling mice were fed to HFD or standard diet for 40 days. In the last 20 days of diets, mice received either MeHg (20 mg/L) or drinking water. Proximal small intestine, cecum, and hair samples were collected. Villus length, crypt depth, villus/crypt length, mucin2 and lysozyme-positive cell counts, ZO-1 and occludin gene expression, and intestinal functional permeability were analyzed to assess the intestinal barrier. Blood samples were drawn to assess lipid parameters. Gut microbiome profiling was conducted with DNA from fecal/cecal samples. In addition, we analyzed ZO-1 immunofluorescence in the colon and small intestine. HFD increased MDA, Mucin2, and reduced villus height, crypt depth, villus/crypt length, lysozyme(+)-cell count, and increased intestinal permeability, regardless of MeHg intoxication. MeHg-HFD combination affected the intestinal barrier, decreasing ZO-1, occludin, and Nrf2 transcription, and increased permeability. HFD increased total plasma cholesterol and triglycerides. Only MeHg-HFD reduced microbiome alpha-diversity along with colonic ZO-1 immunolabeling loss compared to non-intoxicated mice fed a control diet. Regardless of diet, the genera Streptococcus, Psychrobacter, Facklamia, and Corynebacterium were severely depleted following MeHg intoxication. Other groups, such as Atopostipes and Jeotgalicoccus, were not altered by MeHg or HFD alone, but were significantly reduced by the combined HFD-MeHg. Synergistic effects of MeHg-HFD on the mucosa-associated microbiota are more pronounced than their individual effects. Our findings suggest that MeHg intoxication does not cause extensive dysbiosis but led to intestinal barrier disruption.

Inter- and Transgenerational Effects of In Ovo Stimulation with Bioactive Compounds on Cecal Tonsils and cecal Mucosa Transcriptomes in a Chicken Model

Backgroub/Objectives: Exploring how early-life nutritional interventions may impact future generations, this study examines the inter- and transgenerational effects of in ovo injection of bioactive compounds on gene expression in the cecal tonsils and cecal mucosa using a chicken model. Methods: Synbiotic PoultryStar® (Biomin) and choline were injected in ovo on the 12th day of egg incubation. Three experimental groups were established in the generation F1: (1) a control group (C) receiving 0.9% physiological saline (NaCl), (2) a synbiotic group (SYN) receiving 2 mg/embryo, and (3) a combined synbiotic and choline group (SYNCH) receiving 2 mg synbiotic and 0.25 mg choline per embryo. For the generations F2 and F3, the SYN and SYNCH groups were each divided into two subgroups: (A) those injected solely in F1 (SYNs and SYNCHs) and (B) those injected in each generation (SYNr and SYNCHr). At 21 weeks posthatching, cecal tonsil and cecal mucosa samples were collected from F1, F2, and F3 birds for transcriptomic analysis. Results: Gene expression profiling revealed distinct intergenerational and transgenerational patterns in both tissues. In cecal tonsils, a significant transgenerational impact on gene expression was noted in the generation F3, following a drop in F2. In contrast, cecal mucosa showed more gene expression changes in F2, indicating intergenerational effects. While some effects carried into F3, they were less pronounced, except in the SYNs group, which experienced an increase compared to F2. Conclusions: The study highlights that transgenerational effects of epigenetic modifications are dynamic and unpredictable, with effects potentially re-emerging in later generations under certain conditions or fading or intensifying over time. This study provides valuable insights into how epigenetic nutritional stimulation during embryonic development may regulate processes in the cecal tonsils and cecal mucosa across multiple generations. Our findings provide evidence supporting the phenomenon of epigenetic dynamics in a chicken model.

The expression pattern of butyric acid transporter in the large intestine with growth and development of suckling lambs.

The objective of this research was to explore the changes of morphological parameters, short chain fatty acid contents and butyric acid transporter expression levels in the large intestine with growth and development of sucking lambs. A total of 48 newborn male Hu sheep lambs (body weight = 2.94 ± 0.22 kg) were selected in this experiment. At 0, 7, 14, 28 and 42 days of ages, 6 lambs were slaughtered to collect cecal and colonic samples to analyze morphological parameters, short chain fatty acid contents and butyric acid transporter mRNA expression. The organ index of the cecum in d 0, 28 and 42 groups was higher (p<0.05) than that in d 7 and 14 groups. Compared with other age groups, the organ index of the colon was significantly increased (p<0.05) at d 42 group. After 7 days of age, the contents of acetic, propionic and butyric acids in the cecum and colon were significantly increased (p<0.05) as age increasing. A similar trend of mRNA expressions of butyric acid transporters, including monocarboxylate transporter, sodium-coupled monocarboxylate transporter, sodium-hydrogen exchanger, down regulated in adenoma, putative anion transporter-1 and anion exchanger-2, was found. In the cecum, the Escherichia coli count in d 14 group was higher (p<0.05) than that in other age groups, whereas an opposite tendency was found in Lactobacillus count between d 14 and other groups. Additionally, in the cecum and colon, the relative expression of claudin-1 in d 14 group was lower (p<0.05) that in d 42 group. Overall, the current results indicate that the expression levels of butyric acid transporters in the cecum and colon of lambs are significantly influenced by days of age.

