Bioenergy recovery and carbon emissions benefits of short-term bio-thermophilic pretreatment on low organic sewage sludge anaerobic digestion: A pilot-scale study.

Sewage sludge in cities of Yangzi River Belt, China, generally exhibits a lower organic content and higher silt contentdue to leakage of drainage system, which caused low bioenergy recovery and carbon emission benefits in conventional anaerobic digestion (CAD). Therefore, this paper is on a pilot scale, a bio-thermophilic pretreatment anaerobic digestion (BTPAD) for low organic sludge (volatile solids (VS) of 4%) was operated with a long-term continuous flow of 200 days. The VS degradation rate and CH4 yield of BTPAD increased by 19.93% and 53.33%, respectively, compared to those of CAD. The analysis of organic compositions in sludge revealed that BTPAD mainly improved the hydrolysis of proteins in sludge. Further analysis of microbial community proportions by high-throughput sequencing revealed that the short-term bio-thermophilic pretreatment was enriched in Clostridiales, Coprothermobacter and Gelria, was capable of hydrolyzing acidified proteins, and provided more volatile fatty acid (VFA) for the subsequent reaction. Biome combined with fluorescence quantitative polymerase chain reaction (PCR) analysis showed that the number of bacteria with high methanogenic capacity in BTPAD was much higher than that in CAD during the medium temperature digestion stage, indicating that short-term bio-thermophilic pretreatment could provide better methanogenic conditions for BTPAD. Furthermore, the greenhouse gas emission footprint analysis showed that short-term bio-thermophilic pretreatment could reduce the carbon emission of sludge anaerobic digestion system by 19.18%.

Cronobacter sakazakii induced sepsis-associated arrhythmias through its outer membrane vesicles.

Sepsis-induced arrhythmia, linked to sudden cardiac death, is associated with gut microbiota, though the exact relationship is unclear. This study aimed to elucidate the relationship between Cronobacter sakazakii (C. sakazakii) and arrhythmia. The relative abundance of C. sakazakii was increased in cecal ligation and puncture (CLP)-induced septic mice. Live C. sakazakii, supernatant, and outer membrane vesicles (OMVs) resulted in premature ventricular beat (PVB), sinus arrhythmia (SA), and increased arrhythmia and mortality in sepsis model through dysregulated ion channel proteins. Moreover, short-chain fatty acids (SCFAs) showed antibacterial effects in vitro. We confirmed sodium acetate (C2) and sodium butyrate (C4) protect from C. sakazakii-induced arrhythmia, and C2 and C4 protected from septic arrhythmia by activating free fatty acid receptor 2 and 3 (FFAR2 and FFAR3) in mice. These findings point to how C. sakazakii's OMVs trigger arrhythmia, and SCFAs may be a treatment for septic arrhythmia.

Epidemiological, Phylogenetic, and Resistance Heterogeneity Among Acinetobacter baumannii in a Large U.S. Deep South Healthcare system.

Background: Acinetobacter baumannii (Ab) disease in the United States is commonly attributed to outbreaks of 1 or 2 monophyletic carbapenem resistance (CR) Ab lineages that vary by region. However, there is limited knowledge regarding CRAb epidemiology and population structures in the U.S. Deep South, and few studies compare contemporary CR and carbapenem-susceptible (Cs) Ab, despite relative prevalence of the latter. Methods: We performed a multiyear analysis of 2462 Ab cases in a large healthcare system in Birmingham, AL, and 89 post-2021 Ab isolates were sequenced and phenotyped by antibiotic susceptibility tests. Results: Although the cumulative CR rate was 17.7% in our cohort, rates regularly increased in winter months as result of seasonal changes in case incidence of CsAb, specifically. Genotyped CRAb belonged to clonal group (CG) 1, CG2, CG108, CG250, or CG499, with local clones of CG108, CG250, and CG499 persisting over multiple months. There was no clonal expansion of any CsAb lineage. Among CRAb isolates, levels of ß-lactam antibiotic resistance and the repertoire of related genetic resistance determinants, which included the novel CR-conferring FtsI A515V polymorphism, differed according to CG. CG108 and CG499 isolates displayed specific heteroresistance to sulbactam and trimethoprim/sulfamethoxazole, respectively, which resulted in discrepant susceptibility results in microbroth versus agar-based antibiotic susceptibility tests modalities. Conclusions: We report an unusually high degree of CRAb phylogenetic diversity principally driven by emergent U.S. lineages harboring novel resistance elements that must be incorporated into diagnostic, surveillance, and preclinical research efforts.