Reviving diversity: cryoprotectants and culturing methods enhance recovery of mammalian gut microbes from field samples

Abstract The field of microbial ecology is increasingly recognizing the need for methods to isolate and culture gut microbes to better understand how these microorganisms impact animal physiology, especially in mammalian hosts. Currently, there is a lack of clear methods to store microbial samples for cultivability, especially when samples are collected from the field, transported to the laboratory, and preserved under long-term storage for weeks to months compared to mere days in the biomedical field. Here, the cecal contents of groundhogs (Marmota monax) were processed and stored with or without various preservation solutions at −80 °C for at least 2 months. All microbial samples were then grown in distinct nutrient media in liquid and plate conditions and were incubated under anaerobic and aerobic environments. Treatment comparisons revealed that the samples stored in preservation solutions containing 1 or more cryoprotectants provided the greatest and most consistent bacterial densities. To test the long-term storage efficacy of the preservation solutions, we inventoried taxonomic identities and abundances of these cultures using 16S rRNA amplicon sequencing. Our findings highlight that: (1) preserved samples containing cryoprotectants exhibited the highest microbial richness and diversity and resembled the original cecal samples the most when grown under anaerobic conditions; and (2) the effect of individual animal identity was detectable in the membership of cultured communities, irrespective of preservation solutions. Our study is the first to demonstrate the importance of preservation solutions containing multiple cryoprotectants for long-term storage and further microbial culturing and novel isolation. Understanding and improving storage methods that preserve microbial physiology and conserve their compositional diversity is essential for field-collected samples useful in mammalian microbiome and culturomics studies, promoting a better comprehension of the identity and function of wild host-associated microbiomes.

Optimizing mouse metatranscriptome profiling by selective removal of redundant nucleic acid sequences.

Sequencing total RNA from microbiome samples is seriously impaired by the overwhelming proportion of rRNA to mRNA content. As much as 99% of sequencing reads can be assigned to the rRNA content, thus removal of these abundant transcripts is critical to MetaT analysis. The use of Ribo Zero Plus rRNA depletion probes designed for human gut microbiomes proved to be less effective and more inconsistent across mouse cecal donor samples, a common experimental system for microbiome studies. In the present work, we have extended and refined a taxonomically-neutral probe design method for mouse cecal content. The additional probes were carefully chosen to limit the number needed for effective depletion to reduce both the cost and risk of introducing bias to MetaT analysis. Our results demonstrate this method as efficient and consistent for rRNA removal in mouse cecal samples, thus providing a significant increase in the number of mRNA-rich sequencing reads for MetaT analysis.

Hypoglycemic Effect of Ginsenoside Compound K Mediated by N-Acetylserotonin Derived From Gut Microbiota.

Ginsenoside compound K (GCK) has been proved to have great hypoglycemic effect pertinent to gut microbiota. However, the improvement of high-fat-diet (HFD)-induced type 2 diabetes (T2D) as well as the mechanism of GCK mediated by gut microbiota is not well-known. This study aimed to investigate the hypoglycemic effects and mechanism of GCK on a HFD-induced diabetic mouse model. HFD-induced pseudo-germ free (GF) T2D mice model and fecal microbiota transplantation (FMT) experiments were performed to clarify the role of gut microbiota in the hypoglycemic effect of GCK. Differential metabolites were screened by untargeted metabolomics analysis and their functions were verified by suppling to T2D mice. The level of glucagon-like peptide-1 (GLP-1) in plasma was detected by ELISA analysis to explore the potential hypoglycemic mechanism of GCK. The results showed GCK alleviated metabolic disorders and altered gut microbiota in HFD-induced diabetic mice, which was transmitted to pseudo-GF diabetic mice via FMT experiment to reproduce the hypoglycemic effect. Non-targeted metabolites analysis on cecal content samples indicated that N-acetylserotonin (NAS) was markedly increased after GCK treatment. Moreover, gavage with NAS improved insulin sensitivity and increased the secretion of GLP-1 in HFD mice. Our study showed that GCK had hypoglycemic effect through modifying gut microbiota profiling.