Identifying microbiome-based changes and biomarkers prior to disease development in mother and child, with a focus on gestational diabetes mellitus: protocol for the DANish Maternal and Offspring Microbiome (DANMOM) cohort study.

INTRODUCTION: The human gut microbiota is associated with gestational diabetes mellitus (GDM), which imposes a risk of developing long-term health problems for mother and child. Most studies on GDM and microbiota have been cross-sectional, which makes it difficult to make any conclusions on causality. Furthermore, it is important to assess if a dysbiotic microbiota is passed from the mother to the child, and then being at risk of developing metabolic health problems later in life. The DANish Maternal and Offspring Microbiome study aims to identify gut microbiota-related factors involved in metabolic dysfunction in women with GDM and their offspring. Importantly, the study design allows for early detection of biological changes associated with later development of metabolic disease. This could provide us with unique tools to support early diagnosis or implement preventative measures. METHODS AND ANALYSIS: Pregnant women are included in the study after the 11-14 weeks' prenatal ultrasound scan and followed throughout pregnancy with enrolment of the offspring at birth. 202 women and 112 children have been included from North Denmark Regional Hospital and Aalborg University Hospital in Denmark. Mother and child are followed until the children reach the age of 5 years. From the mother, we collect faeces, urine, blood, saliva, vaginal fluid and breast milk samples, in addition to faeces and a blood sample from the child. Microbiota composition in biological samples will be analysed using 16S rRNA gene sequencing and compared with demographic and clinical data from medical charts, registers and questionnaires. Sample and data collection will continue until July 2028. ETHICS AND DISSEMINATION: The study protocol has been approved by the North Denmark Region Committee on Health Research Ethics (N20190007). Written informed consent is obtained from all participants prior to study participation. Study results will be published in international peer-reviewed journals and presented at international conferences. The results will also be presented to the funders of the study and study participants.

Helicobacter pylori in oral cavity: current knowledge.

The oral cavity may play a role as a reservoir and in the transmission and colonization of Helicobacter pylori. The route of transmission for H. pylori is not fully understood. The prevalence of this pathogen varies globally, affecting half of the world's population, predominantly in developing countries. Here, we review the prevalence of H. pylori in the oral cavity, the characteristics that facilitate its colonization and dynamics in the oral microbiome, the heterogeneity and diversity of virulence of among strains, and noninvasive techniques for H. pylori detection in oral samples. The prevalence of H. pylori in the oral cavity varies greatly, being influenced by the characteristics of the population, regions where samples are collected in the oral cavity, and variations in detection methods. Although there is no direct association between the presence of H. pylori in oral samples and stomach infection, positive cases for gastric H. pylori frequently exhibit a higher prevalence of the bacterium in the oral cavity, suggesting that the stomach may not be the sole reservoir of H. pylori. In the oral cavity, H. pylori can cause microbiome imbalance and remodeling of the oral ecosystem. Detection of H. pylori in the oral cavity by a noninvasive method may provide a more accessible diagnostic tool as well as help prevent transmission and gastric re-colonization. Further research into this bacterium in the oral cavity will offer insights into the treatment of H. pylori infection, potentially developing new clinical approaches.

Metformin facilitates viral reservoir reactivation and their recognition by anti-HIV-1 envelope antibodies.

The mechanistic target of rapamycin (mTOR) positively regulates multiple steps of the HIV-1 replication cycle. We previously reported that a 12-week supplementation of antiretroviral therapy (ART) with metformin, an indirect mTOR inhibitor used in type-2 diabetes treatment, reduced mTOR activation and HIV transcription in colon-infiltrating CD4+ T cells, together with systemic inflammation in nondiabetic people with HIV-1 (PWH). Herein, we investigated the antiviral mechanisms of metformin. In a viral outgrowth assay performed with CD4+ T cells from ART-treated PWH, and upon infection in vitro with replication-competent and VSV-G-pseudotyped HIV-1, metformin decreased virion release, but increased the frequency of productively infected CD4lowHIV-p24+ T cells. These observations coincided with increased BST2/tetherin (HIV release inhibitor) and Bcl-2 (pro-survival factor) expression, and improved recognition of productively infected T cells by HIV-1 envelope antibodies. Thus, metformin exerts pleiotropic effects on post-integration steps of the HIV-1 replication cycle and may be used to accelerate viral reservoir decay in ART-treated PWH.