Preventive effects of kefir on colon tumor development in Wistar rats: gut microbiota critical role.

To clarify the effects of kefir in critical periods of development in adult diseases, we study the effects of kefir intake during early life on gut microbiota and prevention of colorectal carcinogenesis in adulthood. Lactating Wistar rats were divided into three groups: control (C), kefir lactation (KL), and kefir puberty (KP) groups. The C and KP groups received 1 mL of water/day; KL dams received kefir milk daily (108 CFU/mL) during lactation. After weaning (postnatal day 21), KP pups received kefir treatment until 60 days. At 67 days old, colorectal carcinogenesis was induced through intraperitoneal injection of 1, 2-dimethylhydrazine. The gut microbiota composition were analyzed by 16S rRNA gene sequencing and DESeq2 (differential abundance method), revealing significant differences in bacterial abundances between the kefir consumption periods. Maternal kefir intake strong anticancer power, suppressed tumors in adult offspring and reduced the relative risk of offspring tumor development. The gut microbiota in cecal samples of the KL group was enriched with Lactobacillus, Romboutsia, and Blautia. In contrast, control animals were enriched with Acinetobacter. The administration of kefir during critical periods of development, with emphasis on lactation, affected the gut microbial community structure to promote host benefits. Pearson analysis indicated positive correlation between tumor number with IL-1 levels. Therefore, the probiotic fermented food intake in early life may be effective as chemopreventive potential against colon tumor development, especially in lactation period.

Effect of carrot intake on glucose tolerance, microbiota, and gene expression in a type 2 diabetes mouse model.

Type 2 diabetes (T2D) pathophysiology involves insulin resistance (IR) and inadequate insulin secretion. Current T2D management includes dietary adjustments and/or oral medications such as thiazolidinediones (TZDs). Carrots have shown to contain bioactive acetylenic oxylipins that are partial agonists of the peroxisome proliferator-activated receptor γ (Pparg) that mimic the antidiabetic effect of TZDs without any adverse effects. TZDs exert hypoglycemic effects through activation of Pparg and through the regulation of the gut microbiota (GM) producing short-chain fatty acids (SCFAs), which impact glucose and energy homeostasis, promote intestinal gluconeogenesis, and influence insulin signaling pathways. This study investigated the metabolic effects of carrot intake in a T2D mouse model, elucidating underlying mechanisms. Mice were fed a low-fat diet (LFD), high-fat diet (HFD), or adjusted HFD supplemented with 10% carrot powder for 16 weeks. Oral glucose tolerance tests were conducted at weeks 0 and 16. Fecal, cecum, and colon samples, as well as tissue samples, were collected at week 16 during the autopsy. Results showed improved oral glucose tolerance in the HFD carrot group compared to HFD alone after 16 weeks. GM analysis demonstrated increased diversity and compositional changes in the cecum of mice fed HFD with carrot relative to HFD. These findings suggest the potential effect of carrots in T2D management, possibly through modulation of GM. Gene expression analysis revealed no significant alterations in adipose or muscle tissue between diet groups. Further research into carrot-derived bioactive compounds and their mechanisms of action is warranted for developing effective dietary strategies against T2D.

Gut microbiota in two chickens' breeds: Characteristics and Dynamic Changes

The gut microbiota has been demonstrated to play an important role in host immunity, metabolism, digestion, and growth. However, studies regarding the gut microbiota in Tibetan chickens remains scarce in comparison with other poultry breeds. Here, we investigated the gut microbial characteristics of Tibetan chickens and Arbor Acres broiler chickens (AA broiler chickens) and compare their gut microbial differences. For this purpose, we collected cecal samples from 10 Tibetan chickens and 10 AA broiler chickens for amplicon sequencing. Results indicated that Tibetan chickens exhibited higher gut microbial diversity and abundance compared with AA broiler chickens. Moreover, PCoA-based scatter plot analysis showed that the gut microbial structure of the both breeds was significantly different. Although the dominant bacterial phyla (Firmicutes, Firmicutes and Bacteroidota) of Tibetan chickens and AA broiler chickens were the same, the abundance of some bacterial phyla and genera changed significantly. Microbial taxonomic analysis indicated that the relative abundance of 876 genera of 20 phylum in Tibetan chickens increased significantly, while the relative abundance of 160 genera of 3 phyla decreased significantly compared with AA broiler chickens. In summary, these results indicated that there are significant differences in the gut microbiota between Tibetan chickens and AA broiler chickens. This is an important exploration of the gut microbial characteristics and distribution of Tibetan chickens. The findings may contribute to promoting the development of the Tibetan chicken's industry and reveal the adaptability of Tibetan chickens to the environment.

Partial replacement of soybean with local alternative sources: effects on behavior, cecal microbiota, and intestinal histomorphometry of local chickens.

Interest in partially replacing soybean meal in poultry diets with alternative protein sources such as agri-industrial by-products and black soldier fly (BSF, Hermetia illucens) has gained significant attention due to sustainability concerns. This study aimed to evaluate the effects of broiler diets in which soybean meal was partially substituted with agri-industrial by-products with or without BSF larvae meal, on the behavior, intestinal histomorphometry, and microbiome profile of a local broiler chicken strain. There were three dietary treatments. (1) A corn-soybean-based diet (Control), (2) a diet in which soybean was partly replaced (SPR) with local agri-industrial by-products, namely sunflower meal, brewers' dried grain, and wheat middlings, and (3) a diet in which BSF (5%) meal was added to SPR (SPR+BSF). Behavior was recorded on days 14, 35, and 49 at the pen level. On day 55, intestinal segments and cecal contents were collected from eight chickens per pen for histomorphometry and microbiome analysis. Dietary manipulations did not affect the behavior of broiler chickens (P > 0.05) suggesting that the experimental diets had no influence on behavior. A significant interaction between the intestinal segment and diets revealed that the SPR and SPR+BSF diets decreased duodenal villus height (VH) compared to the control diet (P < 0.05). However, this effect was not consistent across all of intestinal segments. Diet did not affect villus height to crypt depth ratio (VH/CD; P > 0.05), indicating no significant impact on the absorptive capacity of the digestive system. Firmicutes and Bacteroidetes were the dominant phyla in the cecal samples. Colidextribacter and Oscillibacter spp. were more abundant in chickens fed the SPR diet compared to those fed the control diet. The SPR+BSF diet resulted in higher abundance of Rikenella and Colidextribacter spp. compared to the control diet, while Desulfovibrio, Ruminococcus torques group, and Lachnoclostridium were more abundant in the ceca of birds fed the SPR diet than those fed SPR+BSF. In conclusion, replacement of soybean with agri-industrial by-products and BSF larvae meal could regulate the cecal microbiota composition without negatively affecting the behavior and intestinal histomorphometry of the local chickens.

Effect of Dietary Xylanase Inclusion on Growth Performance, Nutrient Digestibility, and Digesta Viscosity of Weaned Pigs Fed Wheat-Soybean Meal-Based Diets.

(1) Background: This study aimed to evaluate the effects of dietary xylanase addition on growth performance, nutrient digestibility, volatile fatty acids, and digesta viscosity at different digestive sites in weaned pigs fed wheat-soybean meal-based diets with reduced metabolizable energy. (2) Methods: A total of 312 weaned pigs (5.1 ± 0.9 kg, 20 ± 2 days of age) were assigned to one of six dietary treatments. The experimental diets were formulated in a three-phase nursery feeding program: phase 1 (d0-d7), phase 2 (d8-d21), and phase 3 (d22-d42). The experimental diets consisted of a wheat-soybean meal-based diet formulated to meet pig requirements (positive control, PC); the PC diet with a reduction of 100 kcal of metabolizable energy (ME) (negative control, NC); and the NC diet with either 900, 1800, 3600, or 7200 units of xylanase. Feed disappearance and body weight were measured at d7, 14, 21, and 42 in the nursery phase. The pen fecal score was assessed daily from d0 to d14 and three times a week from d15 to d28. On d21-d24 of the experiment (12 pigs per day), one pig per pen was selected for sample collection: ileal, cecal, and mid-colon digesta for viscosity and ileal digesta, feces for nutrient digestibility, and feces and cecal digesta for the measurement of volatile fatty acid. (3) Results: The addition of xylanase to the NC diets did not improve pig growth performance (body weight, feed conversion ratio, and average daily gain; p > 0.10) during the entire nursery phase. In Week 2 and Week 3, pigs fed xylanase had a lower (χ2 < 0.05) incidence of fecal scores 3 and 4 (diarrhea) than the PC and NC diets. In addition, the apparent total tract digestibility of neutral detergent fiber and acid detergent fiber increased linearly (p < 0.1) in response to xylanase addition. Xylanase addition (900 to 7200 U) decreased digesta viscosity in the colon compared to the PC and NC diets. Furthermore, xylanase addition resulted in a lower (p < 0.05) concentration of acetic, propionic, butyric, valeric, and total volatile fatty acid in cecal samples compared to PC. The addition of xylanase resulted in greater acetic and valeric acid concentrations in cecal samples compared to the NC group (p < 0.10). (4) Conclusions: Xylanase addition can improve nutrient digestibility, particularly at the total tract level, and reduce viscosity in the hindgut, which could be related to decreasing the occurrence of looseness. However, its impact on growth performance was minimal in wheat-soybean meal-based diets with a reduction of 100 kcal of ME.