Autographiviridae phage HH109 uses capsular polysaccharide for infection of Vibrio alginolyticus.

Autographiviridae phage HH109 is a lytic Vibrio alginolyticus E110-specific phage, but the molecular mechanism underlying host recognition of this phage remains unknown. In this study, a transposon mutagenesis library of E110 was used to show that several capsular polysaccharide (CPS) synthesis-related genes were linked to the phage HH109 infection. Gene deletion combined with multiple functional assays demonstrated that CPS serves as the receptor for the phage HH109. Deletions of CPS genes caused reduction or loss of capsule and reduced adsorption. Comparative genome analysis revealed that phage-resistant mutants harbored loss-of-function mutations in the previously identified genes responsible for CPS biosynthesis. The tail protein gp02 of phage HH109 was identified as the receptor-binding protein (RBP) on CPS using antibody blocking assay, immunofluorescence staining, and CPS quantification. Additionally, we found that the phage HH109 could degrade approximately 88% of mature biofilms. Our research findings provide a theoretical basis against vibriosis.

Changes in performance, cecal microflora counts and intestinal histology of Japanese quails fed diets containing different fibre sources.

The purpose of this experiment was to investigate how various fiber sources impact the performance, microbial population, and intestinal histology of Japanese quail that was performed in a completely randomized design for 42 days. The dietary treatments involved a fiber-free corn-soybean meal-based diet (control, CTL), and CTL with added levels of sunflower hulls (SFH) and sugar beet pulp (SBP) (20 and 40 g kg-1). Body weight gain (BWG) and feed intake (FI) were recorded weekly. Carcass characteristics, cecal microbial population, blood variables and intestinal histology were measured on the 42 day of age. Adding 40 g kg-1 of SBP led to a significant decrease in body weight gain and an increase in the feed conversion ratio of birds from 1 to 21 days (P < 0.05). The relative weight of the gastrointestinal tract and gizzard increased significantly in birds that consumed SFH. Blood triglyceride concentration decreased with the inclusion of fiber in the diet. However, there was a notable increase in blood cholesterol concentration in the birds that were fed SBP (20 and 40 g kg-1) in comparison to those fed SFH (P < 0.05). The population of E. Coli in the cecum increased significantly in the birds that were fed 4 g kg-1 of SBP as opposed to those fed 20 and 40 g kg-1 of SFH (P < 0.05). The villus height of the jejunum in birds that were fed 20 g kg-1 and 40 g kg-1 of SFH demonstrated a significant increase in comparison to the other treatments (P < 0.05). In general, the findings of this research indicated that the inclusion of 40 g kg-1 of SBP in the diet had a negative impact on performance and other physiological parameters. However, the use of SFH and 20 g kg-1 of SBP yielded similar results to birds in the CTL, and in some cases, even better outcomes.

Meta-analysis of oral microbiome reveals sex-based diversity in biofilms during periodontitis.

Sex is an often overlooked, yet compulsory, biological variable when performing biomedical research. Periodontitis is a common yet progressively debilitating chronic inflammatory disorder affecting the tissues supporting teeth that ultimately leads to tooth loss if left untreated. The incidence of periodontitis is sex biased, with increased prevalence in males compared with females but with unknown etiology. We performed a sex-specific meta-analysis using publicly available oral microbiome data from different sampling sites of patients with periodontitis and periodontally healthy controls; sex balance was established for each periodontal health condition. Our results show sex-based diversity in oral biofilms of individuals with periodontitis but not in their saliva, with increased abundance of several periodontal pathogens in subgingival plaques from females compared with males. We devised a quantitative measure, uniquely defined as the Microsexome Index (MSI), which indicates that sexual dimorphism in subgingival bacterial composition is a distinct feature of reduced microbial diversity during periodontitis but not under healthy conditions. In addition, we found that smoking exacerbates microsexome diversity in supragingival biofilms, particularly during periodontitis. Taken together, we provide insights regarding sex-based diversity in periodontitis, a disease with multiorgan associations, and provide the rationale for further mechanistic, diagnostic, and therapeutic studies.

Protocol to establish an accelerated murine model for Helicobacter-induced gastric cancer.

Helicobacter-induced gastric cancer progresses very slowly, even in animal models, making it difficult to study. Here, we present a protocol to establish an accelerated murine model for Helicobacter-induced gastric cancer. We describe steps for infecting mice with Helicobacter felis, harvesting gastric tissue, assessing disease severity by histopathologic scoring, and performing gene expression studies with RT-qPCR and RNA sequencing. The accelerated model shows rapid progression of the disease, with gastric precancerous lesions developing within 6 months post-infection with Helicobacter. For complete details on the use and execution of this protocol, please refer to Bali et al.1.

Profiling the fecal microbiome and its modulators across the lifespan in the Netherlands.

Defining what constitutes a healthy microbiome throughout our lives remains an ongoing challenge. Understanding to what extent host and environmental factors can influence it has been the primary motivation for large population studies worldwide. Here, we describe the fecal microbiome of 3,746 individuals (0-87 years of age) in a nationwide study in the Netherlands, in association with extensive questionnaires. We validate previous findings, such as infant-adult trajectories, and explore the collective impact of our variables, which explain over 40% of the variation in microbiome composition. We identify associations with less explored factors, particularly those ethnic related, which show the largest impact on the adult microbiome composition, diversity, metabolic profiles, and CAZy (carbohydrate-active enzyme) repertoires. Understanding the sources of microbiome variability is crucial, given its potential as a modifiable target with therapeutic possibilities. With this work, we aim to serve as a foundational element for the design of health interventions and fundamental research.

The effect of organic fruit juices on physicochemical, microbiological and antioxidative aspects of organic goat's and cow's fermented whey beverages produced on laboratory and industrial scale.

Fermented milk beverages have been known for years and are characterized by excellent health-promoting properties. Therefore, consumer attention has been drawn to this product type in recent years. In the presented research, the technology of production in laboratory and industrial scale of controlled fermentation of whey beverages containing sweet and sour organic cow's or goat's whey with the addition of organic fruit juices (apple, blackcurrant juice or Kamchatka berry), has been described. Food production on a laboratory scale involves small batch processes designed for experimentation and refinement, often with precise control over variables and conditions. In contrast, industrial-scale food production in enterprises focuses on large volume output with an emphasis on efficiency, consistency, and adherence to regulatory standards for mass consumption. In this study was examined the amino acid content and nutritional value of the obtained products. Tests were carried out on fermented whey drinks' microbiology and antioxidant properties. The significance was determined using an ANOVA (ANOVA)-each prepared drink was characterized by better antioxidant properties and nutritional values compared with product without juice addition. Microbiological examination proved that only one product was not fit for consumption according to the Polish norm. Using whey (goat and cow) as a base for a fermented beverage with enhanced health benefits is a positive step toward using products commonly regarded as waste.

Surgical site infection in severe trauma patients in intensive care: epidemiology and risk factors.

BACKGROUND: Severe trauma is the leading cause of disability and mortality in the patients under 35 years of age. Surgical site infections (SSI) represent a significant complication in this patient population. However, they are often inadequately investigated, potentially impacting the quality of patient outcomes. The aim of this study was to investigate the epidemiology of SSI and risk factors in severe trauma patients. METHODS: We conducted a multicenter retrospective cohort study screening the severe trauma patients (STP) admitted to two intensive care units of an academic institution in Marseille between years2018 and 2019. Those who underwent orthopedic or spinal surgery within 5 days after admission were included and classified into two groups according to the occurrence of SSI (defined by the Centers for Disease Control (CDC) international diagnostic criteria) or not. Our secondary goal was to evaluate STP survival at 48 months, risk factors for SSI and microbiological features of SSI. RESULTS: Forty-seven (23%) out of 207 STP developed an SSI. Mortality at 48-months did not differ between SSI and non-SSI patients (12.7% vs. 10.0%; p = 0.59). The fractures of 22 (47%) severe trauma patients with SSI were classified as Cauchoix 3 grade and 18 (38%) SSI were associated with the need for external fixators. Thirty (64%) severe trauma patients with SSI had polymicrobial infection, including 34 (72%) due to Gram-positive cocci. Empirical antibiotic therapy was effective in 31 (66%) cases. Multivariate analysis revealed that risk factors such as low hemoglobin, arterial oxygenation levels, hyperlactatemia, high serum creatinine and glycemia, and Cauchoix 3 grade on the day of surgery were associated with SSI in severe trauma patients. The generated predictive model showed a good prognosis performance with an AUC of 0.80 [0.73-0.88] and a high NPV of 95.9 [88.6-98.5] %. CONCLUSIONS: Our study found a high rate of SSI in severe trauma patients, although SSI was not associated with 48-month mortality. Several modifiable risk factors for SSI may be effectively managed through enhanced perioperative monitoring and the implementation of a patient blood management strategy.