Abundance of Tumor-Infiltrating B Cells in Human Epithelial Malignancies.

Cancer is a major global health problem. The type of malignant neoplasm and the potency of the immune response against tumors are two of the key factors influencing the outcome of the disease. The degree of tumor infiltration by lymphocytes plays an important role in antitumor response development, generally correlating with a favorable prognosis of treatment for certain cancers. We analyzed the abundance of tumor-infiltrating B cells (TIBs) in solid tumors of different cancers. TIBs were shown to be more abundant in colon and sigmoid colon cancer samples compared with cecal, rectal, and kidney cancer samples. The median and interquartile range of the TIB fraction were 11.5% and 4-20% in colon cancer, 6% and 3-11% in sigmoid colon cancer, 2.7% and 0.7-3.7% in cecal cancer, 2.5% and 0.9-3.6% in rectal cancer, 1.4% and 1.0-2.3% in kidney cancer, and 3.0% and 1.8-12% in lung cancer, respectively. However, there were no significant differences in the abundance of TIBs among samples at different stages of the cancer. Hence, investigation of the B cell response in colon cancer is of particular interest, since increased quantities of TIBs may indicate the existence of immunogenic tumor markers or the cell-cell interactions involved in disease progression. We believe that studying the diversity of TIBs in colon cancer will increaseour understanding of the mechanisms of the disease, contributing to the identification of new molecular targets for targeted oncotherapy.

Antibiotic Resistance Patterns of Escherichia coli Isolates in Broiler Chickens at Slaughterhouse and Retails

This study aims to identify the antibiotic resistance pattern of Escherichia coli in the broiler food chain at poultry slaughterhouses and outlets. The sampling method used purposive sampling. Each of the 30 cecum and carcass samples was obtained from a Poultry Slaughterhouse, and 40 samples from 1 retail (consisting of 5 outlets), which received supplies from the same Poultry Slaughterhouse, were isolated and identified as E. coli. The results showed that 57 isolates were positively identified as E. coli, and the sensitivity was tested for the Antibiotic Susceptibility Test using the disc diffusion method. Statistical analysis using the Chi-Square method and Fisher's Exact Test (p-value=0.01) showed a pattern of sensitivity of Ciprofloxacin (p=0.89), Colistin (p=0.07), Sulfamethoxazole (p=1.00), Chloramphenicol (p=0.67) and Meropenem (p= &lt;0.001). The highest resistance was found in frozen carcass isolates at the outlet to Colistin (69%), and E. coli isolates from cecum at the poultry slaughterhouse showed resistance to Meropenem (67%). Furthermore, it was discovered that 35% of isolates were only resistant to one type of antibiotic. Resistance bacteria may spread and threaten consumers' health while they get carried from farm to retail.

Prevalence of Campylobacter and antibiotic susceptibility in chickens at slaughterhouses and retail markets in the Mekong Delta

This study aims to determine the prevalence of Campylobacter and its antibiotic susceptibility at slaughterhouses and live poultry markets in the Mekong Delta, Vietnam. A total of 177 samples, including 112 fecal samples, 28 cecal samples, and 37 rectal swab samples were collected. Thirty-nine samples tested positive for Campylobacter spp. by culture method, accounting for 22%. Isolation rates of Campylobacter in rectal swab samples were highest at 29.7% (11/37), followed by cecal samples at 25% (7/28) and fecal dropping samples at 18.8% (21/112). Species identification was performed by multiplex PCR and the result showed that the prevalence of Campylobacter coli (12.4%) was higher than Campylobacter jejuni (3.4%). Antibiotic susceptibility test showed that all isolated Campylobacter were susceptible to imipenem and meropenem (100%). Most Campylobacter were resistant to tetracycline (96.4%), ciprofloxacin (92.9%), nalidixic acid (89.3%), and ampicillin (67.9%). This study highlights the high contamination of Campylobacter and its antibiotic resistance in poultry in the Mekong Delta, Vietnam.