Efficient nitrogen removal via simultaneous ammonium assimilation and heterotrophic denitrification of Paracoccus denitrificans R-1.

Although diverse microorganisms can remove ammonium and nitrate simultaneously, their metabolic mechanisms are not well understood. Paracoccus denitrificans R-1 showed the maximal NH4 + removal rate 9.94 mg L-1·h-1 and 2.91 mg L-1·h-1 under aerobic and anaerobic conditions, respectively. Analysis of the nitrogen balance calculation and isotope tracing experiment indicated that NH4 + was consumed through assimilation. The maximal NO3 - removal rate of strain R-1 was 18.05 and 19.76 mg L-1·h-1 under aerobic and anaerobic conditions, respectively. The stoichiometric consumption ratio of acetate to nitrate was 0.902 and NO3 - was reduced to N2 for strain R-1 through 15NO3 - isotopic tracing experiment, which indicated a respiratory process coupled with the oxidation of electron donors. Genomic analysis showed that strain R-1 contained genes for ammonium assimilation and denitrification, which effectively promoted each other. These findings provide insights into microbial nitrogen transformation and facilitate the simultaneous removal of NH4 + and NO3 - in a single reactor.

TolRad, a model for predicting radiation tolerance using Pfam annotations, identifies novel radiosensitive bacterial species from reference genomes and MAGs.

The trait of ionizing radiation (IR) tolerance is variable between bacterium, with species succumbing to acute doses as low as 60 Gy and extremophiles able to survive doses exceeding 10,000 Gy. While survival screens have identified multiple highly radioresistant bacteria, such systemic searches have not been conducted for IR-sensitive bacteria. The taxonomy-level diversity of IR sensitivity is poorly understood, as are genetic elements that influence IR sensitivity. Using the protein domain (Pfam) frequencies from 61 bacterial species with experimentally determined D10 values (the dose at which only 10% of the population survives), we trained TolRad, a random forest binary classifier, to distinguish between radiosensitive (D10 < 200 Gy) and radiation-tolerant (D10 > 200 Gy) bacteria. On untrained species, TolRad had an accuracy of 0.900. We applied TolRad to 152 UniProt-hosted bacterial proteomes associated with the human microbiome, including 37 strains from the ATCC Human Microbiome Collection, and classified 34 species as radiosensitive. Whereas IR-sensitive species (D10 < 200 Gy) in the training data set had been confined to the phylum Proteobacterium, this initial TolRad screen identified radiosensitive bacteria in two additional phyla. We experimentally validated the predicted radiosensitivity of a Bacteroidota species from the human microbiome. To demonstrate that TolRad can be applied to metagenome-assembled genomes (MAGs), we tested the accuracy of TolRad on Egg-NOG assembled proteomes (0.965) and partial proteomes. Finally, three collections of MAGs were screened using TolRad, identifying further phyla with radiosensitive species and suggesting that environmental conditions influence the abundance of radiosensitive bacteria. IMPORTANCE: Bacterial species have vast genetic diversity, allowing for life in extreme environments and the conduction of complex chemistry. The ability to harness the full potential of bacterial diversity is hampered by the lack of high-throughput experimental or bioinformatic methods for characterizing bacterial traits. Here, we present a computational model that uses de novo-generated genome annotations to classify a bacterium as tolerant of ionizing radiation (IR) or as radiosensitive. This model allows for rapid screening of bacterial communities for low-tolerance species that are of interest for both mechanistic studies into bacterial sensitivity to IR and biomarkers of IR exposure.

Optimization of exopolysaccharide production from the novel Enterococcus species, using statistical design of experiment.