Comparing the Impact of Continuous Endurance and High-intensity Interval Training on Cecal Butyrate and Propionate Levels in Diabetic Rats Induced by High-fat Diet

Background: Dysbiosis and metabolic disorders of the microbiota, often caused by an imbalance in the intestinal microbial composition, are significant issues linked to immobility, obesity, and diabetes. Physical exercise is recognized for its role in managing these symptoms by regulating the composition and metabolites of the intestinal microbiota, thereby improving gut health and overall metabolic function. Objectives: This study aimed to investigate and compare the effects of continuous endurance training (CET) and high-intensity interval training (HIIT) on two key cecal microbiota metabolites, butyrate and propionate, in diabetic rats. Methods: Forty-five male Wistar rats were made diabetic by a high-fat diet and were trained under CET and HIIT exercise protocols. Cecal tissue samples were taken from the rats to evaluate the effect of exercise, and the levels of two microbial metabolites, butyrate and propionate, were measured using the high-performance liquid chromatography (HPLC) method. Results: Among the exercise patterns studied, HIIT significantly improved the concentrations of butyrate and propionate, while CET showed no effect on these metabolites. Conclusions: Our findings suggest that, unlike CET, HIIT may effectively mitigate metabolic disturbances resulting from gut dysbiosis in diabetic patients. However, any definitive conclusion about the effects of CET and HIIT exercises on intestinal microbial metabolites necessitates further comprehensive tests on other metabolites and an examination of additional supporting evidence, such as changes in the composition of the intestinal microbiome.

PSVI-5 Bacteriophage biocontrol of avian pathogenic Escherichia coli in laying hens does not produce collateral effects on the cecal microbiota

Abstract Bacteriophages have shown promise as an alternative to antibiotics for managing or preventing avian pathogenic Escherichia coli (APEC) in the egg industry. In a previous study, a phage cocktail comprising 3 virulent phages was shown to prevent APEC infections in laying hens. The aim of this study was to investigate whether orally and intramuscularly administered phages affect the intestinal barrier function of chickens, with a focus on cecal microbiome. In a 4-d trial, a total of 35 laying hens were randomly assigned to one of four treatment groups: 1) Medium control group (bacterial broth + phage buffer), 2) APEC only control (APEC + buffer); 3) IM group (Phage +APEC), or 4) DW group (phage in drinking water (4 d prior to APEC + APEC). Cecum samples were subjected to 16S rRNA gene sequencing targeting the V4 region to assess the effects of phages on cecal bacterial diversity. A 1-way ANOVA was used to evaluate alpha diversity with respect to treatment and time. Microbial community structure was analyzed using β-dispersion and permutational multivariate analysis of variance (PERMANOVA) to determine the effect of each treatment. Significant (P &amp;lt; 0.05) differentially abundant genera were identified with DESeq2 by fitting a negative binomial model to each treatment vs treatment comparison (“~Treatment”). Across the treatment and control groups, 13 phyla were identified. Firmicutes (53.81%), Bacteroidota (23.83%), Actinobacteriota (1.84%), and Proteobacteria (1.02%) were the most abundant phyla among the amplicon sequence variants (ASVs), with 18.39% being unclassified. Neither richness (P = 0.448) nor Shannon diversity (P = 0.687) exhibited significance in α- diversity metric between treatment and control groups. β-dispersion analysis showed no significant difference (P = 0.239) between treatment and control groups, indicating similar bacterial variability among groups. Moreover, the β-diversity representing microbiota structure was not affected by any phage treatment (P = 0.083). In each treatment group, 76 unique genera were identified, with Megamonas (15.80 %), Lactobacillus (8.76%), Faecalibacterium (7.70 %), Megasphaera (1.58%), Alloprevotella (1.54%), [Ruminococcus] torques group (1.29%), Prevotellaceae UCG-001 (1.14%), Bifidobacterium (1.04%), and Olsenella (0.48%) being the most abundant. Notably, Lactobacillus was more abundant (~ 10 to 20%) in medium control group compared with phage treated and APEC only treatment groups. Furthermore, gene expression profiles of various genera (pairwise) were analyzed using log2 fold change (log2FC), revealing differences (P &amp;lt; 0.05) between control and treatment groups for a total of 58 genera. It was concluded that bacteriophage selectively killed APEC population without comprising the population and structure of cecal microbiome in laying hens.

489 Antibiotic Resistance of cecal Escherichia coli isolates from Lohmann LSL lite pullets and laying hens fed omega-3 fatty acids or yeast bioactives, reared at different spacing allowance

Abstract Poultry diets provide components that alter gut microbiota composition. We investigated antimicrobial resistance (AMR) phenotypes of E. coli isolates from ceca digesta of pullets and hens reared at different spacing allowance (SA) and dietary source of omega-3 fatty acids (N-3 FA) and yeast bioactives (YB) from placement to 17 wk of age (woa). A total of 2,632-day old chicks were placed in cages at two SA (24 cages each): High SA (HSA, 348 cm2/bird; ~50 birds/cage) and Low SA (LSA, 284 cm2/bird; ~68 birds/cage). Birds were fed corn and soybean-based diets (C), C + 3% co-extruded full fat flaxseed and pulse mixture (N-3 FA) and C + 0.05% YB processed with β-1,3-glucan hydrolase. At 4, 16 and 35 woa, ceca samples were collected to isolate E. coli using Chromocult agar. Their susceptibility to 14 antibiotics were determined by automated Sensititre system according to CLSI and CIPARS guidelines to determine their antimicrobial susceptibility profile. Number of antibiotics resistant E. coli isolates were analyzed using general linear mixed model procedure of SAS with diets, SA, sampling age and associated interactions as fixed factors. Cochran-Mantel-Haenszel test was used to determine the relationship between these treatments, and differences between treatments were considered significant at P &amp;lt; 0.05. Out of 428 presumptive E. coli isolated, 153 of 428 isolates (35.7%) were resistant to one antimicrobial class but no resistance against ciprofloxacin, azithromycin and nalidixic acid were detected. The prevalence of antimicrobial resistant E. coli decreased as the birds aged from wk 4–35 (P &amp;lt; 0.05). At wk 4 (P = 0.002) and 16 (P = 0.011), the least prevalence of ampicillin (AMP) resistance was in ceca digesta from N-3 FA-fed birds. At 35 woa, no AMP and cefoxitin (FOX) resistance were detected in birds fed YB (Table 2). At 16 woa, the prevalence of streptomycin resistance was greater in N-3 FA-fed than in YB-fed birds (P = 0.02), which was less than control-fed birds. Of the 428 isolates, 59 (13.8%) were resistant to more than two antimicrobial classes, with most from TIO-FOX-AMP-TET and STR-SULF-TET. At wk-4, SA had a significant effect on multiple resistance spectrum with LSA having the lowest MDR E. coli (P = 0.02) mainly from N-3 FA-fed pullets. This study showed an overall low AMR prevalence which decreases with age of the bird. This study is the first to report that feed additives (N-3 FA or YB) influence the AMR profile of cecal E. coli in pullets and layers. Prevalence of AMP resistance was least in N-3 FA-fed birds, whereas least STEP- and SULF resistance was observed in YB-fed birds. Our next study would help understand the molecular and evolutionary aspect of this resistant E. coli and their potential to cause colibacillosis.

476 Induced hindgut acidosis in sheep affected gut permeability and ruminal fermentation

Abstract Leaky gut may be caused by acidotic conditions in the hindgut and lead to systemic inflammation and negative effects on the gastrointestinal tract. The objective was to determine the effects of induced hindgut acidosis in sheep on cecal pH, ruminal fermentation, and gut permeability. Ruminally and cecally cannulated ewes [n = 11; body weight (BW) = 49 ± 4 kg] were assigned to one of two treatments: control (CON; n = 5) or induced hindgut acidosis (HGA; n = 6). To induce hindgut acidosis, 3 g wheat starch/kg BW per 24 h was continuously infused via the cecal cannula for 4 d. Control ewes received a constant infusion of deionized water. The diet contained 40% grass hay pellets, 35% whole shelled corn, 15% dried distillers grains, and 10% alfalfa cubes. Chromium EDTA was dosed once daily via the cecal cannula as a marker of gut permeability. Rumen, cecal, and fecal samples were collected to determine pH and volatile fatty acids (VFA). Rumen fluid was collected on d 4 for an ex vivo fermentation. Flasks were incubated for 24 h to determine pH, VFA, ammonia and in vitro dry matter digestibility. Tissue samples from the ileum, cecum, and colon were mounted in Ussing chambers to measure transepithelial electrical resistance (TEER). There was a treatment × time effect (P = 0.05) for cecal pH with HGA ewes having lesser cecal pH after d 1. By d 4, cecal pH had declined to 5.07 for HGA ewes compared with CON ewes which remained above 6.40 throughout the experiment. A treatment × time interaction was also observed (P &amp;lt; 0.01) for fecal pH and followed the same trend as cecal pH. Rumen pH was not affected (P = 0.87) by the interaction of treatment and time but was affected (P &amp;lt; 0.01) by treatment as ewes on the HGA treatment had a lesser rumen pH than CON ewes. Control ewes had lesser ruminal VFA and ammonia concentrations than HGA ewes (P &amp;lt; 0.01). Urinary Cr recovery was not affected by the interaction of treatment and time, or treatment (P ≥ 0.13). In vitro dry matter disappearance was not affected by cecal infusion treatment (P = 0.60). There was no effect (P = 0.78) of treatment on ex vivo pH. There were no effects (P ≥ 0.11) of treatment, time, or their interaction on ex vivo ammonia or total VFA concentration. In cecal tissue, TEER tended (P = 0.09) to be greater in CON ewes than HGA ewes. In contrast, TEER was not different (P ≥ 0.83) in ileal or colonic tissues between treatments. Induced hindgut acidosis in sheep altered rumen fermentation and tended to increase ex vivo measures of cecal permeability, but did not affect ex vivo rumen fermentation.