The Exopolysaccharide (EPS) producing novel strains of Enterococcus previously isolated from the vaginal source of pregnant women were selected based on ropy structure formation. The two selected strains, E.villorum SB-2 and E.rivorum S22-3, were found to be producing 2.87 g/l and 3.14 g/l EPS, respectively, in the minimal media (M17 media) after 24-hour fermentation under anaerobic condition. Both the strains have probiotic properties and have the potential to be used for industrial applications. The production media and fermentation conditions were optimized to enhance the EPS production using the one-factor method, Placket-Burman factorial designing and Central composite design (CCD) of Response surface methodology (RSM). The most relevant factors affecting the EPS yield were sucrose, yeast extract and pH for E.villorum SB2 and sucrose, yeast extract and magnesium sulfate for the E.rivorum S22-3 as determined by Placket-Burman design, whose concentrations were further optimized using CCD. The optimized fermentation conditions gave the total EPS of 9.76 g/l (4 times the initial production) from E.villorum SB-2 and 7.74 g/l (2.5 times the initial production) from E.rivorum S22-3, respectively, after 36-hour incubation at 37 °C. These optimization studies might be helpful during scale-up process for the industrial scale production of these exopolysaccharide.

Protocol for rapid evaluation of antibody-mediated protection in macrophages using immunofluorescence techniques.

Antibodies have been shown to provide protection against tuberculosis (TB). It is important to identify the qualitative and quantitative protective effects of antibodies before a clinical study. Here, we present a protocol to evaluate antibody-mediated protection in macrophages using confocal fluorescence microscopy and flow cytometry. We describe steps for bacteria and macrophage preparation, cell infection, phagocytosis, and phagosome maturation analysis. For complete details on the use and execution of this protocol, please refer to Zeng et al.1.

Protocol for lipid mediator profiling and phenotyping of human M1- and M2-monocyte-derived macrophages during host-pathogen interactions.

Here, we present a protocol for primary, human immune cell isolation and stimulation for lipid mediator profiling. We describe steps for the isolation of monocytes from human leukocyte concentrates via density centrifugation and differentiation/polarization toward M1- or M2-monocyte-derived macrophages (MDMs). We detail stimulation approaches of MDMs with live bacteria or influenza A virus for lipid mediator profiling and sample preparation for subsequent analysis, such as enzyme expression, mRNA analysis, or surface marker determination. For complete details on the use and execution of this protocol, please refer to Jordan et al.1.

Nano-protected form of rosemary essential oil has a positive influence on blood biochemistry parameters, haematological indices, immune-antioxidant status, intestinal microbiota and histomorphology of meat-type chickens.

This study aimed to investigate the potential of free and nanoprotected rosemary essential oil (REO) as an antibiotic alternative in blood biochemistry, hematology, immune-antioxidant status, intestinal microbiology, and histomorphology of broilers. A total of 420 1-wk-old broiler chicks (Cobb) were randomly allotted into 7 treatments, each having 4 replicates of 15 chicks. The first group served as control received basal diet, while the second group was fed a basal diet plus 100 mg/kg enramycin and third group basal diet plus 150 mg/kg chitosan nanoparticles (CNPs). The fourth and fifth groups received diets supplemented with 100 mg and 200 mg free REO (F-REO)/kg diet. The sixth and seventh groups were supplemented with 100 mg and 200 mg nanoprotected REO (N-REO)/kg diet. Results revealed that supplementing nanoprotected REO significantly (P < 0.05) decreased the levels of blood cholesterol and low-density lipoproteins (LDL) compared to control and enramycin groups. The kidney and liver function parameters were not altered by adding free or nanoprotected REO to the diet. Both levels of nanoprotected REO significantly (P < 0.05) reduced heterophil: lymphocyte (H: L) ratio compared to all other groups. Birds receiving nanoprotected REO at 200 mg/kg diet had significantly (P ˂ 0.05) raised serum levels of immunoglobulin G (IgG) and immunoglobulin M (IgM) compared to control and other birds. Anti-SRBC titre and cell-mediated immunity improved significantly (P < 0.05) in nanoprotected REO groups. Supplementation of nanoprotected REO resulted in significantly (P < 0.05) higher values for superoxide dismutase (SOD), glutathione (GSH) and total antioxidant status (TAS). The caecal microbiota was improved in broiler birds fed diets supplemented with nanoprotected REO. The 200 mg nanoprotected REO/kg diet supplementation resulted in significantly (P < 0.05) better villus height (VH) and villus height: crypt depth (VH: CD) ratio in all the segments of the small intestine. In conclusion, feeding REO in nanoprotected form in a 200 mg/kg diet could be used as an antibiotic substitute to improve broiler chicken's lipid profile, immune-antioxidant status, and intestinal health.