Towards the identification of transmission pathways and early detection of Enterococcus cecorum infection in broiler chickens

Enterococcus cecorum (EC) infection is an emerging endemic disease in UK and global broiler poultry with major economic impact and welfare concerns. There are significant research gaps with regards to EC pathogenesis, source of infection, transmission routes and early detection of disease, which this study aimed to address. In this prospective study, 725 environmental samples were collected from 4 broiler farms (A-D) the day before chick placement (d 1) and through the subsequent crop (d 7, 14, and 21). Cecal swabs were collected from birds that died of natural causes during the study period. A sample of birds that had been found dead or were culled for health reasons, were presented for post-mortem and samples were taken from lesions for EC culture. DNA was extracted from all environmental samples and EC detected using a qPCR and MALDI-TOF. Two EC isolates from diseased birds were inoculated on concrete slabs and incubated at 23°C and 32°C followed by swabbing of concrete culturing and determination of EC cfu at defined time points. Alongside environmental and bird sampling commercially available, smart camera systems were installed in selected houses on each farm to monitor bird activity and distribution. No EC outbreak occurred during the study, however, it was detected by qPCR in 215/725 (29.7 %) of all samples collected. Also, EC DNA was detected on average in 37% of samples collected on d 1, with approx. 88% of samples from chick paper being positive. Despite this, it was only cultured from 3 ceca samples and joint fluids of two infected birds from farm B on d 14 and 21. The survival experiments using isolates from infected chickens showed EC can survive on concrete for at least 21 d. This study provides invaluable insights into transmission pathways and tenacity of EC. Further studies are needed to determine strain characteristics in relation to their ability to cause disease and to further elucidate the sources of infection on poultry farms.

Persistence of Salmonella and Campylobacter on Whole Chicken Carcasses under the Different Chlorine Concentrations Used in the Chill Tank of Processing Plants in Sri Lanka.

The persistence of non-typhoidal Salmonella and Campylobacter in chicken meat is a considerable public health risk and a future challenge. This study aimed to determine the prevalence of Salmonella and Campylobacter in poultry processing lines where different chlorine concentrations were used in the chill tank. The samples were collected from four types of processing plants in Sri Lanka, considering the chlorine concentration used in the chill tank, which ranged from 2 ppm to 50 ppm. Salmonella and Campylobacter were isolated from whole carcass washings, neck skin, and cecal samples. Subsequently, an antimicrobial susceptibility test was performed for the isolates. The results revealed the overall prevalence of Salmonella and Campylobacter was 78.25% and 63.5%, respectively. Positive percentages of Salmonella and Campylobacter were high in the carcasses compared to the neck skin and ceca. The Campylobacter counts on the whole carcasses were significantly low (p < 0.001), at higher chlorine concentrations ranging from 20 to 30 ppm and 40 to 50 ppm. The pathogen prevalence in the whole carcasses was 84.7% Campylobacter coli, 39.1% Campylobacter jejuni, 71.1% Salmonella Typhimurium, and 28.8% Salmonella Infantis. The highest resistance was observed for tetracycline (63.8%) in Salmonella, while it was for gentamicin (87.8%) in Campylobacter. The prevalence percentage of multidrug-resistant Campylobacter was 51.2%, while it was 2.12% for Salmonella. The persistence of multidrug-resistant Salmonella and Campylobacter on the post-chill carcasses was highlighted in the present study as a significant public health threat that has to be addressed urgently.